Shear-reversible clusters of HIV-1 in solution: stabilized by antibodies, dispersed by mucin.

IMPORTANCE: The phenomenon of reversible clustering is expected to further nuance HIV immune stealth because virus surfaces can escape interaction with antibodies (Abs) by hiding temporarily within clusters. It is well known that mucin reduces HIV virulence, and the current perspective is that mucin aggregates HIV-1 to reduce infections. Our findings, however, suggest that mucin is dispersing HIV clusters. The study proposes a new paradigm for how HIV-1 may broadly evade Ab recognition with reversible clustering and why mucin effectively neutralizes HIV-1.

Duration of seroprotection of the live attenuated SA-14-14-2 Japanese encephalitis vaccine in children in India.

BACKGROUND: Acute encephalitis syndrome (AES) is a major public health concern in India, and the Japanese Encephalitis (JE) virus is the most common cause of viral encephalitis in Asia affecting children under the age of 15 years. In India, despite the introduction of the JE vaccine (SA-14-14-2) in the immunization programme, JE continues to account for 15-20% of AES cases to date. This study evaluates the immunogenicity of live attenuated SA-14-14-2 JE vaccine in terms of persistence of the humoral response after two doses. METHODS: A cross-sectional study was conducted among 266 children belonging to one of the JE endemic regions of Uttar Pradesh, India. Blood samples were taken from children (2-10 years) and grouped according to the duration (in years) after two doses of the vaccine (5 groups with a class interval of 2 years). Informed written consent was obtained from the parents/guardians. All the samples collected were tested for the presence of anti-JEV-specific IgG antibodies by enzyme-linked immunosorbent assay (ELISA) and further confirmed by micro neutralization test (MNT) and immunofluorescence assays. RESULTS: Of the 266 samples tested by ELISA for anti-JEV-specific IgG antibodies, 260 (97.74%) were negative and 6 (2.26%) were equivocal. The geometric mean immune status ratio across the five groups, 0-2 years (n = 59), 2-4 years (n = 73), 4-6 years (n = 65), 6-8 years (n = 48) and 8-10 years (n = 21) post-two doses of SA-14-14-2 JE vaccine was 1.143, 1.059, 1.138, 1.075 and 1.130, respectively, and the geometric mean titre obtained from MNT across the five groups was 10.77, 8.400, 8.453, 9.517 and 9.674, respectively. CONCLUSION: The study showed a decreasing trend of anti-JEV specific IgG antibody titres across the five groups based on the duration following two doses of SA-14-14-2 vaccine. The results emphasize the significance of booster doses of vaccine for children living in endemic areas.

Shells of compacted DNA as nanocontainers transporting proteins in multiplexed delivery.

Polyethyleneimine (PEI) polymers are known to compact DNA strands into spheroid, toroid, or rod structures. A formulation with mannose-grafted PEI (PEIm), however, was reported to compact DNA into ~100 nm spheroids that indented like thin-walled pressurized shells. The goal of the study is to understand why mannose bristles divert the traditional pathway of PEI-DNA compaction to produce shell-like structures, and to manipulate the process so that proteins can be packed into the core of the assembling shells for co-delivering DNA and proteins into cells. DLS, AFM, and TEM imaging provide a consistent picture that BSA proteins can be packed into the shells without altering the shell architecture, as long as the proteins were added during the time course of shell assembly. Force spectroscopy studies reveal that DNA shells that buckle also have a rich surface-coating of mannose, indicating that a micelle-like partitioning of hydrophobic and hydrophilic layers governs shell assembly. When HEK293T cells are spiked with BSA-laden DNA shells, co-transfection of DNA and BSA is observed at higher levels than control formulations. Distinct micron-sized features appear having both green fluorescence from BSA-FITC and blue fluorescence from NucBlue DNA stain, suggesting BSA release in nucleus and secretory granules. With DNA nanocontainers, proteins can take advantage of the efficiency of PEI-based DNA transfection for hitchhiking into cells while being shielded from the challenges of the intracellular route. DNA nanocontainers are rapid to assemble, not dependent on the DNA sequence, and can be adapted for different protein types; thereby having potential to serve as a high-throughput platform in scenarios where DNA and protein have to be released at the same site and time within cells (e.g., theranostics, multiplexed co-delivery, gene editing).

Therapeutic use of Guggulsterone in COVID-19 induced obesity (COVIBESITY) and significant role in immunomodulatory effect.

COVID-19 has emerged as a major cause of health crisis around the world. Psychosocial, Behavioral and metabolic changes especially weight gain, among variety of population was produced in this pandemic, through variety of mechanisms. Hyperlipidemia is one of the major issues that result in serious cardiovascular complications. Governmental strategies to minimize the spread of COVID-19 through closures, lockdowns, and alterations in social interaction have complicated weight management efforts. And immunity being the need of the hour has to be improved to prevent the infection. Guggulsterone (GS) isomers are major bioactive compounds present in Commiphora mukul and Commiphora wighti. Guggulsterone shows anti-hyperlipidemic, anti-oxidant, anti-inflammatory, immunomodulatory and appetite regulating activity due to its peculiar characteristics. On the basis of clinical evidence, Guggulsterone seems to possess good cholesterol lowering, appetite regulating as well as immunomodulatory activity which can be beneficial during the pandemic of COVID-19.

Establishing Rules for Self-Adhesion and Aggregation of N-Glycan Sugars Using Virus Glycan Shields.

The surfaces of cells and pathogens are covered with short polymers of sugars known as glycans. Complex N-glycans have a core of three mannose sugars with distal repeats of N-acetylglucosamine and galactose sugars terminating with sialic acid (SA). Long-range tough and short-range brittle self-adhesions were observed between SA and mannose residues, respectively, in ill-defined artificial monolayers. We investigated if and how these adhesions translate when the residues are presented in N-glycan architecture with SA at the surface and mannose at the core and with other glycan sugars. Two pseudotyped viruses with complex N-glycan shields were brought together in force spectroscopy (FS). At higher ramp rates, slime-like adhesions were observed between the shields, whereas Velcro-like adhesions were observed at lower rates. The higher approach rates compress the virus as a whole, and the self-adhesion between the surface SA is sampled. At the lower ramp rates, however, the complex glycan shield is penetrated and adhesion from the mannose core is accessed. The slime-like and Velcro-like adhesions were lost when SA and mannose were cleaved, respectively. While virus self-adhesion in forced contact was modulated by glycan penetrability, the self-aggregation of the freely diffusing virus was only determined by the surface sugar. Mannose-terminal viruses self-aggregated in solution, and SA-terminal ones required Ca2+ ions to self-aggregate. Viruses with galactose or N-acetylglucosamine surfaces did not self-aggregate, irrespective of whether or not a mannose core was present below the N-acetylglucosamine surface. Well-defined rules appear to govern the self-adhesion and -aggregation of N-glycosylated surfaces, regardless of whether the sugars are presented in an ill-defined monolayer, or N-glycan, or even polymer architecture.

The PET-Tracer 89Zr-Df-IAB22M2C Enables Monitoring of Intratumoral CD8 T-cell Infiltrates in Tumor-Bearing Humanized Mice after T-cell Bispecific Antibody Treatment.

CD8-expressing T cells are the main effector cells in cancer immunotherapy. Treatment-induced changes in intratumoral CD8+ T cells may represent a biomarker to identify patients responding to cancer immunotherapy. Here, we have used a 89Zr-radiolabeled human CD8-specific minibody (89Zr-Df-IAB22M2C) to monitor CD8+ T-cell tumor infiltrates by PET. The ability of this tracer to quantify CD8+ T-cell tumor infiltrates was evaluated in preclinical studies following single-agent treatment with FOLR1-T-cell bispecific (TCB) antibody and combination therapy of CEA-TCB (RG7802) and CEA-targeted 4-1BB agonist CEA-4-1BBL. In vitro cytotoxicity assays with peripheral blood mononuclear cells and CEA-expressing MKN-45 gastric or FOLR1-expressing HeLa cervical cancer cells confirmed noninterference of the anti-CD8-PET-tracer with the mode of action of CEA-TCB/CEA-4-1BBL and FOLR1-TCB at relevant doses. In vivo, the extent of tumor regression induced by combination treatment with CEA-TCB/CEA-4-1BBL in MKN-45 tumor-bearing humanized mice correlated with intratumoral CD8+ T-cell infiltration. This was detectable by 89Zr-IAB22M2C-PET and γ-counting. Similarly, single-agent treatment with FOLR1-TCB induced strong CD8+ T-cell infiltration in HeLa tumors, where 89Zr-Df-IAB22M2C again was able to detect CD8 tumor infiltrates. CD8-IHC confirmed the PET imaging results. Taken together, the anti-CD8-minibody 89Zr-Df-IAB22M2C revealed a high sensitivity for the detection of intratumoral CD8+ T-cell infiltrates upon either single or combination treatment with TCB antibody-based fusion proteins. These results provide further evidence that the anti-CD8 tracer, which is currently in clinical phase II, is a promising monitoring tool for intratumoral CD8+ T cells in patients treated with cancer immunotherapy. SIGNIFICANCE: Monitoring the pharmacodynamic activity of cancer immunotherapy with novel molecular imaging tools such as 89Zr-Df-IAB22M2C for PET imaging is of prime importance to identify patients responding early to cancer immunotherapy.

Tumor-targeted 4-1BB agonists for combination with T cell bispecific antibodies as off-the-shelf therapy.

Endogenous costimulatory molecules on T cells such as 4-1BB (CD137) can be leveraged for cancer immunotherapy. Systemic administration of agonistic anti-4-1BB antibodies, although effective preclinically, has not advanced to phase 3 trials because they have been hampered by both dependency on Fcγ receptor-mediated hyperclustering and hepatotoxicity. To overcome these issues, we engineered proteins simultaneously targeting 4-1BB and a tumor stroma or tumor antigen: FAP-4-1BBL (RG7826) and CD19-4-1BBL. In the presence of a T cell receptor signal, they provide potent T cell costimulation strictly dependent on tumor antigen-mediated hyperclustering without systemic activation by FcγR binding. We could show targeting of FAP-4-1BBL to FAP-expressing tumor stroma and lymph nodes in a colorectal cancer-bearing rhesus monkey. Combination of FAP-4-1BBL with tumor antigen-targeted T cell bispecific (TCB) molecules in human tumor samples led to increased IFN-γ and granzyme B secretion. Further, combination of FAP- or CD19-4-1BBL with CEA-TCB (RG7802) or CD20-TCB (RG6026), respectively, resulted in tumor remission in mouse models, accompanied by intratumoral accumulation of activated effector CD8+ T cells. FAP- and CD19-4-1BBL thus represent an off-the-shelf combination immunotherapy without requiring genetic modification of effector cells for the treatment of solid and hematological malignancies.

Complex-type N-glycans on VSV-G pseudotyped HIV exhibit 'tough' sialic and 'brittle' mannose self-adhesions.

The complex-type glycan shields of eukaryotic cells have a core layer of mannose residues buried under tiers of sugars that end with sialic acid (SA) residues. We investigate if the self-latching of mannose residues, earlier reported in pure monolayer studies, also manifests in the setting of a complex-type glycan shield. Would distal SA residues impede access to the mannose core? The interactions of mannobiose-, SA-, and lactose-coated probes with the complex-type VSV-G glycan shield on an HIV pseudovirus were studied with force-spectroscopy and gold-nanoparticle solutions. In force spectroscopy, the sugar probes can be forced to sample the depths of the glycan shield, whereas with sugar-coated nanoparticles, only interactions permitted by freely-diffusive contact occur. Deep-indentation mechanics was performed to verify the inferred structure of the engineered virus and to isolate the glycan shield layer for subsequent interaction studies. The adhesion between the sugar-probes and complex-type glycan shield was deconvoluted by comparing against the cross- and self- adhesions between the sugars in pure monolayers. Results from complementing systems were consistent with mannobiose-coated probes latching to the mannose core in the glycan shield, unhindered by the SA and distal sugars, with a short-range 'brittle' release of adhesion resulting in tightly coated viruses. SA-Coated probes, however, adhere to the terminal SA layer of a glycan shield with long-range and mechanically 'tough' adhesions resulting in large-scale virus aggregation. Lactose-coated probes exhibit ill-defined adherence to sialic residues. The selection and positioning of sugars within a glycan shield can influence how carbohydrate surfaces of different composition adhere.

Capturing 3D large-strain Euler-bending filament dynamics in fibrous media simulations; sample case of compression collapse in dendritic actin network.

Cytoskeletal networks to transmission towers are comprised of slender elements. Slender filaments bend and buckle more easily than stretch. Therefore a deforming network is expected to exhaust all possible bending-based modes before engaging filament stretch. While the large-strain bending critically determines fibrous-media response, simulations use small-strain and jointed approximations. At low resolution, these approximations inflate bending resistance and delay buckling onset. The proposed string-of-continuous-beams (SOCB) approach captures 3D nonlinear Euler bending of filaments with high fidelity at low cost. Bending geometry (i.e. angles and its differentials) is solved as primary variables, to fit a 5th order polynomial of the contour angle. Displacement, solved simultaneously as length conservation, is predicted with C3 and C6 smoothness between and within segments, using only 2 nodes. In the chosen analysis frame, in-plane and out-plane moments can be decoupled for arbitrarily-curved segments. Complex crosslink force-transfers can be specified. Simulations show that when a daughter branch is appended, the buckling resistance of a filament changes from linear to nonlinear before reversible collapse. An actin outcrop with 8 generations of mother-daughter branching produced the linear, nonlinear, and collapse regimes observed in compression experiments. 'Collapse' was a redistribution of outcrop forces following the buckling of few strands.

Microscale mapping of extracellular matrix elasticity of mouse joint cartilage: an approach to extracting bulk elasticity of soft matter with surface roughness.

Cartilage is composed of cells and an extracellular matrix, the latter being a composite of a collagen mesh interpenetrated by proteoglycans responsible for tissue osmotic swelling. The matrix composition and structure vary through the tissue depth. Mapping such variability requires tissue sectioning to gain access. The resulting surface roughness, and concomitant proteoglycan loss contribute to large uncertainties in elastic modulus estimates. To extract elasticity values for the bulk matrix which are not obfuscated by the indeterminate surface layer, we developed a novel experimental and data analysis methodology. We analyzed the surface roughness to optimize the probe size, and performed high-resolution (1 µm) elasticity mapping on thin (∼12 µm), epiphyseal newborn mouse cartilage sections cut parallel to the bone longitudinal axis or normal to the articular surface. Mild fixation prevented the major proteoglycan loss observed in unfixed specimens but not the stress release that resulted in thickness changes in the sectioned matrix. Our novel data analysis method introduces a virtual contact point as a fitting parameter for the Hertz model, to minimize the effects of surface roughness and corrects for the finite section thickness. Our estimates of cartilage elasticity converge with increasing indentation depth and, unlike previous data interpretations, are consistent with linearly elastic material. A high cell density that leaves narrow matrix septa between cells may cause the underestimation of elastic moduli, whereas fixation probably causes an overestimation. The proposed methodology has broader relevance to nano- and micro-indentation of soft materials with multiple length scales of organization and whenever surface effects (including roughness, electrostatics, van der Waals forces, etc.) become significant.

A Study on Doped Heterojunctions in TiO2 Nanotubes: An Efficient Photocatalyst for Solar Water Splitting.

The two important factors that affect sunlight assisted water splitting ability of TiO2 are its charge recombination and large band gap. We report the first demonstration of nitrogen doped triphase (anatase-rutile-brookite) TiO2 nanotubes as sun light active photocatalyst for water splitting with high quantum efficiency. Nitrogen doped triphase TiO2 nanotubes, corresponding to different nitrogen concentrations, are synthesized electrochemically. Increase in nitrogen concentration in triphase TiO2 nanotubes is found to induce brookite to anatase phase transformation. The variation in density of intra-band states (Ti3+ and N 2p states) with increase in nitrogen doping are found to be critical in tuning the photocatalytic activity of TiO2 nanotubes. The presence of bulk heterojunctions in single nanotube of different nitrogen doped TiO2 samples is confirmed from HRTEM analysis. The most active nitrogen doped triphase TiO2 nanotubes are found to be 12 times efficient compared to pristine triphase TiO2, for solar hydrogen generation. The band alignment and charge transfer pathways in nitrogen doped TiO2 with triphase heterojunctions are delineated. Bulk heterojunctions among the three phases present in the nanotubes with intra-band defect states is shown to enhance the photocatalytic activity tremendously. Our study also confirms the theory that three phase system is efficient in photocatalysis compared to two phase system.

Mannose Surfaces Exhibit Self-Latching, Water Structuring, and Resilience to Chaotropes: Implications for Pathogen Virulence.

Several viral and fungal pathogens, including HIV, SARS, Dengue, Ebola, and Cryptococcus neoformans, display a preponderance of mannose residues on their surface, particularly during the infection cycle or in harsh environments. The innate immune system, on the other hand, abounds in mannose receptors which recognize mannose residues on pathogens and trigger their phagocytosis. We pose the question if there is an advantage for pathogens to display mannose on their surface, despite these residues being recognized by the immune system. The surface properties and interactions of opposing monolayers of mannobiose (disaccharide of mannose) were probed using atomic force spectroscopy. Unlike its diastereoisomer lactose, mannobiose molecules exhibited lateral packing interactions that manifest on the surface scale as a self-recognizing latch. A break-in force is required for opposing surfaces to penetrate and a breakout (or self-adhesion force) of similar magnitude is required for penetrated surfaces to separate. A hierarchy of self-adhesion forces was distinguished as occurring at the single residue (∼25 pN), cluster (∼250 pN), monolayer (∼1.1 nN), and supramonolayer level. The break-in force and break-out force appear resilient to the presence of simple chaotropes that attenuate a layer of structured water around the mannose surface. The layer of structured water otherwise extends to distances several times longer than a mannobiose residue, indicating a long-range propagation of the hydrogen bonding imposed by the residues. The span of the structured water increases with the velocity of an approaching surface, similar to shear thickening, but fissures at higher approach velocities. Our studies suggest that mannose residues could guide interpathogen interactions, such as in biofilms, and serve as a moated fortress for pathogens to hide behind to resist detection and harsh environments.

An engineering design approach to systems biology.

Measuring and modeling the integrated behavior of biomolecular-cellular networks is central to systems biology. Over several decades, systems biology has been shaped by quantitative biologists, physicists, mathematicians, and engineers in different ways. However, the basic and applied versions of systems biology are not typically distinguished, which blurs the separate aspirations of the field and its potential for real-world impact. Here, we articulate an engineering approach to systems biology, which applies educational philosophy, engineering design, and predictive models to solve contemporary problems in an age of biomedical Big Data. A concerted effort to train systems bioengineers will provide a versatile workforce capable of tackling the diverse challenges faced by the biotechnological and pharmaceutical sectors in a modern, information-dense economy.

Unusual Salt and pH Induced Changes in Polyethylenimine Solutions.

Linear PEI is a cationic polymer commonly used for complexing DNA into nanoparticles for cell-transfection and gene-therapy applications. The polymer has closely-spaced amines with weak-base protonation capacity, and a hydrophobic backbone that is kept unaggregated by intra-chain repulsion. As a result, in solution PEI exhibits multiple buffering mechanisms, and polyelectrolyte states that shift between aggregated and free forms. We studied the interplay between the aggregation and protonation behavior of 2.5 kDa linear PEI by pH probing, vapor pressure osmometry, dynamic light scattering, and ninhydrin assay. Our results indicate that: At neutral pH, the PEI chains are associated and the addition of NaCl initially reduces and then increases the extent of association.The aggregate form is uncollapsed and co-exists with the free chains.PEI buffering occurs due to continuous or discontinuous charging between stalled states.Ninhydrin assay tracks the number of unprotonated amines in PEI.The size of PEI-DNA complexes is not significantly affected by the free vs. aggregated state of the PEI polymer. Despite its simple chemical structure, linear PEI displays intricate solution dynamics, which can be harnessed for environment-sensitive biomaterials and for overcoming current challenges with DNA delivery.

DNA nanoparticles with core-shell morphology.

Mannobiose-modified polyethylenimines (PEI) are used in gene therapy to generate nanoparticles of DNA that can be targeted to the antigen-presenting cells of the immune system. We report that the sugar modification alters the DNA organization within the nanoparticles from homogenous to shell-like packing. The depth-dependent packing of DNA within the nanoparticles was probed using AFM nano-indentation. Unmodified PEI-DNA nanoparticles display linear elastic properties and depth-independent mechanics, characteristic of homogenous materials. Mannobiose-modified nanoparticles, however, showed distinct force regimes that were dependent on indentation depth, with 'buckling'-like response that is reproducible and not due to particle failure. By comparison with theoretical studies of spherical shell mechanics, the structure of mannobiosylated particles was deduced to be a thin shell with wall thickness in the order of few nanometers, and a fluid-filled core. The shell-core structure is also consistent with observations of nanoparticle denting in altered solution conditions, with measurements of nanoparticle water content from AFM images, and with images of DNA distribution in Transmission Electron Microscopy.

Improving water quality communications at beaches: input from stakeholders.

OBJECTIVES: Water quality communication practices vary widely and stakeholder input has not played a role in defining acceptable levels of risk. Although the 2012 Recreational Water Quality Criteria (RWQC) emphasize the importance of promptly notifying the public about hazardous conditions, little is known about the public's understanding of notifications, or about levels of risk deemed acceptable. We sought to address these gaps. METHODS: A mixed methods approach was used. Focus groups (FGs) provided qualitative data regarding the understanding of surface water quality, awareness, and use, of currently available water quality information, and acceptability of risk. Intercept interviews (INTs) at recreation sites provided quantitative data. RESULTS: INTs of 374 people and 15 FG sessions were conducted. Participants had limited awareness about water quality information posted at beaches, even during swim bans and advisories. Participants indicated that communication content should be current, from a trusted source, and describe health consequences. Communicating via mobile electronics should be useful for segments of the population. Risk acceptability is lower with greater outcome severity, or if children are impacted. CONCLUSIONS: Current water quality communications approaches must be enhanced to make notification programs more effective. Further work should build on this initial effort to evaluate risk acceptability among US beachgoers.

Structural mechanism for alteration of collagen gel mechanics by glutaraldehyde crosslinking.

Soft collagenous tissues that are loaded in vivo undergo crosslinking during aging and wound healing. Bioprosthetic tissues implanted in vivo are also commonly crosslinked with glutaraldehyde (GA). While crosslinking changes the mechanical properties of the tissue, the nature of the mechanical changes and the underlying microstructural mechanism are poorly understood. In this study, a combined mechanical, biochemical and simulation approach was employed to identify the microstructural mechanism by which crosslinking alters mechanical properties. The model collagenous tissue used was an anisotropic cell-compacted collagen gel, and the model crosslinking agent was monomeric GA. The collagen gels were incrementally crosslinked by either increasing the GA concentration or increasing the crosslinking time. In biaxial loading experiments, increased crosslinking produced (1) decreased strain response to a small equibiaxial preload, with little change in response to subsequent loading and (2) decreased coupling between the fiber and cross-fiber direction. The mechanical trend was found to be better described by the lysine consumption data than by the shrinkage temperature. The biaxial loading of incrementally crosslinked collagen gels was simulated computationally with a previously published network model. Crosslinking was represented by increased fibril stiffness or by increased resistance to fibril rotation. Only the latter produced mechanical trends similar to that observed experimentally. Representing crosslinking as increased fibril stiffness did not reproduce the decreased coupling between the fiber and cross-fiber directions. The study concludes that the mechanical changes in crosslinked collagen gels are caused by the microstructural mechanism of increased resistance to fibril rotation.

Structure and biological activity of pathogen-like synthetic nanomedicines.

Here we characterize the structure, stability and intracellular mode of action of DermaVir nanomedicine that is under clinical development for the treatment of HIV/AIDS. This nanomedicine comprises pathogen-like pDNA/PEIm nanoparticles (NPs) having the structure and function resembling spherical viruses that naturally evolved to deliver nucleic acids to the cells. Atomic force microscopy demonstrated spherical 100 - 200 nm NPs with a smooth polymer surface protecting the pDNA in the core. Optical absorption determined both the NP structural stability and biological activity relevant to their ability to escape from the endosome and release the pDNA at the nucleus. Salt, pH and temperature influence nanomedicine shelf-life and intracellular stability. This approach facilitates the development of diverse polyplex nanomedicines where the delivered pDNA-expressed antigens induce immune responses to kill infected cells. FROM THE CLINICAL EDITOR: The authors investigated DermaVir nanomedicine comprised of pathogen-like pDNA/PEIm nanoparticles with structure and function resembling spherical viruses. DermaVir delivery of pDNA expresses antigens that induce immune responses to kill HIV infected cells.