Customer Relationship Management (CRM) And Social Community

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UK Authorizes Pfizer Coronavirus Vaccine For Emergency Use

British officials authorized a COVID-19 vaccine for emergency use on Wednesday, greenlighting the world’s first shot against the virus that’s backed by rigorous science and taking a major step toward eventually ending the pandemic.
The go-ahead for the vaccine developed by American drugmaker Pfizer and Germany’s BioNTech comes as the virus surges again in the United States and Europe, putting pressure on hospitals and morgues in some places and forcing new rounds of restrictions that have devastated economies.
The Medicines and Healthcare Products Regulatory Agency, which licenses drugs in the U.K., recommended the vaccine could be used after it reviewed the results of clinical trials that showed the vaccine was 95% effective overall — and that it also offered significant protection for older people, among those most at risk of dying from the disease. But the vaccine remains experimental while final testing is done.

“Help is on its way,″ British Health Secretary Matt Hancock told the BBC, adding that the situation would start to improve in the spring.
“We now have a vaccine. We’re the first country in the world to have one formally clinically authorized but, between now and then, we’ve got to hold on, we’ve got to hold our resolve,” he said.
MORE ON THE PANDEMIC
Other countries aren’t far behind: Regulators in the United States and the European Union also are vetting the Pfizer shot along with a similar vaccine made by competitor Moderna Inc. British regulators also are considering another shot made by AstraZeneca and Oxford University.
Hancock said Britain expects to begin receiving the first shipment of 800,000 doses “within days,″ and people will begin receiving shots as soon as the National Health Service gets the vaccine.
Doses everywhere are scarce, and initial supplies will be rationed until more is manufactured in the first several months of next year.
A government committee will release details of vaccination priorities later Wednesday, but Hancock said nursing home residents, people over 80, and healthcare workers and other care workers will be the first to receive the shot.
Pfizer said it would immediately begin shipping limited supplies to the U.K. — and has been gearing up for even wider distribution if given a jsimilar nod by the U.S. Food and Drug Administration, a decision expected as early as next week.
Pfizer CEO Albert Bourla called the U.K. decision “a historic moment.”
“We are focusing on moving with the same level of urgency to safely supply a high-quality vaccine around the world,” Bourla said in a statement.

While the U.K. has ordered 40 million doses of the Pfizer vaccine, enough for 20 million people, it’s not clear how many will arrive by year’s end. Hancock said the U.K. expects to receive “millions of doses” by the end of this year, adding that the actual number will depend on how fast Pfizer can produce the vaccine.
One concern about the Pfizer-BioNTech vaccine is that it must be stored and shipped at ultra-cold temperatures of around minus 70 degrees Celsius (minus 94 degrees Fahrenheit), adding to the challenge of distributing the vaccine around the world.
Pfizer says it has developed shipping containers that use dry ice to keep the vaccine cool. GPS-enabled sensors will allow the company to track each shipment and ensure they stay cold, the company says.
“Pfizer has vast experience and expertise in cold-chain shipping and has an established infrastructure to supply the vaccine worldwide, including distribution hubs that can store vaccine doses for up to six months,” the company said in a statement.
The company also says it has agreed to work with other vaccine makers to ensure there is sufficient supply and a range of vaccines, “including those suitable for global access.”
Every country has different rules for determining when an experimental vaccine is safe and effective enough to use. Intense political pressure to be the first to roll out a rigorously scientifically tested shot colored the race in the U.S. and Britain, even as researchers pledged to cut no corners. In contrast, China and Russia have offered different vaccinations to their citizens ahead of late-stage testing.
The shots made by U.S.-based Pfizer and its German partner BioNTech were tested in tens of thousands of people. And while that study isn’t complete, early results suggest the vaccine is 95% effective at preventing mild to severe COVID-19 disease. The companies told regulators that of the first 170 infections detected in study volunteers, only eight were among people who’d received the actual vaccine and the rest had gotten a dummy shot.
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“This is an extraordinarily strong protection,” Dr. Ugur Sahin, BioNTech’s CEO, recently told The Associated Press.
The companies also reported no serious side effects, although vaccine recipients may experience temporary pain and flu-like reactions immediately after injections.
Final testing must still be completed. Still to be determined is whether the Pfizer-BioNTech shots protect against people spreading the coronavirus without showing symptoms. Another question is how long protection lasts.
The vaccine also has been tested in only a small number of children, none younger than 12, and there’s no information on its effects in pregnant women.

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About Data Sonification – A New Cosmic Triad of Sound

A new trio of examples of ‘data sonification’ from NASA missions provides a new method to enjoy an arrangement of cosmic objects. Data sonification translates information collected by various NASA missions — such as the Chandra X-ray Observatory, Hubble Space Telescope, and Spitzer Space Telescope — into sounds.
This image of the Bullet Cluster (officially known as 1E 0657-56) provided the first direct proof of dark matter, the mysterious unseen substance that makes up the vast majority of matter in the Universe. X-rays from Chandra (pink) show where the hot gas in two merging galaxy clusters has been wrenched away from dark matter, seen through a process known as “gravitational lensing” in data from Hubble (blue) and ground-based telescopes. In converting this into sound, the data pan left to right, and each layer of data was limited to a specific frequency range. Data showing dark matter are represented by the lowest frequencies, while X-rays are assigned to the highest frequencies. The galaxies in the image revealed by Hubble data, many of which are in the cluster, are in mid-range frequencies. Then, within each layer, the pitch is set to increase from the bottom of the image to the top so that objects towards the top produce higher tones.
This image of the Bullet Cluster (officially known as 1E 0657-56) provided the first direct proof of dark matter, the mysterious unseen substance that makes up the vast majority of matter in the Universe. X-rays from Chandra (pink) show where the hot gas in two merging galaxy clusters has been wrenched away from dark matter, seen through a process known as “gravitational lensing” in data from Hubble Space Telescope (blue) and ground-based telescopes. In converting this into sound, the data pan left to right, and each layer of data was limited to a specific frequency range. Data showing dark matter are represented by the lowest frequencies, while X-rays are assigned to the highest frequencies. The galaxies in the image revealed by Hubble data, many of which are in the cluster, are in mid-range frequencies. Then, within each layer, the pitch is set to increase from the bottom of the image to the top so that objects towards the top produce higher tones.
Credits: NASA/CXC/SAO/K.Arcand, SYSTEM Sounds (M. Russo, A. Santaguida)
The Crab Nebula has been studied by people since it first appeared in Earth’s sky in 1054 A.D. Modern telescopes have captured its enduring engine powered by a quickly spinning neutron star that formed when a massive star collapsed. The combination of rapid rotation and a strong magnetic field generates jets of matter and anti-matter flowing away from its poles, and winds outward from its equator. For the translation of these data into sound, which also pans left to right, each wavelength of light has been paired with a different family of instruments. X-rays from Chandra (blue and white) are brass, optical light data from Hubble (purple) are strings, and infrared data from Spitzer (pink) can be heard in the woodwinds. In each case, light received towards the top of the image is played as higher pitched notes and brighter light is played louder.
The Crab Nebula has been studied by people since it first appeared in Earth’s sky in 1054 A.D. Modern telescopes have captured its enduring engine powered by a quickly spinning neutron star that formed when a massive star collapsed. The combination of rapid rotation and a strong magnetic field generates jets of matter and anti-matter flowing away from its poles, and winds outward from its equator. For the translation of these data into sound, which also pans left to right, each wavelength of light has been paired with a different family of instruments. X-rays from Chandra X-ray Observatory (blue and white) are brass, optical light data from Hubble Space Telescope (purple) are strings, and infrared data from Spitzer (pink) can be heard in the woodwinds. In each case, light received towards the top of the image is played as higher pitched notes and brighter light is played louder.
Credits: NASA/CXC/SAO/K.Arcand, SYSTEM Sounds (M. Russo, A. Santaguida)
On February 24, 1987, observers in the southern hemisphere saw a new object in the Large Magellanic Cloud, a small satellite galaxy to the Milky Way. This was one of the brightest supernova explosions in centuries and soon became known as Supernova 1987A (SN 87A). This time lapse shows a series of Chandra (blue) and Hubble (orange and red) observations taken between 1999 and 2013. This shows a dense ring of gas, which was ejected by the star before it went supernova, begins to glow brighter as the supernova shockwave passes through. As the focus sweeps around the image, the data are converted into the sound of a crystal singing bowl, with brighter light being heard as higher and louder notes. The optical data are converted to a higher range of notes than the X-ray data so both wavelengths of light can be heard simultaneously. An interactive version lets the user play this astronomical instrument for themselves.
On February 24, 1987, observers in the southern hemisphere saw a new object in the Large Magellanic Cloud, a small satellite galaxy to the Milky Way. This was one of the brightest supernova explosions in centuries and soon became known as Supernova 1987A (SN 87A). This time lapse shows a series of Chandra X-ray Observatory (blue) and Hubble Space Telescope (orange and red) observations taken between 1999 and 2013. This shows a dense ring of gas, which was ejected by the star before it went supernova, begins to glow brighter as the supernova shockwave passes through. As the focus sweeps around the image, the data are converted into the sound of a crystal singing bowl, with brighter light being heard as higher and louder notes. The optical data are converted to a higher range of notes than the X-ray data so both wavelengths of light can be heard simultaneously.
Credits: NASA/CXC/SAO/K.Arcand, SYSTEM Sounds (M. Russo, A. Santaguida)
The data sonification project is led by the Chandra X-ray Center (CXC) as part of the NASA’s Universe of Learning (UoL) program. NASA’s Science Activation program strives to enable NASA science experts and to incorporate NASA science content into the learning environment effectively and efficiently for learners of all ages. The collaboration was driven by visualization scientist Kimberly Arcand (CXC), astrophysicist Matt Russo and musician Andrew Santaguida (both of the SYSTEMS Sound project.)
NASA’s Marshall Space Flight Center manages the Chandra program. The Smithsonian Astrophysical Observatory’s Chandra X-ray Center controls science from Cambridge Massachusetts and flight operations from Burlington, Massachusetts. NASA’s Universe of Learning materials are based upon work supported by NASA under cooperative agreement award number NNX16AC65A to the Space Telescope Science Institute, working in partnership with Caltech/IPAC, Center for Astrophysics | Harvard & Smithsonian, Jet Propulsion Laboratory, and Sonoma State University.

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By George Soros – An Effective Response to Europe’s Fiscal Paralysis

With Hungary and Poland vetoing the European Union’s budget and COVID-19 recovery fund, the case for issuing perpetual bonds has never been stronger. While the EU cannot currently do so, given uncertainty about its future, many of its member states can and should.
NEW YORK – I have written a lot in the past about the desirability of the European Union issuing perpetual bonds. But today I am proposing that individual member states should do so.
Right now, it would be impossible for the EU to issue perpetual bonds, because the member states are too divided. Poland and Hungary have vetoed the next EU budget and the COVID-19 recovery fund, and the so-called Frugal Five (Austria, Denmark, Germany, the Netherlands, and Sweden) are more interested in saving money than in contributing to the common good. Investors will buy perpetual bonds only from an entity that they believe will continue to exist for the foreseeable future. That was true of Britain in the eighteenth century (when it issued Consols) and of the United States in the nineteenth century (when it consolidated individual states’ debt). Sadly, it is not true of the EU today.
The EU finds itself in a very difficult situation. It is experiencing a second wave of COVID-19 that threatens to be even more devastating than the first. Member states used up most of their financial resources fighting the first wave. Providing health care and resuscitating the economy will require much more than the €1.8 trillion ($2.2 trillion) in the new budget and recovery fund, called Next Generation EU. In any case, the availability of those funds has been delayed by Hungary and Poland’s veto.
Hungarian Prime Minister Viktor Orbán is concerned that the EU’s new rule-of-law provision would impose practical limits on his personal and political corruption. He is so worried that he has concluded a binding cooperation agreement with Poland, dragging that country down with him.
It turns out that there is an easy way to overcome the veto: employ the so-called enhanced cooperation procedure. It was formalized in the Lisbon Treaty with the express purpose of creating a legal basis for further eurozone integration, but it was never used for that purpose. Its great merit is that it can be used for fiscal purposes. A sub-group of member states can set a budget and agree on a way to fund it – say, through a joint bond.
At this point, perpetual bonds could come in very useful. They would be issued by member states whose continued existence would be readily accepted by long-term investors such as life-insurance companies.
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Perpetual bonds offer the great advantage that the principal never has to be repaid; only the annual interest is due. The discounted present value of future interest payments diminishes over time – it will approach, but never reach, zero. A certain amount of financial resources – say, the €1.8 trillion currently planned – would go several times further if it were used to issue perpetual bonds rather than ordinary bonds. This would largely solve Europe’s financial problems.
If one country issued perpetual bonds, it would have the additional advantage that other European countries would find it an example worth following. The Frugal Five should find perpetual bonds particularly attractive. After all, they like to save money.
There is a lot of unsatisfied demand in Europe from insurance companies and other long-term investors for long-term bonds. At first, they may demand a premium for perpetual bonds because they are not familiar with the instrument. But the premium is likely to disappear as they acquaint themselves with it.
Italy is not among the countries fortunate enough to be able to issue perpetual bonds in their own name; yet it needs the benefits more than others. Italy is the EU’s third largest economy – what would the EU be without it?

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Tracking the Reopening of New York City – What You Need To Know

After months in an economically devastating lockdown, America’s largest metropolis is taking its first steps in the long slog back to normal. New York City, hobbled by what was once the biggest and deadliest Covid-19 outbreak in the country, began reopening on June 8, despite lingering concerns about the virus and against a backdrop of civil unrest over police brutality that led to protests, and at times, looting and violence.
Yet as the city’s economic engine revs up, questions abound: What will business in post-Covid New York look like? How will commuters manage in a social-distancing world? How will New York’s major industries, from finance to real estate and the arts, meet the needs of their customers and employees, all while trying to keep everyone safe?
Key Coverage
• NYC’s Virus Threat Has Faded, But Its Future Is Stuck in Limbo
• New York City Reopening Splits Along Lines of Wealth and Race
• New Yorkers Anxiously Exit Covid Lockdowns After 80 Days of Hell
By The Numbers
• $1.8TSize of New York metro area’s economy, which rivals that of many countries, including Brazil, Canada and South Korea
• 63MVisitors to New York City annually before the pandemic hit
• 900,000Jobs that the city may lose during lockdown
Why It Matters
No city is more important to America’s economic recovery than New York.
As the capital of American capitalism, it’s a global hub for business and commerce. The economic output of the New York metro area, estimated at $1.8 trillion, rivals that of entire nations. The city of 8.6 million is also the country’s center for banking and finance, real estate, media, retail, advertising, tourism and the arts.
100K New York Businesses Will ‘Disappear’: Bankruptcy Lawyer
100K New York Businesses Will ‘Disappear’: Bankruptcy Lawyer
Dozens of the world’s biggest companies are headquartered in New York City, employing hundreds of thousands of people across America and the world. It’s also home to Wall Street, a vital part of the city’s economy and the go-to destination for global capital.
So understanding how New York City emerges from the pandemic — through the lens of business and finance — is crucial to understanding the prospects of the U.S. economy as a whole.

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Multi-Dimension Monitoring – The Software Is Real-Time Capable And Reacts Self-adaptively

flexis Multi Dimension Monitoring visualizes the supply situation and provides reliable transparency of bottlenecks, ranges of coverage, and inventories. This SCM software enables an objective, forward-looking identification of problems before they become critical and can even network different hierarchies. The program includes deviation management and transmits all information in real-time.
Outcome
A guaranteed ability to deliver while simultaneously reducing inventories. As a result, productivity increases considerably.
Benefit
The software is real-time capable and reacts self-adaptively, even when changes occur.

Detail Description
With the flexis Monitoring Solution, users can monitor processes in a global environment. For example, the software recognizes critical parts with long supply chains; it creates comprehensive transparency for requirements, inventories, and transports for the entire planning period. Using the alert function, supply risks or bottleneck situations are detected early, allowing immediate measures to be taken.
The program monitors the supply of parts in the short-term horizon against the planned sequence and compares inventories, shipments, and production. Parts that are responsible for bottleneck situations are defined. The program provides an overview of the saved stocks for planned production and filters the orders that are affected by a parts bottleneck.
The program checks the ability to deliver and identifies which requirements are covered and which cannot be met. It creates Shop FloorTransparency for the planner and displays the status of the products in the process in a clear and concise manner. The SCM system automatically sifts through planning alternatives.
Added Value
• Decisions can be made with real-time knowledge with “turbo transparency”
• Linked knowledge: holistic view of production, warehouse, and transport
• Secure basis for decision-making with a controlling rather than a reactive role
• Early detection of imminent bottlenecks through alarm functions
• Clear presentation like critical parts or critical orders

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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
Discussion

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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.

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“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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