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On The Shuttling Across The Blood-brain Barrier Via Tubule Formation: Mechanism And Cargo Avidity Bias

Abstract
The blood-brain barrier is made of polarized brain endothelial cells (BECs) phenotypically conditioned by the central nervous system (CNS). Although transport across BECs is of paramount importance for nutrient uptake as well as ridding the brain of waste products, the intracellular sorting mechanisms that regulate successful receptor-mediated transcytosis in BECs remain to be elucidated. Here, we used a synthetic multivalent system with tunable avidity to the low-density lipoprotein receptor–related protein 1 (LRP1) to investigate the mechanisms of transport across BECs. We used a combination of conventional and super-resolution microscopy, both in vivo and in vitro, accompanied with biophysical modeling of transport kinetics and membrane-bound interactions to elucidate the role of membrane-sculpting protein syndapin-2 on fast transport via tubule formation. We show that high-avidity cargo biases the LRP1 toward internalization associated with fast degradation, while mid-avidity augments the formation of syndapin-2 tubular carriers promoting a fast shuttling across.
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INTRODUCTION
The human brain accounts for about 2 to 3% of the total body mass, and yet, it consumes up to 50% of the total intake of oxygen and glucose (1). Such a high energy demand is only possible because of a controlled gating of mass exchange with the body across a network of barriers that are phenotypically regulated by the brain cells. The most important of all gateways is the blood-brain barrier (BBB). This is the richest capillary network in the body that can effectively feed the brain components with about one capillary per neuron and about 10 to 15 μm of the average distance between one capillary to another (2, 3). Capillaries are made of polarized endothelial cells connected via tight junctions. Brain endothelial cells (BECs) are conditioned by the neighboring brain cells to limit passive transport by forming impermeable tight junctions, lacking fenestrations, and expressing efflux transporters that protect the brain from harmful compounds (3–5). BBB dysfunctions are at the core of aging, neurological degeneration, stroke, and multiple sclerosis (4). The BBB makes the brain impermeable to most therapeutics, leading to a bottleneck in drug development (5).
BECs control the transport of small molecules, such as glucose and amino acids, by expressing specialized solute carrier transporters on both apical (blood) and basal (brain) membranes that pump molecules across one by one (6). BECs overexpress transferrin (7), insulin receptors (8), and low-density lipoprotein receptor–related protein 1 (LRP1) (9–11), and these receptors are often involved in shuttling their respective ligands into trafficking membrane–enveloped carriers across the cell via a process collectively known as transcytosis (6, 12). Among these receptors, LRP1 is a critical motif highly expressed by neurons (13) and astrocytes (14), and it has been reported to bind to more than 40 ligands (10) undergoing rapid endocytosis with a half-life of less than 30 s (10, 15). LRP1 has been associated with the blood-to-brain efflux of lactoferrin (16), receptor-associated protein (RAP) (17), and Kunitz protease inhibitor (KPI) domain–containing proteins (18). Transcytosis is an active transport involving the rearrangement of large membrane volumes, and although it has been investigated in detail in other barrier tissues (such as the epithelium), little is known about it in endothelial cells (8, 12, 19). Epithelial, and by analogy endothelial, transcytosis involves three steps: (i) endocytosis, a vesicular carrier emerges from one side of the membrane, typically involving clathrin or caveolin; (ii) trafficking, the carrier moves toward and fuses with the endolysosome network; and eventually (iii) exocytosis, a new vesicular carrier emerges from endolysosome, moves toward, and fuses with the opposite side of the plasma membrane (8). This sequence of events is viable in thick epithelial cells but often endothelium can be as thin as few hundreds of nanometers (19), and as such, the internal volume is too small to house the machinery associated with the three transcytosis steps. Furthermore, although there is growing evidence supporting the role of transcytosis at the BBB, particularly via LRP1, the mechanism that determines whether the receptor is to be sorted for transcytosis or for degradation in lysosomes remains still enigmatic.
One of the parameters that appear to influence the mechanism of transcytosis at BECs is avidity of the cargo (20–23). Using a Brain Shuttle platform targeting transferrin receptor at BECs, it has been shown that a monovalent construct is successfully sorted for transcytosis and colocalizes with narrow intracellular tubules, while a bivalent one is sorted for degradation exhibiting impaired transport along such tubules (22). Previous ultrastructural observations also reported the formation of “pores” or “channels” spanning endothelial cells referred to as transendothelial channels (TECs) (24). Bundgaard (25) reconstructed three-dimensional (3D) projections from serial sections of transmission electron micrographs of hagfish BECs, showing that intracellular membranes arising from transcytosis were rarely single vesicles but, instead, part of large multidimensional dendritic networks or “tubes.” Tubular networks and chains of vesiculo-vacuolar organelles (VVOs) were also reported in fenestrated endothelium (26). Despite being widely observed using electron microscopy, the molecular identity and the mechanism regulating the formation of these tubular structures are still not completely understood, especially on transcytosis mediated by LRP1. A piece of essential information missing from all these studies is the role of the membrane-sculpting proteins, most notably those comprising a Bin/amphiphysin/Rvs (BAR) domain (27). Syndapin-2 is a Fer-CIP4 homology–BAR (F-BAR) protein that senses and induces positive curvature on membranes (i.e., invaginations) and thus stabilizes tubular carriers (28, 29) through the BAR domain. Apart from the BAR domain, syndapin-2 also contains an Src homology 3 (SH3) domain that binds to dynamin-2 and to the WASP/Scar family proteins that, ultimately, regulate actin filaments (27). Although syndapin-2 is ubiquitously expressed and associated with fundamental endocytic trafficking proteins, its functions in transcytosis at BECs are still to be unraveled.
Here, we elucidate the trafficking mechanism of LRP1 in BECs and correlate its transcytosis mechanism with syndapin-2 using both in vitro and in vivo models of the BBB. We use synthetic vesicles, polymersomes (POs), functionalized with LRP1 targeting moieties established to transverse the BBB to assess how multivalency, and hence binding avidity, controls LRP1-mediated transcytosis. We demonstrate that binding avidity controls transcytosis of LRP1 and further shed light on the mechanisms and dynamics of a unique mechanism of tubulation regulated by syndapin-2 on BECs.
RESULTS AND DISCUSSION
LRP1 trafficking across brain endothelium
To study the mechanism of LRP1-mediated transcytosis across BECs, we used a well-established 3D model of the BBB consisting of confluent mouse brain endothelioma cells (bEnd3) cultured onto collagen-coated porous transwell inserts (fig. S1A). We have established the barrier properties of this BBB model (30, 31) by measuring both the transendothelial resistance (TEER) and an apparent permeability coefficient of different molecular mass dextran (P), calculated as
P=
(1)
where C0 is the initial cargo concentration, A is the total surface area of the transwell membrane, and
dQdt
is the transport rate calculated as the gradient of mass over time. bEnd3 monolayers presented TEER values of ∼40 ohm·cm2, and for 4- and 70-kDa dextrans, we measured a permeability of P4kDa = 19.6 and P70kDa = 4.7 nm s−1, respectively (fig. S1B). bEnd3 monolayer presented a classical morphology with the expression of platelet endothelial cell adhesion molecule (PECAM-1) and tight junction proteins, claudin-5, and zona occludens 1 (ZO-1) (fig. S1C). Most relevant to the present study, we confirmed the expression of LRP1 in BECs using both Western blot (WB) (Fig. 1A) and immunofluorescence (Fig. 1B) targeting the cytosolic and extracellular domains, respectively. The micrographs collected across different monolayer regions show the wide expression of LRP1 in BECs (Fig. 1, B and C). Moreover, 3D reconstructions evidence that LRP1 is expressed on both the apical and basal cell surfaces as well as in the perinuclear area (Fig. 1D).

Fig. 2 Ligand avidity versus BBB crossing. (A) Heatmap showing the experimental measurement of % of AL-P crossing as a function of incubation time and ligand number per particle (L). (B) Ex vivo fluorescent photographs of whole murine brains imaged 2 hours after intravenous injection of PBS, pristine POs (L = 0), free angiopep-2 peptide (L = 1), A22-P, or A110-P. Violin plots showing the quantification in the brain parenchyma of the various preparations tested. **P < 0.01, ***P < 0.001, and ****P < 0.0001, one-way ANOVA (n = 6). (C) Concentration of angiopep-2 functionalized cargo expressed as percentage of injected dose (% ID) per gram of tissue as a function of the number of ligands. (D) Heatmap of the apparent permeability, P, obtained from agent-based simulations as a function of the ligand number per particle and the single ligand dissociation constant, Kd, with the LRP1 receptor. (E) Comparison between apparent permeability, P, across BBB experimental data (red markers and solid line) and simulation (blue markers and dashed lines) calculated for two different receptor densities and single ligand dissociation constant, Kd = 300 nM. Note that the control pristine PO apparent permeability was subtracted to the other formulations to remove passive diffusion. (F) Phase diagram showing different regimes of nanoparticle aggregation across the receptor densities and nanoparticle-receptor affinities expressed in kBT (with kB being the Boltzmann constant and T the temperature) as observed in MD simulations. Nanoparticle distributions are illustrated MD simulations using a coarse-grained membrane surface patch.
From a theoretical standpoint, transcytosis involves five major stages: binding, endocytosis, trafficking, exocytosis, and unbinding. Efficient transcytosis requires the formation of ligand/receptor bonds that last enough for it to be trafficked across; yet, the higher the ligand binding energy, the lower is its ability to detach once across to the other side. Therefore, a balance is required to form and maintain not only sufficiently strong bonds to enable binding and endocytosis but also a sufficiently weak bond to allow unbinding and release. Such an approximation allows the creation of an in silico model to stimulate transcytosis (see the Supplementary Materials). We used flexible large-scale agent-based modeling environment (FLAME), a generalized agent-based modeling platform, that models the behavior of individual POs undergoing Brownian motion, binding to endothelial cells, crossing the cells by transcytosis, and being released into the basal compartment (45). We designed the model based on the geometry of the transwell insert used in the in vitro experiments (fig. S1A), and the BECs were modeled as a uniform 2-μm-thick layer at the top of the insert. We also modeled POs with different ligand numbers and different individual ligand-receptor dissociation constants, starting them at time zero in the aqueous apical phase. POs were subjected to Brownian motion and bound to cells according to the multivalent-avidity binding model described in the Supplementary Materials. The particles were allowed to go through the different stages of transcytosis as described in fig. S5A, and the number of POs that crossed the BECs was measured. We thus used Eq. 1 to calculate the apparent permeability and plotted it as a function of both ligand number per particle (L) and the single ligand/receptor dissociation constant (Kd). We used models for nanoparticles with radius R= 20 and 50 nm, as well as receptor densities RD= 15 and 30 μm−2, respectively. According to the simulations, there is a nonlinear dependence between ligand number and binding strength (fig. S5B), whereby the optimal transcytosis is obtained in a “Goldilocks” regime of avidity, i.e., not too strong and not too weak, and it is independent of the particle size or receptor density. We selected both size and receptor density to match our in vitro experimental data, and within such a range, our simulations suggest that bigger particles and larger receptor density lead to improved transcytosis. In Fig. 2D, we plot the apparent permeability across the BBB as a function of ligand numbers per particle (L) and the dissociation constant of the ligand/receptor binding (Kd) for particles with radius R = 50 nm and receptor density RD = 30 μm−2, which is very close to what we recently estimated using the super-selective theory (46). We know from previous work that angiopep-2 has a dissociation constant Kd = 313 nM (47), and using this, we can thus compare the simulations at similar dissociation constant with the experimental data. In Fig. 2E, we plot the experimental apparent permeability (in red), measured from the data in Fig. 2A and the simulations with Kd = 300 nM, particle with size R = 50 nm, and receptor densities RD = 30 μm−2 or RD = 15 μm−2. The experimental and simulation data show broad agreement, and the Goldilocks avidity effect is reproduced experimentally at similar values to those we observed computationally.
Last, we complemented both computational and experimental permeability measurements by performing molecular dynamics (MD) simulations to capture the effect of avidity on membrane topological changes and nanoparticle aggregation dynamics. We used a well-established coarse-grained membrane surface patch (fig. S5C) on an equilibrated spherical membrane and varied the receptor density and the nanoparticle to membrane binding energy (ϵ) expressed in kBT, with kB being the Boltzmann constant and T the temperature. The latter represents the depth of the potential well in the attractive interaction between nanoparticles and “receptor” membrane beads (see Eq. 3 in the Supplementary Materials). A different initial nanoparticle distribution was randomly chosen for each simulation, and each parameter pair used the same set of six different initial nanoparticle distributions. The receptor density was represented in the model by the ratio of receptor membrane beads to the total number of membrane beads. The simulation results are summarized in Fig. 2F, and it is evident that across all receptor densities, no clear binding of the nanoparticles was observed for low binding energy. Some receptor beads clustered around individual adsorbed nanoparticles, but the nanoparticle-receptor adhesion was too weak to drive any interaction. As the binding energy increases, the nanoparticles bind to the membrane. While their relative adhesion energy is converted into membrane deformation, this is not sufficiently strong to induce full endocytosis. Nonetheless, progressively more particles bind the associated membrane deformations forming linear aggregates. The anisotropic aggregation is the consequence of the trade-off between nanoparticle-receptor adhesion and the membrane’s resistance to deformation (48). Higher binding energy results in the linear aggregates that can be internalized as tubular aggregates. These can coexist on a membrane together with membrane-bound tubular aggregates and internalized tubular aggregates. At higher receptor densities, lower binding energies are required for the nanoparticles to form tubular and linear aggregates. However, at high receptor density and high binding energy, the particles have sufficient adhesion to create singular deformation and enter via discrete endocytic events. The pseudo-phase diagram in Fig. 2F shows the limits of the different regimes observed in the simulations and the collective processes leading to different outcomes. As shown in fig. S5D, tubulation results in a high number of cargo units transported per single event, while higher binding energy and receptor density correspond to fewer number of particles per internalized carrier. The latter process is more efficient in internalizing the nanoparticles reaching almost 100%, while the collective process that occurs at lower binding energy and receptor density achieves only a much lower percentage of internalization (fig. S5E). Together, the MD simulations add another dimension to the avidity effect, showing that different binding energies drive alternative membrane deformations including tubulation and that these lead to a different endocytic initiation.
Involvement of syndapin-2 in transcytosis as a function of avidity
The data in Fig. 1 showed that LRP1 is associated with several endocytic and trafficking elements, suggesting that the receptor is physiologically processed in endosomes and lysosomes, but it is also shuttled in tubular structures stabilized by syndapin-2. In addition, we demonstrated that transcytosis of LRP1 is driven by avidity and that fast shuttling across the BBB is associated with tubular structures. To further understand whether syndapin-2 is implicated into tubulation at BECs, we repeated the PLA assay between LRP1 and the endocytic proteins and syndapin-2, but this time, BECs were exposed to angiopep-2 peptide (L = 1) and AL-P formulations, L = 22 and L = 110. The data reported in Fig. 3A as a variation between the treated and untreated cells reveal how the avidity of the ligand for LRP1 affects the localization of the receptor within the cells. At an early incubation time (0.25 hour), the single peptide (L = 1) reduces the proximity events between LRP1 and clathrin by more than five times, while prolonged incubation promotes the association of the receptor with the late endosome marker Rab7. The two AL-P formulations have a more marked effect. Incubation with L = 22 prevents the interaction of LRP1 with all the endolysosomal compartments at any time point, with Rab5 showing the most notable decrease. The presence of L = 22 at 0.25 hour also increases the interaction of the receptor with actin, tubulin, and clathrin. On the other hand, incubation with L = 110 increases LRP1 interaction with both Rab7 and Rab11 in the short term, while it constantly reduces the association with syndapin-2. At later time points, incubation with L = 110 also decreases the proximity between LRP1 and tubulin or clathrin. Note that oscillations of the values between −5 and +5 were considered as physiological fluctuations. Our data suggest two trends: one is the association of LRP1 with syndapin-2 and one is with Rab5. We then plotted the ratio of the relative interactions between LRP1/Rab5 and LRP1/syndapin-2 (Rab5/syndapin-2) as a function of incubation time and ligand number (Fig. 3B). While angiopep-2 peptide does not alter the Rab5/syndapin-2 ratio, both L = 22 and L = 110 do but with opposite trends. L = 22 pushes the interaction of LRP1 toward syndapin-2 for all time points, while L = 110 biases LRP1 toward the endosomal protein Rab5. As we expected that LRP1 association with endolysosomal markers should result in its degradation, we assessed its levels of expression over time following incubation with L = 1, L = 22, and L = 110 (Fig. 3C). The WB results show that LRP1 is unaltered after up to 1-hour incubation with angiopep-2 and L = 22. In contrast, exposure to L = 110 results in a fast reduction of LRP1 expression, which then recovers to physiological levels after 2 hours of incubation. We observed a twofold increase in LRP1 expression after 2 hours of incubation with L = 22. Overall, both PLA and WB analyses suggest that LRP1 can follow two different intracellular pathways across BECs and their schematics are shown in Fig. 3D. One pathway is mediated by syndapin-2, β-actin, and, possibly, clathrin. Here, LRP1 shuttles across tubular carriers from apical to basal and vice versa, avoiding endolysosomal degradation and sorting. The other pathway is a conventional endocytosis where LRP1 enters the cells and gets trafficked to endosomes and lysosomes where it is degraded. On the basis of our findings, these pathways are driven by cargo avidity: Intermediate ligand numbers push more to the syndapin-2 pathway associated with tubular deformations, while the higher number of ligands and avidity pushes the cargo more toward endosomal sorting.

Fig. 3 LRP1 subcellular localization and expression as a function of avidity. (A) Deviation of the number of proximity events measured by a PLA between untreated endothelial cells and treated for 0.25, 1, and 2 hours of incubation with free angiopep-2 peptide, L = 1, and AL-P, with L = 22 and L = 110. Note that zero corresponds to no variation, while positive and negative values indicate up- and down-regulation, respectively. (B) Ratio between LRP1/Rab5 and LRP1/syndapin-2 number of proximity events for the different treatments with free angiopep-2 peptide, L = 1, and AL-P, where L = 22 and L = 110, with Rab5/syndapin-2 being 10 for the untreated cells. (C) WB measuring the LRP1 expression relative to the untreated cells for free angiopep-2 peptide, L = 1, and AL-P, with L = 22 and L = 110 measured at different incubation times with 0.25, 1, and 2 hours. *P < 0.05, **P < 0.01, and ***P < 0.001, one-way ANOVA (n = 6). Note that LRP1 expression is normalized to the loading control. (D) Diagram showing the syndapin-2–mediated transcellular route and the intracellular degradation of LRP1.
Tubular transcytosis mechanism
To further shed light on the novel shuttling mechanism, we used a combination of qualitative and quantitative confocal microscopy in conjunction with antibodies and small-molecule inhibitors against proteins of interest. First, we coincubated the A22-P with the free peptide to provide an insight on whether transcytosis is more efficient when angiopep-2 is alone or when attached to the POs. Quantification of fluorescence is shown in fig. S6A. After 10 min of coincubation, A22-P fluorescence is of similar intensity of angiopep-2 and much lower than that of A22-P after 10 min with no competing ligand. Free peptide fluorescence remains similar to levels without competition. Such results show that angiopep-2 and A22-P compete for LRP1 binding and endocytosis, as expected, but also that the free peptide inhibits PO internalization more than vice versa. When coincubated, the intensities of A22-P and angiopep-2 are both markedly higher at 60 min compared to when added without competition. However, competition for A22-P shows a biphasic shift in behavior compared to the A22-P only control: decreased endocytosis at 10 min and increased intracellular residence, i.e., decreased exocytosis at 60 min. The biased inhibition of A22-P transcytosis rather than angiopep-2 may be due to more rapid or efficient endocytosis, intracellular trafficking, and exocytosis pathway occurring for A22-P than for angiopep-2.
We subsequently studied the mechanisms of endo- and exocytosis of Cy5-labeled A22-P during transcytosis. Confocal studies suggested that clathrin, but not caveolin, is involved in the mechanism of internalization of A22-P (fig. S6, B and C). High-magnification confocal images in fig. S6B demonstrate that A22-P fluorescence is closely associated with clathrin after 60 min of incubation. However, these data are qualitative and are thus only an indication that clathrin is involved in transcytosis of A22-P. We performed similar experiments to evaluate the association of Cy5-labeled A22-P with caveolin-1, and as shown in fig. S6C, a partial overlap was observed initially at 10 min of incubation. However, 3D z-stack projections in fig. S6D display no apparent colocalization at 10 min. A few cytoplasmic puncta with fluorescence overlap were observed at 60 min. However, r values for A22-P and caveolin-1 remained low along the time with r = 0.2 and −0.02 at 10 and 60 min, respectively. Overall, these findings fail to show a role for caveolae as essential structures for apical and basal transcytosis, particularly, as a higher colocalization would be anticipated at 10 min when the majority of transcytosis is occurring. Cytoskeletal motor proteins can quickly transport cargo from one side of a cell to another and were therefore of particular interest for their potential involvement in transcytosis. We thus investigated the role of actin in BEC transcytosis by colocalization of Cy5-labeled A22-P with phalloid-488 (an established marker for F-actin). Confocal images are displayed in fig. S6E, with a magnification of an area of interest (fig. S6E1), along with r values at 10, 30, and 60 min for A22-P and F-actin. The data suggest that actin has a role in transporting POs from the apical to basal membrane within the first few minutes of endocytosis. The time scale of BEC transcytosis and unconventional intracellular trafficking pathways prompted us to further explore the identity of intracellular transport vesicles as well as membrane deformation mediators in transcytosis. Small-molecule inhibitors of endocytosis or exocytosis were used in conjunction with live-cell imaging to obtain transwell z stacks. Incubation with dynasore, a cell-permeable inhibitor of dynamin, impaired transcytosis and caused Cy5-labeled A22-P to remain stuck on the BEC surface (fig. S6, F and G). These effects were reversible upon removal of the inhibitor, as the A22-P were visible both inside cells and in transwell membrane pores. Dynamin may, therefore, be a required cellular component of the internalization stage of transcytosis in BECs. In a separate experiment, N-ethylmaleimide (NEM) was used to inhibit NEM soluble factor (NSF) to inhibit exocytosis indirectly. A22-P remained aggregated on top of the cells after incubation for 60 min (fig. S6, F and G). Thus, NSF may participate not only in exocytosis of cargos once inside the cell but also in endocytosis. To further explore the role of NSF and soluble NSF attachment receptors (SNAREs) in transcytosis, a cell membrane cholesterol depletion method was used to disrupt lipid raft containing SNAREs (49). Cells were preincubated for 60 min with methyl-β-cyclodextrin (CD) added to either the apical or basal compartment of the transwell. A cholesterol quantification assay revealed a slight asymmetry in measured free cholesterol in the medium in the apical and basal compartments (fig. S7A). Depletion of cholesterol in the apical or basal membrane resulted in an approximately twofold or three- to fourfold increase in cholesterol released into the apical or basal side of the transwell, respectively (fig. S7B). Such an effect may be indicative of a stronger effect of cholesterol depletion on the basal membrane. Confocal images were acquired from Cy5-labeled A22-P incubated for 60 min in BECs with CD added to the apical or basal side of the transwell (fig. S7C). Basal membrane cholesterol depletion showed an increase in intracellular A22-P after 60 min compared to untreated cells, which may be due to the ability of cells to do endocytose but not exocytose the cargo. Together, these findings suggest the involvement of dynamin and also NSF in the LRP1-mediated cargo internalization stage of BEC transcytosis. Depletion of cholesterol in the basal side of BECs inhibited exocytosis but not endocytosis, which may suggest a role for cholesterol in transcytosis. We next assessed whether the trafficking from apical to basal involves sorting into endosomes and acidification, as we already demonstrated (30). A22-P do not colocalize with endosome and lysosomes crossing the BECs without losing integrity. Here, we represent confocal images acquired from A22-P in BECs fixed and stained for Rab guanosine triphosphatases of endosomal organelles in fig. S7D. There was no colocalization between POs and any of the markers at any time investigated. Colocalization quantification (fig. S7E) indicated no association between A22-P and Rab5, Rab7, Rab11, and LAMP-1. On the contrary, r values displayed a negative trend implicating negative association, i.e., exclusion of A22-P from these organelles.
Last, we confirmed the colocalization between A22-P and syndapin-2 in our in vitro BBB model. In Fig. 4A, 3D rendering of polarized BECs imaged 30 min after incubation with the Cy5-labeled A22-P (red) shows very effectively that A22-P cross the cell through tubular structures coated with syndapin-2 (in green). To further show the involvement of syndapin-2 on the transcytosis of A22-P, we modulated the expression of syndapin-2 on BECs and assessed the transport of A22-P across an in vitro BBB model (fig. S8). Specifically, we performed short hairpin RNA (shRNA) on bEnd3 to knock down syndapin-2, generating a stable cell line expressing significantly less syndapin-2, as confirmed by WB (fig. S8A). When cultured onto collagen-coated transwells, these syndapin-2 knockdown bEnd3 showed permeability P4kDa = 25.6 and P70kDa = 5.4 nm s−1, which are similar to the values obtained for bEnd3 transfected with a control shRNA (fig. S8B). We then assessed the transport of A22-P across BECs expressing different levels of syndapin-2. In fig. S8C, we observe a twofold decrease in the apparent permeability of A22-P from apical to basal side when compared to bEnd3 expressing normal endogenous levels of syndapin-2. These results further indicate the involvement of syndapin-2 in the transport across BECs. We complemented the colocalization of syndapin-2 and A22-P with animal studies where we injected either A22-P or pristine POs loaded with PtA2. In Fig. 4B, the ex vivo fluorescent photographs of whole brains extracted from healthy mice 30 min after injection show the effective delivery of the dye by functionalized POs. PtA2 has unique fluorescence characteristics with a wide Stoke shift and extremely bright emission, allowing us to visualize the PO penetration with high sensitivity. The metallic nature of the dye allows quantification of its biodistribution by ICP-MS. The graph in Fig. 4C shows an extremely effective delivery of the dye into the brain with a staggering brain/liver ratio of about 8.2 opposite to the pristine POs, where the majority of the dye is found in liver and spleen. Such a high concentration of dye allows us to visualize A22-P penetration in the brain capillary by TEM (fig. S9A) and STED. The histology in Fig. 4D demonstrates that A22-P cross the brain endothelium (stained with lectin in green) via the formation of tubules as shown in regions of interest 1 and 2. We then imaged brain sections collecting 30 optical slides, and the corresponding 3D renderings are shown in Fig. 4E, where the A22-P loaded with PtA2 (red) are imaged alongside the capillary walls (green) and syndapin-2 (blue) with improved spatial resolution. The rendering showed very well that A22-P colocalize into tubular structures coated by the F-BAR protein syndapin-2, with dimensions in agreement of what we observed in vitro and by the simulations.

Fig. 3 LRP1 subcellular localization and expression as a function of avidity. (A) Deviation of the number of proximity events measured by a PLA between untreated endothelial cells and treated for 0.25, 1, and 2 hours of incubation with free angiopep-2 peptide, L = 1, and AL-P, with L = 22 and L = 110. Note that zero corresponds to no variation, while positive and negative values indicate up- and down-regulation, respectively. (B) Ratio between LRP1/Rab5 and LRP1/syndapin-2 number of proximity events for the different treatments with free angiopep-2 peptide, L = 1, and AL-P, where L = 22 and L = 110, with Rab5/syndapin-2 being 10 for the untreated cells. (C) WB measuring the LRP1 expression relative to the untreated cells for free angiopep-2 peptide, L = 1, and AL-P, with L = 22 and L = 110 measured at different incubation times with 0.25, 1, and 2 hours. *P < 0.05, **P < 0.01, and ***P < 0.001, one-way ANOVA (n = 6). Note that LRP1 expression is normalized to the loading control. (D) Diagram showing the syndapin-2–mediated transcellular route and the intracellular degradation of LRP1.
Tubular transcytosis mechanism
To further shed light on the novel shuttling mechanism, we used a combination of qualitative and quantitative confocal microscopy in conjunction with antibodies and small-molecule inhibitors against proteins of interest. First, we coincubated the A22-P with the free peptide to provide an insight on whether transcytosis is more efficient when angiopep-2 is alone or when attached to the POs. Quantification of fluorescence is shown in fig. S6A. After 10 min of coincubation, A22-P fluorescence is of similar intensity of angiopep-2 and much lower than that of A22-P after 10 min with no competing ligand. Free peptide fluorescence remains similar to levels without competition. Such results show that angiopep-2 and A22-P compete for LRP1 binding and endocytosis, as expected, but also that the free peptide inhibits PO internalization more than vice versa. When coincubated, the intensities of A22-P and angiopep-2 are both markedly higher at 60 min compared to when added without competition. However, competition for A22-P shows a biphasic shift in behavior compared to the A22-P only control: decreased endocytosis at 10 min and increased intracellular residence, i.e., decreased exocytosis at 60 min. The biased inhibition of A22-P transcytosis rather than angiopep-2 may be due to more rapid or efficient endocytosis, intracellular trafficking, and exocytosis pathway occurring for A22-P than for angiopep-2.
We subsequently studied the mechanisms of endo- and exocytosis of Cy5-labeled A22-P during transcytosis. Confocal studies suggested that clathrin, but not caveolin, is involved in the mechanism of internalization of A22-P (fig. S6, B and C). High-magnification confocal images in fig. S6B demonstrate that A22-P fluorescence is closely associated with clathrin after 60 min of incubation. However, these data are qualitative and are thus only an indication that clathrin is involved in transcytosis of A22-P. We performed similar experiments to evaluate the association of Cy5-labeled A22-P with caveolin-1, and as shown in fig. S6C, a partial overlap was observed initially at 10 min of incubation. However, 3D z-stack projections in fig. S6D display no apparent colocalization at 10 min. A few cytoplasmic puncta with fluorescence overlap were observed at 60 min. However, r values for A22-P and caveolin-1 remained low along the time with r = 0.2 and −0.02 at 10 and 60 min, respectively. Overall, these findings fail to show a role for caveolae as essential structures for apical and basal transcytosis, particularly, as a higher colocalization would be anticipated at 10 min when the majority of transcytosis is occurring. Cytoskeletal motor proteins can quickly transport cargo from one side of a cell to another and were therefore of particular interest for their potential involvement in transcytosis. We thus investigated the role of actin in BEC transcytosis by colocalization of Cy5-labeled A22-P with phalloid-488 (an established marker for F-actin). Confocal images are displayed in fig. S6E, with a magnification of an area of interest (fig. S6E1), along with r values at 10, 30, and 60 min for A22-P and F-actin. The data suggest that actin has a role in transporting POs from the apical to basal membrane within the first few minutes of endocytosis. The time scale of BEC transcytosis and unconventional intracellular trafficking pathways prompted us to further explore the identity of intracellular transport vesicles as well as membrane deformation mediators in transcytosis. Small-molecule inhibitors of endocytosis or exocytosis were used in conjunction with live-cell imaging to obtain transwell z stacks. Incubation with dynasore, a cell-permeable inhibitor of dynamin, impaired transcytosis and caused Cy5-labeled A22-P to remain stuck on the BEC surface (fig. S6, F and G). These effects were reversible upon removal of the inhibitor, as the A22-P were visible both inside cells and in transwell membrane pores. Dynamin may, therefore, be a required cellular component of the internalization stage of transcytosis in BECs. In a separate experiment, N-ethylmaleimide (NEM) was used to inhibit NEM soluble factor (NSF) to inhibit exocytosis indirectly. A22-P remained aggregated on top of the cells after incubation for 60 min (fig. S6, F and G). Thus, NSF may participate not only in exocytosis of cargos once inside the cell but also in endocytosis. To further explore the role of NSF and soluble NSF attachment receptors (SNAREs) in transcytosis, a cell membrane cholesterol depletion method was used to disrupt lipid raft containing SNAREs (49). Cells were preincubated for 60 min with methyl-β-cyclodextrin (CD) added to either the apical or basal compartment of the transwell. A cholesterol quantification assay revealed a slight asymmetry in measured free cholesterol in the medium in the apical and basal compartments (fig. S7A). Depletion of cholesterol in the apical or basal membrane resulted in an approximately twofold or three- to fourfold increase in cholesterol released into the apical or basal side of the transwell, respectively (fig. S7B). Such an effect may be indicative of a stronger effect of cholesterol depletion on the basal membrane. Confocal images were acquired from Cy5-labeled A22-P incubated for 60 min in BECs with CD added to the apical or basal side of the transwell (fig. S7C). Basal membrane cholesterol depletion showed an increase in intracellular A22-P after 60 min compared to untreated cells, which may be due to the ability of cells to do endocytose but not exocytose the cargo. Together, these findings suggest the involvement of dynamin and also NSF in the LRP1-mediated cargo internalization stage of BEC transcytosis. Depletion of cholesterol in the basal side of BECs inhibited exocytosis but not endocytosis, which may suggest a role for cholesterol in transcytosis. We next assessed whether the trafficking from apical to basal involves sorting into endosomes and acidification, as we already demonstrated (30). A22-P do not colocalize with endosome and lysosomes crossing the BECs without losing integrity. Here, we represent confocal images acquired from A22-P in BECs fixed and stained for Rab guanosine triphosphatases of endosomal organelles in fig. S7D. There was no colocalization between POs and any of the markers at any time investigated. Colocalization quantification (fig. S7E) indicated no association between A22-P and Rab5, Rab7, Rab11, and LAMP-1. On the contrary, r values displayed a negative trend implicating negative association, i.e., exclusion of A22-P from these organelles.
Last, we confirmed the colocalization between A22-P and syndapin-2 in our in vitro BBB model. In Fig. 4A, 3D rendering of polarized BECs imaged 30 min after incubation with the Cy5-labeled A22-P (red) shows very effectively that A22-P cross the cell through tubular structures coated with syndapin-2 (in green). To further show the involvement of syndapin-2 on the transcytosis of A22-P, we modulated the expression of syndapin-2 on BECs and assessed the transport of A22-P across an in vitro BBB model (fig. S8). Specifically, we performed short hairpin RNA (shRNA) on bEnd3 to knock down syndapin-2, generating a stable cell line expressing significantly less syndapin-2, as confirmed by WB (fig. S8A). When cultured onto collagen-coated transwells, these syndapin-2 knockdown bEnd3 showed permeability P4kDa = 25.6 and P70kDa = 5.4 nm s−1, which are similar to the values obtained for bEnd3 transfected with a control shRNA (fig. S8B). We then assessed the transport of A22-P across BECs expressing different levels of syndapin-2. In fig. S8C, we observe a twofold decrease in the apparent permeability of A22-P from apical to basal side when compared to bEnd3 expressing normal endogenous levels of syndapin-2. These results further indicate the involvement of syndapin-2 in the transport across BECs. We complemented the colocalization of syndapin-2 and A22-P with animal studies where we injected either A22-P or pristine POs loaded with PtA2. In Fig. 4B, the ex vivo fluorescent photographs of whole brains extracted from healthy mice 30 min after injection show the effective delivery of the dye by functionalized POs. PtA2 has unique fluorescence characteristics with a wide Stoke shift and extremely bright emission, allowing us to visualize the PO penetration with high sensitivity. The metallic nature of the dye allows quantification of its biodistribution by ICP-MS. The graph in Fig. 4C shows an extremely effective delivery of the dye into the brain with a staggering brain/liver ratio of about 8.2 opposite to the pristine POs, where the majority of the dye is found in liver and spleen. Such a high concentration of dye allows us to visualize A22-P penetration in the brain capillary by TEM (fig. S9A) and STED. The histology in Fig. 4D demonstrates that A22-P cross the brain endothelium (stained with lectin in green) via the formation of tubules as shown in regions of interest 1 and 2. We then imaged brain sections collecting 30 optical slides, and the corresponding 3D renderings are shown in Fig. 4E, where the A22-P loaded with PtA2 (red) are imaged alongside the capillary walls (green) and syndapin-2 (blue) with improved spatial resolution. The rendering showed very well that A22-P colocalize into tubular structures coated by the F-BAR protein syndapin-2, with dimensions in agreement of what we observed in vitro and by the simulations.

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About – This $1.7 Million Motor Home With Its Own Garage

For most people, the term ‘motor home’ doesn’t exactly scream ‘luxury.’ One German company, however, is begging to differ, and they’ve just rolled out a $1.7m beast-on-wheels that makes your dad’s dingy 1973 GMC look like a Hot Wheels truck. The Performance S is the latest model in Volkner Mobil’s lauded Performance series of motorhomes, and much like its predecessors, it’s basically a 5-star hotel that you can park wherever you like. The sleek and stunning 40-foot vehicle contains a double bed, a fully-equipped kitchen, a spacious lounge area and a heated bathroom. In case all of that just isn’t enough high society for you, the classy caravan even has a garage with an electrohydraulic lift, and it suits everything from a Ferrari to a Mercedes.
So, if you’re itching to set off on the open road, but you’re not ready to leave behind the comforts of your penthouse and your beloved Lambo, the Performance S just might be the perfect adventure vessel for you – provided you have about 2 million bucks laying around to spare. Scroll down to see it for yourself.
This is the Volkner Mobil Performance S, the most luxurious motorhome you’ve ever seen
Inside the 40-foot, 1.7 million-dollar vehicle, you can find a 5-star hotel on wheels

A comfy double bed…

A heated bathroom…

And best of all, an hydroelectric garage big enough to fit anything from a Ferrari to a Mercedes

Itching to hit the road but can’t do it without your ‘extra’ lifestyle? This is the ride for you
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About – What is Firebase? – DEV

Firebase is a Backend-as-a-Service – BaaS – that’s currently owned and developed by Google. Firebase frees developers to focus on crafting fantastic user experiences, free from the burdens and horrors of writing, deploying, and maintaining server clusters.

No more scary servers
Yes, there are maybe some specific functions that you’ll have to use Google Cloud or other Cloud services for, but for the most part, Firebase is written generically that it can be used everywhere for everything.
So what is Firebase?

We’re going to be covering that first third (‘Build Better Apps’) in depth, and leave the other two for you to mull over on your own time.
Realtime Database
Realtime data is the way of the future. Gone are the days or REST API’s that rely on HTTP to get and sync data, leading to slow async behavior as you wait for data, and constant calls to the server to get up to date data.
When you use the Firebase database, you’re not connecting through normal HTTP, but instead a WebSocket. Web sockets create a two way connection that is real time. That means no waiting for a HTTP call to finish, you get data updates as fast as the network can carry them. When updating data in an application, it’s synced to the database and other users practically instantly.
Even better, all of this is already setup with the easy ability to add authentication rules to protect your data how you see fit.
File Storage
Firebase Storage provides a simple way to save files, from images to archives, to Google Cloud Storage directly from the client. Firebase Storage comes with its own set of security rules to ensure you control what’s being uploaded to your server.
Authentication
Firebase Auth has a variety of authentication providers, from traditional email/password to Github, Google, Twitter, and even phone verification. You are free from having to write convulted authentication systems that may or may not be secure.
Firebase Authentication also works seamlessly with the rest of Firebase, such as the Realtime Database and Storage to ensure that you can easily control who’s accessing your data.
Hosting
Firebase has an easy-to-use hosting service for your static files, served from a global CDN with HTTP2. You can easily deploy your app from the command line across the world.
Serverless Functions
Firebase Functions provide an easy way to write and deploy serverless functions. What are serverless functions, well in a nutshell they’re functions that only run when called, that means no Express HTTP server running 24/7 in the cloud, saving cost. Chron jobs, HTTP calls, and triggers from other Firebase actions are all built in, making it super easy to integrate with your system.
ML Kit
Firebase also includes an SDK for common ML tasks, such as image recognition. A bunch of ML applications are provided out of the box, but you can also upload a custom Tensorflow model.
A Bunch of Others
I’m not going to cover the whole suite, but there’s also:
Analytics — understand your users, and how they use your app

Predictions — apply machine learning to analytics to predict user behavior

Cloud Messaging — send messages and notifications to users

Remote Config — customize your app without deploying a new version; monitor the changes

A/B Testing — run marketing and usability experiments to see what works best

Dynamic Links — enable native app conversions, user sharing, and marketing campaigns

App Indexing — re-engage users with Google Search integration

In-App Messaging — engage your active users with targeted messages
Test Lab — scalable and automated app testing on cloud-hosted devices

Crashlytics — get clear, actionable insight into your app’s crashes

Performance Monitoring — gain insight into your app’s performance issues
AHHH That’s a lot of features, I’ve yet to use all the features in one app, but I’m looking forward to that day.
Who’s Firebase For?
Anyone who needs a backend! It’s designed to integrate really well with web and mobile applications with SDK’s for tons of different languages. And the best part, it’s super duper cheap. The free tier is extremely generous, so you can play around to your hearts content with the various features that Firebase offers.
Quick Summary:
What Firebase Is
• Firebase is Google’s mobile application development platform
• You’re going to save tons of time and money using Firebase products instead of building them yourself
• You can use all of it, parts of it, or a single piece of it
• All those parts work together like a well oiled engine
Cross Posted from Comet Code
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What’s Next With The Latest In App Analytics

Steve Ganem Senior Product Manager
Last year we announced that app developers could upgrade their Firebase projects to the next generation of app analytics. Upgrading enables0 them to view their app analytics data in Analytics and unlocks additional analysis capabilities.
Since then, we’ve expanded more Google Analytics features like automated and custom insights to also include app data so that you can more quickly identify key trends and anomalies from your app reporting. Earlier this year we introduced a gaming-specific Analytics experience to help mobile game developers more easily see how players move through the lifecycle. And to bring predictive insights to your site and app, we rolled out new predictive capabilities in Analytics – not only helping you reach customers most likely to purchase, but also giving you new ways to retain those less likely to return to your app via App campaigns in Google Ads.
We are continuing our investment in the app ecosystem and today, we are introducing new updates to Google Analytics that will help you get the insights you need to be ready for what’s next. Let’s take a look at some new features you can use when you upgrade.
View your app’s revenue sources together
The ability to measure all your revenue sources helps you monetize and grow your apps business. Soon, you’ll be able to view impression-level revenue data in Analytics from AdMob mediated revenue and from other third-party app advertising platforms – giving you a holistic view of your customers’ lifetime value.

You can now view revenue from MoPub and ironsource in the new Analytics.
To get started, use the Google Analytics for Firebase SDK to log the ad_impression event whenever users see an ad impression. Be sure to include details such as the ad platform, source, currency and value.
With this revenue data now in Analytics, you can build audiences of high-value users and reach them for re-engagement campaigns. Third-party ad revenue will also soon be available as an experiment objective in A/B testing with Firebase. This way, you can test changes in user experience and see which drive more revenue through third-party platforms like MoPub or ironSource.
Use new custom dimensions and metrics
In the past, custom parameter reporting in Analytics for Firebase required you to register parameters for each event individually, which is time intensive and quickly uses up your quota. With event-scoped custom dimensions and metrics in the new Analytics, you only need to register each event once at the property level. You can also create and edit custom dimensions and metrics in the “All Events” section for your entire property. Plus, custom parameters you’ve previously created will automatically be upgraded to custom dimensions and metrics.

Create a custom dimension for your entire property.
Let’s say you’re a game app developer and you want your Analytics reports to show the levels at which users are starting, quitting, retrying, and ending your game. Previously, you’d need to register a custom event parameter for every single event. So with four events (starting, quitting, retrying, and ending) you’d have to register a parameter, “level,” four times. With the new Analytics, one single metric, “level,” is applied at the property level across all events — reducing the number of custom metrics your property uses.
Reach people with signed-in user insights
When users are signed in on your Android or iOS app, Analytics can help you connect the customer journey across platforms and devices with a special view in your reporting. Now, you can use those signed-in user insights to create relevant audiences and reach them with personalized messages in remarketing campaigns. And with the new Analytics, we’ve provided you with more granular controls for ads personalization so that you can choose when to use your data to optimize ads and when to limit your data use for measurement.
Let’s say you’re a lifestyle retail brand with a conversion rate on your mobile app that surpasses the rate on your website. Taking a closer look, you might notice a cohort of returning customers who visit your website for lifestyle content but never make a purchase. You can group the signed-in visitors into an audience and reach out to them with a marketing promotion, driving them to your app, where they have a higher likelihood to convert. For those who convert within the app, you can understand their complete customer journey across platforms and more effectively analyze the success of your promotion and adjust from there.
Upgrade to the new Analytics
The enhanced intelligence of Analytics provides additional revenue data to help improve your advertising strategy, simplified and efficient event measurement, and tailored experiences for increased conversion opportunities. If you aren’t already using the new Google Analytics, upgrade to the new Google Analytics from the Firebase console today.

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India Needs More Transparency In Its COVID-19 Vaccine Trials, Critics Say

Workers at a Hindustan Syringes & Medical Devices factory in India. The nation hopes to begin to administer a COVID-19 vaccine later this year. SAJJAD HUSSAIN/AFP VIA GETTY IMAGES
Nov. 25, 2020
Science’s COVID-19 reporting is supported by the Pulitzer Center and the Heising-Simons Foundation.
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Last month, Anil Hebbar, a health entrepreneur, spoke to the media about his experience volunteering for a COVID-19 vaccine trial at King Edward Memorial Hospital in Mumbai, India. He says he wanted to demystify the process of volunteering in a trial. But the hospital’s dean, Hemant Deshmukh, responded with a threat, telling The Times of India the hospital may “be forced to not give this volunteer the second shot” in the study.
Hebbar ultimately did receive his second dose. But the exchange highlighted ongoing concerns about the transparency of India’s COVID-19 vaccine trials. The nation now has five vaccine candidates in various stages of human testing. But the design, conduct, and regulation of these trials is often opaque, said researchers, bioethicists, journalists, lawyers, and others who participated in webinars hosted this month by the nonprofit Sama Resource Group for Women and Health.

Related With more data on its COVID-19 vaccine, Russian institute offers new evidence of success For scientists studying ‘disaster fatigue,’ this has been a year like no other Another COVID-19 vaccine success? Candidate may prevent further coronavirus transmission, too See all of our coverage of the coronavirus outbreak
“Don’t ask the public to trust you blindly,” says Amar Jesani, editor of the Indian Journal of Medical Ethics. “Come and gain the trust of the people, that is what I would ask.”
“Much of what we’re learning about the trials now is from bits and pieces of information being deposited in the press,” says Anant Bhan, an independent policy and bioethics researcher. That’s a problem because that doesn’t give a clear picture of who’s involved in the vaccine trials and their roles, or the study designs and timelines, he says.
The trial Hebbar took part in has helped highlight such concerns. The study, sponsored by the Serum Institute of India, is testing Covishield, the institute’s version of the University of Oxford’s and AstraZeneca’s COVID-19 vaccine candidate. But the institute does not appear to be using AstraZeneca’s phase III testing protocol, say analysts who have examined the few details provided on the Clinical Trials Registry-India (CTRI). The institute’s phase II/III clinical trial, which aims to recruit 1600 healthy volunteers, does not seem to be studying efficacy, but is instead focused on safety and quantifying the immune response the experimental vaccine provokes, Jesani says. That’s not necessarily an unusual approach, vaccine specialists say. But in India, “It’s difficult to make any judgement because [the institute’s] protocol is not in the public domain, although AstraZeneca has published theirs,” Jesani says. “That makes one wonder why there’s a double standard.”
In general, most nations do not require vaccinemakers to publicly share their testing protocols, but several companies have taken that step with COVID-19 vaccine candidates in order to bolster public trust. In India, however, no company testing a COVID-19 vaccine has released complete trial protocols, Jesani and others say. By law, the companies have to register basic trial details on the CTRI. But researchers have found that registry entries are often incomplete, and not regularly updated (a problem found in other national trial registries as well). And even when Indian companies do update their trial details, it’s hard to glean why they made changes, Bhan says.
“Everyone is very hopeful about the vaccines and vaccine science,” Bhan says. “But in the interest of trust in the way these trials are being run, it is good to have the protocol out there.”
Other researchers, however, see rigorous safety reviews as even more important than the publication of protocols. “I understand the demand for publishing protocols, but I don’t necessarily see the need for every information about trials to be out in the public domain,” Vineeta Bal, an immunologist at the Indian Institute of Science, Education and Research, said last month. Instead, Bal would like to see frequent evaluation of safety and adverse events by the relevant authorities. Just this week, Bharat Biotech, one company testing a COVID-19 vaccine, confirmed there was a serious adverse event during its phase I trial in August. The company noted that its protocol was followed and that the “adverse event was investigated thoroughly and determined as not vaccine related.”
Some critics would also like to see India’s regulators to do more to promote transparency. They note that the national government does not disclose the names and institutional relationships of the experts present during each clinical trial proposal meeting for COVID-19 vaccines and drugs. These subject expert committees review the proposals and send recommendations to the government’s Central Drugs Standard Control Organisation (CDSCO), which decides on their approval. The opacity makes it impossible to evaluate any potential conflicts of interest. “The committee is representing the public, and people have the right to know who these experts are,” says Santanu Tripathi, a clinical trials specialist at the Calcutta School of Tropical Medicine.
The All India Drug Action Network (AIDAN), an alliance of advocacy groups, has sent CDSCO three letters asking it to release details about review panel members, but has met with little success. After AIDAN’s first letter in June, the panel did start to release meeting minutes, but they were brief and lacked detail explaining decisions, Jesani says.
The drugs controller general of India, who oversees CDSCO, did not respond to requests for comment. However, A. K. Pradhan, deputy drugs controller, said during a webinar this month that Indian regulators are considering adopting more transparent data-sharing practices, such as those used by the U.S. Food and Drug Administration (FDA) and the European Medicines Agency. “But there are issues,” he said. “If we cannot adopt [such policies] properly, the companies can sue us.”
The secrecy surrounding CDSCO’s advisory panels and the agency’s regulatory decisions has previously come under fire. For example, after Indian regulators approved restricted emergency use of repurposed drugs such as itolizumab, remdesivir, and favipiravir to treat COVID-19, outside researchers argued the moves were based on poorly designed studies and inadequate data.
Unlike FDA, which also issues emergency use authorizations, the Indian regulator has not provided clarity on what restricted emergency use means. And India’s laws and regulations make no specific mention of emergency use, Tripathi says. Now, with the Serum Institute announcing this week that it could soon apply for an emergency use authorization for its vaccine, critics hope the regulator will be more open about the basis of its approvals.
Research institutes and hospitals involved in vaccine testing also need to be more transparent, critics say, as do those institutions’ ethics review committees. Some sites are small hospitals with no prior experience in such studies, they note. “It would be confidence building to have more information out there,” Bhan says.
Even the publicly funded Indian Council of Medical Research (ICMR), which is both supporting research and co-sponsoring some of the vaccine trials, has not been forthcoming, observers argue. “There’s a lack of transparency around the terms of ICMR’s engagement, involvement, and quantum of public funds involved,” says Malini Aisola, co-convener of AIDAN.
Samiran Panda, head of epidemiology at ICMR, says the institute has taken steps to ensure open communication with the public. It has, for example, created a vaccine portal that provides information about ICMR’s COVID-19 research. Panda says ICMR has also published the results of animal studies involving vaccines, and notes that a “decision on whether any vaccine comes into the market is the prerogative of the regulatory authority, not ICMR.”
With India touted by some media outlets as a major international vaccine manufacturer and supplier, trust in the country’s testing and regulatory processes is important, Aisola says. And greater transparency would also help build public confidence within India, Jesani adds. There are signs of vaccine hesitancy in the country—a survey by LocalCircles, a community social media engagement platform, found that more than 60% of the respondents would be reluctant to take a vaccine when it arrived.
Many critics hope Indian officials will be more open and not repeat attacks like the one made on Hebbar, the study volunteer. The threat to withhold his second dose, they say, represented a potential breach of medical ethics. And it was very poor public relations, too, Bhan says. “If research participants, of their own volition, want to talk about their experiences, I don’t think you can stop them,” he says. “What is there to hide?”
Reporting for this story was supported by a journalism grant from the Thakur Family Foundation, which has not exercised any editorial control over its content.

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By Bill Notes – Is inequality inevitable?

Melinda and I started our foundation twenty years ago because we believed that everyone everywhere deserved an equal opportunity to thrive. But in order to address inequities, you need to know exactly where—and for whom—they exist. Few people understand these issues in the United States better than Harvard economist Raj Chetty. Rashida and I were pleased he could join us to talk about his groundbreaking research on economic mobility, which measures a person’s ability to move up the income ladder.
Raj’s data provides fascinating and often surprising insights into where opportunity exists on the neighborhood level. I was particularly interested in his findings about two areas in central Los Angeles: Watts and Compton. Even though their neighborhoods are mere miles apart and demographically similar, a child born in a very low-income family in Compton is much more likely to earn a decent income, avoid incarceration, and escape poverty than one born in Watts.
We asked Compton’s mayor, Aja Brown, to come on the podcast and help us understand why. I am so impressed by the work she’s done to make her city better, especially her gang intervention program. Even though many of the initiatives Mayor Brown has created might not work in every city, civic leaders and policymakers can learn a lot from her about assessing and addressing a community’s specific needs. Compton’s success gives me hope for the future.

Behind The Scenes – Bonus Content 2
Three things

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About – Rank Math General Settings

In this tutorial, we are going to learn about the General Settings in Rank Math. The General Settings contain the settings that affect the overall SEO efforts of your website, and your links, images, and many other things are affected by what the General Settings.
That is why it is important to understand what the General Settings are, what they do, and how to configure them for the best results.
Let us begin.
You can reach Rank Math’s General settings through the WordPress menu. Hover over the Rank Math’s menu entry, and from the options that appear, select the General Settings.

Once you’re there, you’ll notice that the General Settings are divided into many other settings or sections. This is to help find the exact setting you’re looking for, faster.

To find settings, you can use the sections on the left, and we’ve also included a handy search bar, which is even faster if you know what you’re looking for. Here is how it works.

Anyways, you should use the method you prefer. Here are all the settings in the General Settings of Rank Math.
You can click any of the links to be taken to that section of the settings easily.
Let us get started with the Links Section.
Links
As you probably have guessed by now, the links section has all the settings that modify the behaviour of links on your website.

Here are all the settings you’d find in the Links Tab.

Let us understand each of the settings.
Strip Category Base
By default, the URLs of your post can contain the post name (if you configure it so), and the post category as well. For example, a post called best SEO plugin in the SEO category could be named as:
https://yourwebsite.com/SEO/best-seo-plugin
If you enable this option, Rank Math will strip the “category” part of the URL and make the URL shorter. So, the URL will become:
https://yourwebsite.com/best-seo-plugin
This option only affects aesthetics, and there is no research or evidence to show that removing the category from the URL helps your SEO in any way. However, it does make the URL shorter and more appealing. The choice is yours, if you like shorter URLs, then enable this option.

Redirect Attachments
Enabling this setting redirects URLs of your attachments to the post that are used in. For example, let us take a post with the URL yourwebsite.com/post-with-image. Let us assume that the post has an image with the name image-in-post.jpg.
Now, the direct URL of the image would be similar to yourwebsite.com/uploads/image-in-post.jpg.
If you enable this option, then if anyone (even search engines) try to access the image with the URL.
yourwebsite.com/uploads/image-in-post.jpg,
they will be redirected to the post with the URL yourwebsite.com/post-with-image.
Doing this might seem insignificant, but it has plenty of SEO benefits, including distributing the SEO value of the links that your images and other attachments build over time. For those reasons, we recommend that you enable this setting.

Redirect Orphan Media
Images and attachments that are not associated with any post on your website are called orphan media. In the section above, you chose whether you want to redirect attachment URLs to the post that contained them, but what about the attachments that do not have any posts attached to them?
That is where this setting comes in. Here you can enter the URL where you’d like the URLs of Orphan Media to be redirected to. The preferred setting is your homepage URL.

Remove Stopwords from Permalinks
This option removes stop words (a, and, the, or, he, him, etc.) from your WordPress URLs resulting in a cleaner, keyword rich URLs which are better looking and perform better too. We highly recommend that you enable this setting.

An important thing to note about this feature is that it only affects URLs auto-generated by WordPress. If you over-ride the WordPress URL and create one of your own, then stop words from those URLs will not be affected.
Also, this option will only affect URLs for new posts, and posts with existing URLs will also not be affected by this.
When you enable this option, a new setting should pop up below it like this.

Stopwords List
To offer you the most control over stop words, Rank Math lets you configure which stop words it should remove from the URLs. To configure this, simply paste the list of stop words that you’d like removed inside the text area, similar to what we’ve shown below. Here is a link to a comprehensive list of stop words in the English language for reference.

NoFollow External Links
This option automatically no-follows external URLs on all your posts, preserving the SEO of your website. We recommend that you enable this setting only if all external links on your website are affiliate links or sponsored links. You can also take advantage of the exclusion and inclusion lists to selective nofollow of follow certain domains.

NoFollow Image File Links
This option works similar to the NoFollow External Links option above, but this option nofollows any external links to images. We recommend that you enable this setting.

NoFollow Domains
This setting works in conjunction with the NoFollow External Links option listed above and acts like a blacklist for domains. Simply put, links from your website to any domains that you enter in this section will always be nofollowed.

NoFollow Exclude Domains
This option also works in conjunction with the NoFollow External Links option, but it acts as a whitelist instead. What that means is that links from your website to any of the domains you enter in this section will be followed.

How NoFollow Whitelist and Black List Actually Work
Rank Math has 3 sections to manage the link properties of external links.
• NoFollow External Links
• NoFollow Domains
• NoFollow Exclude Domains
This has been done to give you the maximum amount of control over your links. However, it can be a little confusing to understand exactly how it works. Here is a breakdown of different scenarios which will help you understand how the 3 settings work together.
NoFollow External Links is Disabled
In this case, the NoFollow Domains and NoFollow Exclude Domains sections can be ignored, as they will have no effect, even if you add domains to either of the sections.

NoFollow External Links is Enabled
In this case, there are again 2 possibilities.
• NoFollow Domains is Blank: In this case, all external links will be no-followed, except for the domains that are included in the NoFollow Exclude Domains section.
• NoFollow Domains has domains listed: In this case, only the links to the domains listed in the NoFollow Domains will be no-followed, rest of the links will be followed. In this case, the NoFollow Exclude Domains holds no weight.
To explain simply, if you disable the NoFollow External Links setting, then links will keep working for you as normal. By default, all links would be created followed, and you would have to manually no-follow them.
However, if you enable the NoFollow External Links section, then all external links will be no-followed. You can then use the NoFollow Domains (Blacklist) or the NoFollow Exclude Domains (Whitelist) to narrow the domains you want to no-follow. Note that you can either use the blacklist or the whitelist to manage external links, but you cannot use them at the same time.
Open External Links in New Tab
This option applies the target = “_blank” attribute to external links which opens them in new tabs or windows. We recommend that you enable this setting.

This concludes the Links Section of the General Settings in Rank Math. Let us move on to the next section on the list, the Images section.
Affiliate Link Prefix (Pro)
Sometimes, affiliates need to differentiate cloaked links from regular internal links. Traditionally, all links with your domain as prefix are counted as internal links. This causes problems when you wish to nofollow all external links but automation tools skip cloaked links thinking they are internal links.
With this link prefix option, you can choose the URL base for your cloaked links so Rank Math can automatically count them as outgoing links and apply nofollow/open-in-new-tab to them if the option is enabled via Rank Math.
If you are using domain.com/go/ as the base for cloaked URLs, then enter /go/ like in this screenshot:

Images
When you open the images section, you will notice that there are only 2 settings in it.

But, if you enable any of the options, another setting will open up below it.

Let us explore each of the settings.
Add Missing Alt Attributes
Enabling this setting will add Alt Attributes to all the images that do not have their alt text set. Since the alt text is valuable to search engines in understanding your images, we recommend that you enable this setting.

Alt Attribute Format
If you’ve enabled the Add missing Alt Attribute option, the next question is, how does Rank Math know what an image is about? Actually, Rank Math does not know, and it relies on you to help answer the question with the Alt Attribute Format.
The Alt Attribute Format helps you create a pre-defined format which will be used to create alt text for images. Instead of relying on generic names, Rank Math lets you use from the many supported variables to create a useful format.

Selecting a variable will update the live preview below it, which will help you understand how the final alt text will be.

Add Missing Title Attributes
This option automatically adds the Title attributes to the images without a title. A title also helps search engines understand your images better, and we recommend that you enable this setting.

Title Attribute Format
The Title Attribute Format option works similar to the Alt Attribute format, the only difference being that it helps define a format to create the image’s title. Use the variables and live preview to create a format.

This covers all the settings in the Image tab, let’s move on to the Breadcrumbs Tab.
BreadCrumbs
Breadcrumbs are navigational elements which the user understand where they are on a certain website, and also help them navigate the website in an easy to understand manner.
In simple terms, they can be compared to “You’re here” signs in large parks, as they both achieve similar things.
Here is an example of how breadcrumbs appear on a website.

Yes, these are breadcrumbs from the article you’re reading right now.
The Breadcrumbs setting in Rank Math lets you configure Breadcrumbs. When you open the settings up, you only see one setting.

That’s because Breadcrumbs are turned off, and there is no need to show any of the other settings. Once you enabled breadcrumbs, all the other settings will appear on the screen.

Let us understand each of the options one by one.
Enable Breadcrumbs Function
This setting enables breadcrumbs on your website. Breadcrumbs help users navigate your website and also helps search engines index your content efficiently. We recommend that you enable this setting.

Separator Character
This option helps you set a Separator Character for your breadcrumbs. There are many options available, so choose the one you prefer. You can also set a custom separator character by typing it in the last field.

You might have missed it, but guess which separator character are we using?

Show Homepage Link
If you want your homepage to be included in the breadcrumbs, enable this option. This is more of personal preference, but we’ve seen most websites do include the homepage in their breadcrumbs.

If you enable the Homepage Link, 2 new settings will pop up under it.
Homepage Label
Here you can configure what label to give the homepage of your website. All you have to do is type in the label that you want to assign your homepage.

For example, we have included the homepage link in our breadcrumbs, and the label we’ve assigned is home.

Homepage Link
This option lets you configure where will the homepage link take the visitor. This is particularly useful when you have many sections of your website like a blog, a store, or others. Using this option, you can configure the breadcrumb to take your visitor anywhere you prefer.

Prefix Breadcrumbs
If you’d like to add a prefix before your breadcrumbs, you can use this option to do so. Popular options would be to add “You are here”, “Navigation”, “Index”, etc.

Archive Format
This option lets you set a breadcrumb format for the archive pages. You can customize it completely, but we recommend that you don’t remove the “%s” from it, as it represents the taxonomy.

Search Result Format
This option lets you set a breadcrumb format for search result pages. Popular options are, “You searched for”, “Here is what I found for”, etc. We would recommend that you don’t remove the “%s” from the field.

404 Label
This option lets you set a breadcrumb format for your 404 pages.

Hide Post Title
Enabling this option lets you hide the title of the post from the breadcrumb.

For example, here is the breadcrumb of our post with the title.

If you enable this setting, the breadcrumb will change to:

Show Category
This option enables ancestor categories in the breadcrumbs. What that does is that if your current post is part of a child category, then the breadcrumb will include the parent categories as well. We recommend that you keep this disabled to keep your breadcrumbs small and precise.

Hide Taxonomy Name
If you use any other taxonomy other than categories, then you can use this option to enable or disable the inclusion of the taxonomy in the breadcrumb. Our recommended setting is “Off”.

This covers all the settings in the Breadcrumbs tab. Let us move on to the next tab, the Webmaster Tools.
Webmaster Tools
The Webmaster tools is the section where you can take the necessary steps to verify your website with various webmaster tools. There are many tools supported, and here is an image that shows all the supported webmaster tools.

Before getting into the verification process, let us understand how the verification process works.i
How Does Webmaster Verification Work
Almost all webmaster tools have similar verification processes. The specifics might be different, but the overall principle is the same. Here are the steps that usually take place.
• You tell a search engine (or another service) about a “property” and claim to own it
• The webmaster tools generate a piece of code that you need to place on your website
• You place the code on your website, proving that you have access to the site and your ownership
• Your property is verified and stays verified till the code is present
The reason why we specify the process is because we won’t be discussing the specifics of getting your website verified with all the webmaster tools. Rather, we will demonstrate the process on one of the webmaster tools, and you can learn the process there. Actually, you don’t even have to verify your website with all the webmaster tools, only for the ones that you want to.
Here are all the currently supported Webmaster Tools.
• Google Search Console
• Bing Webmaster Tools
• Baidu Webmaster Tools
• Alexa Verification ID
• Yandex Verification ID
• Pinterest Verification ID
• Norton Safe Web Verification ID
Let us demonstrate how to verify your website for Google Search Console (formerly known as Google Webmaster Tools).
Start by going to the Google Search Console Website, and click the “Start Now” button.

You’ll need to log in with a Google Account. Once you’re in, you should see a screen similar to this. Note that your Search Console will look different if you don’t have any websites connected.

To add a new property, click the Property Selection dropdown, and select the new property option. Again, if you don’t have any properties added, your view will be a little different.

Google Search Console offers 2 types of properties. A domain property, and a URL property. For simplicity, we will demonstrate the process using the URL property. Enter your website’s URL in the URL prefix field, and click “Continue”.

You should see a screen similar to the below. Here Google is showing you all the ways you can verify your website ownership and also recommending their preferred method. Click the highlighted drop-down indicator to see all the available methods.

Here are all the methods that you should see. Since Rank Math already does the heavy lifting for you, you don’t have to choose a complicated verification method. Instead, we will use the HTML Tag method. Click the dropdown next to the HTML Tag option to see its details.

The settings will expand, and you will see the verification meta tag. Copy it, and click “done”.
In the Webmaster Tool settings, paste the Meta Code in the Google Search Console Field.

For the code to be applied, you’ll need to update your settings by saving your changes. Head over to the bottom of the page, and click “Save Changes”.

The page will refresh, and you should see a confirmation message on top. Your website will now be verified in Google Search Console.
As we mentioned, the process for verifying with other tools is quite similar, and here are some resources that should help you in verifying your website with other webmaster tools. Remember, you don’t have to follow all the steps from these resources. Once you’ve generated a meta-tag, all you have to do is paste it in the appropriate field.
Use these resources for reference.
That covers the Webmaster Section Completely. Let us move onto the next section.
Edit Robots.txt
Once you open the Edit robots.txt tab, you’ll see that it has only one setting.

In the text area, you can configure your virtual robots.txt file.
If you don’t know, robots.txt is a text file present in the root of your website which helps search engines understand which parts of your website you want them to crawl and index. For example, you wouldn’t want Google to index your website’s admin area, right? You can set this up in the robots.txt file.
We mentioned that you could edit your virtual robots.txt file here. We say virtual because here you are not editing your actual robots.txt file, but a virtual one.
Rank Math already configures some default settings that should be useful. But, any changes you make here will not have an effect on your website if the actual robots.txt file already exists. If it does exist, you would have to edit the file using FTP or any other method, or delete the file completely, and make all the changes through Rank Math.
Edit .htaccess
The .htaccess file is a configuration file that helps in users and search engines navigate your website. Often, changes or updates are needed to be made to the .htaccess file, for which you can use this section.
Inside the section, you will see that there is a single text area which can be used to edit the .htaccess file. The important thing to note is the warning above the text area.

The notice warns you that changing the .htacess file can break your website. Even if you are technically capable, mistakes can happen. To prevent issues, Rank Math automatically backs up your .htacess before you modify it.
So, if you accidentally make any changes that mess up your site, you can roll the changes back using the backup. But remember, the backup will only contain the last save. That means if you make multiple changes that cause problems, then the backup will not help you. Proceed with caution.
If you understand the risks and want to edit the .htacess file, then click the checkbox below the text. The text area below will become active, and you can then change your .htacess file.

This concludes the .htaccess section. Let us move on to the next section.
WooCommerce
This section deals with all the options related to WooCommerce. Needless to say, this section will only be relevant to you if you actually use WooCommerce and sell products online. In fact, if you don’t have WooCommerce installed, then this section won’t even be visible to you in the settings.
With that out of the way, here are all the settings in the WooCommerce Section.

Let us discuss each of sections one by one.
Remove Base
WordPress URLs have a “shop” base added to them when WooCommerce is enabled. So, every product URL follows this format.

Home


Notice the ‘/shop/’ part in the URL? That’s what we’re talking about.
Enabling the Remove Base option lets you remove the /shop/ from the URL, so the resulting URL can become.

Home


This is personal preference, and you can enable or disable it.

Remove Category Base
If you have a large base of products and use multiple categories to manage them, then your WooCommerce URLs will also contain the product category. An example URL would be:

Home


This option lets you remove the “product-category” section from the URL, so the resulting URL becomes.

Home


This option is also a preference thing, and you should make a decision to enable it or disable it accordingly.

Remove Parent Slugs
Categories in WordPress are hierarchical, that means a category can have a parent category. If you have products that have a parent category, then WooCommerce’s default behavior is to list the product with the parent category in the URL. An example URL would be:
https://yourwebsite.com/parent-category/sub-category/product/
Using this option, you can remove the parent category slug from the URL, resulting in a cleaner and shorter URL. Like the other settings, how you set it up is a personal preference.

Remove Generator Tag
When you’re using WooCommerce, it includes plenty of meta information in the source code. While it can be useful for diagnostic purposes, it also presents a security risk. Here is how the Meta Generator tag appears in the source code.

When you enable this option, Rank Math disables the output of this meta information in your website’s source. Since this essentially is a security feature, we recommend that you enable this option.

Remove Snippet Data
This option works similar to the option discussed above, and it removes the Snippet data from the WooCommerce data as a security measure. We recommend that you enable this feature as well.

Brand
Rank Math adds a lot of rich snippet data to your website and your product pages. One of the key components of that rich data is the Brand of products you’re selling. In this setting, you select the taxonomy that you’ve used to configure brand listings on your website so that Rank Math can set it up accordingly. Use the drop-down menu and choose the appropriate taxonomy from the available options.

404 Monitor
A 404 error occurs when the user tries to access a URL that does not exist. During the regular working of a website, it is normal to update and remove content, and as a result, every website will have a few 404 errors. Having 404 errors is not necessarily bad, but the important thing is to find them and resolve them actively. Rank Math’s 404 monitor helps with exactly that. Here are all the options available in the 404 Monitor.

Let’s look at all the options one by one.
Mode
The 404 Monitor has 2 modes. Simple and Advanced. The Simple monitor records only the URL where the error occurred and the time of the error. The Advanced Monitor captures a lot of additional information such as the referring URL of the URL with the error.
The Advanced Mode is pretty powerful, but it does come at a cost. It does require additional resources to capture and store the detailed logs.
If you don’t have many 404 errors, then we would recommend that you use the Simple mode. However, if you’re constantly seeing 404 errors, then it might be useful to enable the Advanced Mode for some time to diagnose the source of the problem.

Log Limit
This setting controls the maximum number of rows that the monitor will generate. The default setting of 100 is good enough for most sites, and you can increase or decrease the number depending on your website. For example, for a large ecommerce website with thousands of products may have thousands of broken links at any time (due to new products being introduced and old ones being removed) and might need to remove the limit altogether. In another example, if you’ve just changed domains names, you might need to increase the log limit temporarily to make sure all your links perform as intended.

Exclude Paths
If you’re making some changes to your website, then the 404 monitor can fill the up the log with unimportant URLs while the real errors get buried. To avoid this from happening, you can set exclusions where the 404 monitor will not check for errors.
The exclusion tool is versatile and offers plenty of options to set up an exclusion path. You can enter exact URLs, categories, keywords, or even use RegEx to define an exclusion path. The image below shares all the options that are available to you. You can also stack multiple exceptions by creating more exclusion rules.

Ignore Query Parameters
This option controls whether the 404 monitor ignores the query parameters when logging the 404 errors. Whether you should enable or disable this setting will depend on your website. We recommend that you keep this disabled at first, then enable it if you see a lot of errors in the log file that have the same URL but different query parameters.

Redirections
Rank Math has a built-in redirection manager that helps you get rid of 404 and other errors. The redirection tab has a few settings that control how the redirections take place. Here is a preview of all the settings in this tab.7

Let us understand what each of the settings does.
Debug Redirections
Redirects can be tricky business, especially when you’re Regex and other complex techniques to create redirects. The more complex the method, the more chances that something might go wrong. If you see redirects on your website that can’t place, then Debug Redirections is here to help.
The Debug Redirection feature places an interstitial window between the source URL and the destination URL. The window contains basic diagnostic information along with the rule that caused the redirection. This helps in finding out the rules that are triggering certain redirects.
And, the interstitial window is visible only to administrators, and your website visitors won’t know a thing about it.
We would recommend that you keep this feature disabled, and only enable it when you have to perform some diagnostics on redirects.

Fallback Behaviour
What happens when the destination URL in a redirect isn’t found? With the Fallback Behaviour setting, you can define it just the way you like. You can set it to a 404, redirect to the homepage, or redirect to a custom URL.
If you select the custom URL option, then another field opens up where you can enter the fallback URL.

You can set this up any way you like; there is no single recommendation.
Custom URL
This field only appears when the Custom URL option is selected. Here you enter the custom fallback URL that your visitors will be taken to if the actual redirect URL is not found.
Redirection Type
Redirections can be of many types, and each type serves a specific purpose. The Redirection Type setting helps configure the default redirection type that Rank Math will use when creating a new redirection. You can also over-ride the redirection type when you’re actually creating a redirect. Here is a detailed article that explains the different redirect types and their uses.

Auto Post Redirect
If you change the URL of a post in WordPress, nothing happens—no redirects are created. As you can probably understand, that is not ideal. The Auto Post Redirect feature fixes that behavior by automatically creating a redirect from the old URL to the new URL whenever you change the URL (slug) of a post. We recommend that you enable this feature.

That covers all the sections in the Redirections Tab. Let us move on to the next tab, the Search Console Tab.
Search Console
The Search Console option is different from the Webmaster Tools that we’ve discussed above. The Webmaster options helped you verify the ownership of your website with different search engines, and the Search Console option connects with Google Search Console to fetch and display information inside your WordPress setup. Here are all the options in it.

Let us start discussing the various options.
Search Console
In the Search Console option, we will authorize Rank Math to fetch data from Google Search Console. A pre-requisite to this is that your website should already be verified in Search Console.
We already how to do that in the Webmaster Tools section. If your website isn’t verified, we recommend that you follow our instructions there before attempting to configure Search Console options. Once your website is verified, follow these steps.
Click the “Get Authorization Code” button.

Google’s OAuth window will open. Click on the account you used to log into Search Console and verify your website.

You will see a confirmation screen. Click “Allow” to connect Rank Math with Google Search Console.

An authorization code will be generated. Copy the code to your clipboard and close the window.

Paste the code in the Search Console setting and click “Authorize”.

Rank Math will take a few seconds to verify the code, after which your screen should look like this.

When you see this screen, that means Rank Math is now authorized to fetch information from your Search Console account. The next step is to select your property.
Search Console Profile
Search Console fetches data on “properties”. A property can be a complete domain, a website, and even a single URL. Here you will select the appropriate property that you want Rank Math to fetch information for. Click the drop-down button to see a list of all the properties.
If this is your first website, then you won’t see multiple entries, just the one. On the contrary, if you don’t see any websites or properties here, you might have missed the part where you need to verify your website with Google Search Console. Head over to the Webmaster Tools Section section and follow the instructions there to verify your website.

Once you select the right property, move to the next setting.
Cache Limit
The cache limit is where you set the number of days that the Search Console data will be stored for. Rank Math will automatically fetch data from Search Console at a defined interval, but you can also fetch the data manually.
When you connect your website, Rank Math has obviously fetched no data from the Search Console, hence it shows this warning message, which you can safely ignore.

In the Cache Limit settings, you can configure the number of days for which you want the data to be visible inside WordPress. The default of 90 days is sufficient in most cases, but you can also change it.
After setting the cache limit, we would recommend that you manually fetch the data for the first time.

Rank Math will fetch the data, and you should see a progress bar at the bottom.
Once the fetch is complete, the meta-data should appear updated.
Others
The Others section is home for miscellaneous settings that could not be placed in any of the categori6es. Here are all the settings in the Other section.

Let us talk about each of the settings.
Usage Tracking
If you’d like to help us improve Rank Math, you can enable the Usage Tracking option. All the data we collect is completely anonymous, is never used for marketing purposes, or even shared with 3rd parties. You can read more about Privacy Policy here.
Enabling this is completely upto you, but we encourage you to do so.o

Show SEO Score
If you have a ton of posts on your website and you just started using Rank Math, then it can be difficult to open each post to check how well they are optimized. This option can come in handy in those situations. When you enable this setting, Rank Math will start displaying the SEO Score of each of the posts on the frontend, which will make it easy for you to identify the posts that need the most work.
This option is useful, but it isn’t mandatory for everyone. Only enable this if you have the need.

If you enable this setting, a bunch of new settings opens up below it.
SEO Score Post Types
This setting lets you select the types of posts that the SEO score will be displayed. Rank Math will detect and display all types of posts available on your website, including custom post types, and you can select one or more of them.

SEO Score Template
The SEO score can be displayed in 2 visual formats—Circular, or Square—and this setting lets you select your preferred method.

SEO Score Position
This option lets you pick the position of the SEO score. You can place it above the content, below the content, or both. You can also choose a custom location by using a shortcode.
The shortcode you need to use is . If you’re editing your template and making the changes there, you’ll need to use this code chunk.

Support Us with A Link
We’ve put in years of hard to bring Rank Math to you, and if you want to help us spread the word, we’ll be delighted. When you enable this option, Rank Math will include a hyperlink to the Rank Math from your website to your website. The link will be no-followed, and no SEO value will be passed from your website to Rank Math’s.

RSS Before Content
This option lets you add additional content at the beginning of your RSS feed. You can enter any content in the text field, and also use the variable listed below to customize the text here.

RSS After Content
This option lets you add content after your RSS feed. You can use the same variables as in the before content option.

Available Variables
This section isn’t configurable, and it just lists all the variables that you can use in the RSS Before and RSS After content options.

Here is a sample text you can create with variables and use with your RSS settings.
The post %POSTLINK% first appeared on %BLOGLINK% and is written by %AUTHORLINK%.
Saving Your Settings
Once you’ve made all the changes to the General Settings, it’s important that you save your settings. Rank Math does not save your settings automatically to avoid accidental changes, but the downside is that you have to save your settings manually.
If you haven’t saved your changes and you accidentally navigate away from the General Settings, Rank Math will attempt to notify you of your unsaved changes.

To save your settings, click the “Save Changes” button that is present in the bottom right of the screen.

It might take a couple of seconds, but the page will refresh, and your settings will be saved. You will also see a confirmation message on the screen.

Resetting Your Settings
If there arises a need that you need to go back to the default settings, then you don’t need to remember the default settings—Rank Math has the capability built-in.
To “Reset” or go back to your default settings, just click the “Reset” button that is present at the bottom of any page.

Since this is a big step, Rank Math will ask for a confirmation before it resets your setting. Click “Ok” on the confirmation dialogue box that appears on the screen.

The page will refresh, and in a few seconds, your settings will be reset. There is no confirmation message on the screen, so take the page refresh as your cue.
Important Thing To Understand About the Reset Process
When you reset your settings, it affects the entire section. To put that into context, if you reset your settings while you were navigating the General Settings, then all your general settings will be reset. Without a backup, all your custom settings will be lost. If you have the slightest of the doubts, make sure to take a backup by following the instructions in this article. You’ve been warned.

Conclusion
Finally, we’ve covered all the sections in the General Settings in Rank Math. There were a lot of settings, so we hope that you’ve understood them all. If you haven’t, go back to that section again. If you still have doubts or have issues while using Rank Math, feel free to open a support ticket and the support team will help you out in the best way they can.

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About – Las Vegas, Nevada, America

The history of Las Vegas covers both the city of Las Vegas, Nevada, and the Las Vegas Valley.

The name Las Vegas was given to the city in 1829 by Rafael Rivera, a member of the Spanish explorer Antonio Armijo trading party that was traveling to Los Angeles, and stopped for water there on the Old Spanish Trail from New Mexico.

At that time, several parts of the valley contained artesian wells surrounded by extensive green areas; Las Vegas means “the meadows” in Spanish. The flows from the wells fed the Las Vegas Wash, which runs to the Colorado River.

The settlement of Las Vegas was founded in 1905 after opening of a railroad that linked Los Angeles and Salt Lake City.

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Science About Light Pollution – Too Bright To Breed

Night light from coastal cities overpowers natural signals for coral spawning from neighboring reefs.
PHOTO: NOKURO/ALAMY STOCK PHOTO
Most coral species reproduce through broadcast spawning. For such a strategy to be successful, coordination has had to evolve such that gametes across clones are released simultaneously. Over millennia, lunar cycles have facilitated this coordination, but the recent development of bright artificial light has led to an overpowering of these natural signals. Ayalon et al. tested for the direct impact of different kinds of artificial light on different species of corals. The authors found that multiple lighting types, including cold and warm light-emitting diode (LED) lamps, led to loss of synchrony and spawning failure. Further, coastal maps of artificial lighting globally suggest that it threatens to interfere with coral reproduction worldwide and that the deployment of LED lights, the blue light of which penetrates deeper into the water column, is likely to make the situation even worse.

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