Effects of Cell-Adhesive Ligand Presentation on Pentapeptide Supramolecular Assembly and Gelation: Simulations and Experiments.

The extracellular matrix (ECM) is a complex, hierarchical material containing structural and bioactive components. This complexity makes decoupling the effects of biomechanical properties and cell-matrix interactions difficult, especially when studying cellular processes in a 3D environment. Matrix mechanics and cell adhesion are both known regulators of specific cellular processes such as stem cell proliferation and differentiation. However, more information is required about how such variables impact various neural lineages that could, upon transplantation, therapeutically improve neural function after a central nervous system injury or disease. Rapidly Assembling Pentapeptides for Injectable Delivery (RAPID) hydrogels are one biomaterial approach to meet these goals, consisting of a family of peptide sequences that assemble into physical hydrogels in physiological media. In this study, we studied our previously reported supramolecularly-assembling RAPID hydrogels functionalized with the ECM-derived cell-adhesive peptide ligands RGD, IKVAV, and YIGSR. Using molecular dynamics simulations and experimental rheology, we demonstrated that these integrin-binding ligands at physiological concentrations (3-12 mm) did not impact the assembly of the KYFIL peptide system. In simulations, molecular measures of assembly such as hydrogen bonding and pi-pi interactions appeared unaffected by cell-adhesion sequence or concentration. Visualizations of clustering and analysis of solvent-accessible surface area indicated that the integrin-binding domains remained exposed. KYFIL or AYFIL hydrogels containing 3 mm of integrin-binding domains resulted in mechanical properties consistent with their non-functionalized equivalents. This strategy of doping RAPID gels with cell-adhesion sequences allows for the precise tuning of peptide ligand concentration, independent of the rheological properties. The controllability of the RAPID hydrogel system provides an opportunity to investigate the effect of integrin-binding interactions on encapsulated neural cells to discern how hydrogel microenvironment impacts growth, maturation, or differentiation.

Stakeholder preferences for attributes of digital health technologies to consider in health service funding.

OBJECTIVES: Health service providers are currently making decisions on the public funding of digital health technologies (DHTs) for managing chronic diseases with limited understanding of stakeholder preferences for DHT attributes. This study aims to understand the community, patient/carer, and health professionals' preferences to help inform a prioritized list of evaluation criteria. METHODS: An online best-worst scaling survey was conducted in Australia, New Zealand, Canada, and the United Kingdom to ascertain the relative importance of twenty-four DHT attributes among stakeholder groups using an efficient incomplete block design. The attributes were identified from a systematic review of DHT evaluation frameworks for consideration in a health technology assessment. Results were analyzed with multinomial models by stakeholder group and latent class. RESULTS: A total of 1,251 participants completed the survey (576 general community members, 543 patients/carers, and 132 health professionals). Twelve attributes achieved a preference score above 50 percent in the stakeholder group model, predominantly related to safety but also covering technical features, effectiveness, ethics, and economics. Results from the latent class model supported this prioritization. Overall, connectedness with the patient's healthcare team seemed the most important; with "Helps health professionals respond quickly when changes in patient care are needed" as the most highly prioritized of all attributes. CONCLUSIONS: It is proposed that these prioritized twelve attributes be considered in all evaluations of DHTs that manage chronic disease, supplemented with a limited number of attributes that reflect the specific perspective of funders, such as equity of access, cost, and system-level implementation considerations.

Engineering human liver fatty acid binding protein for detection of poly- and perfluoroalkyl substances.

Per- and polyfluoroalkyl substances (PFAS) are a large group of synthetic fluorinated chemicals with surface active and water-repellent properties. The combination of wide-spread use in numerous consumer and industrial products and extended biological half-lives arising from strong carbon-fluorine bonds has led to significant accumulation of PFAS in humans. As most human interaction with PFAS comes from ingestion, it is important to be able to detect PFAS in drinking water as well as in agricultural water. Here we present an approach to designing a fluorescence-based biosensor for the rapid detection of PFAS based on human liver fatty acid binding protein (hLFABP). Introduction of solvatochromic fluorophores within the ligand binding pocket (L50) allowed for intrinsic detection of perfluorooctanoic acid (PFOA), perfluorooctanesulfonic acid (PFOS), and perfluorohexanesulfonic acid (PFHxS) via blue-shifts in fluorescence emission spectra. Initially, a single tryptophan mutation (L50W) was found to be able to detect PFOA with a limit of detection (LOD) of 2.8 ppm. We improved the sensitivity of the biosensor by exchanging tryptophan for the thiol reactive fluorophore, acrylodan. The acrylodan conjugated C69S/F50C hLFABP variant is capable of detecting PFOA, PFOS, and PFHxS in PBS with LODs of 112 ppb, 345 ppb, and 1.09 ppm, respectively. The protein-based sensor is also capable of detecting these contaminants at similar ranges in spiked environmental water samples, including samples containing an interfering anionic surfactant sodium dodecyl sulfate. Overall, this study demonstrates engineered hLFABP is a useful platform for detection of PFAS in environmental water samples and highlights its ease of use and versatility in field applications.

Impact of fish consumption on all-cause mortality in older people with and without dementia: a community-based cohort study.

BACKGROUND: Increased fish consumption reduces the risk of dementia. However, it is unknown whether fish consumption reduced all-cause mortality in people with dementia. The purpose of the study is to investigate the association of fish consumption with all-cause mortality in older people with dementia versus those without dementia. METHODS: Using a standard method of the Geriatric Mental State, we interviewed 4165 participants aged ≥ 60 years who were randomly recruited from five provinces in China during 2007-2009 to collect the baseline data of socio-demography, disease risk factors, histories of disease, and details of dietary intakes, and diagnosed dementia (n = 406). They were followed up for vital status until 2012. RESULTS: The cohort follow-up documented 329 deaths; 61 were in participants with dementia (55.3 per 1000 person-years) and 224 were those without dementia (22.3). In all participants, the risk of all-cause mortality was reduced with fish intake at " ≥ twice a week" (multivariate-adjusted hazard ratio 0.58, 95% CI 0.34-0.96) and at "once a week or less" (0.79, 0.53-1.18) compared to "never eat" over the past two years. In participants without baseline dementia, the corresponding HRs for all-cause mortality were 0.57 (0.33-0.98) and 0.85 (0.55-1.31), while in participants with dementia were 1.36 (0.28-6.60) and 1.05 (0.30-3.66), respectively. CONCLUSION: This study reveals that consumption of fish in older age reduced all-cause mortality in older people without dementia, but not in people with dementia. Fish intake should be increased in older people in general, prior to the development of dementia in the hope of preventing dementia and prolonging life.

Perspectives of solid organ transplant recipients on medicine-taking: Systematic review of qualitative studies.

Medicine-taking among transplant recipients is a complex and ubiquitous task with significant impacts on outcomes. This study aimed to describe the perspectives and experiences of medicine-taking in adult solid organ transplant recipients. Electronic databases were searched to July 2020, and thematic synthesis was used to analyze the data. From 119 studies (n = 2901), we identified six themes: threats to identity and ambitions (impaired self-image, restricting goals and roles, loss of financial independence); navigating through uncertainty and distrust (lacking tangible/perceptible benefits, unprepared for side effects, isolation in decision-making); alleviating treatment burdens (establishing and mastering routines, counteracting side effects, preparing for the unexpected); gaining and seeking confidence (clarity with knowledge, reassurance through collective experiences, focusing on the future outlook); recalibrating to a new normal posttransplant (adjusting to ongoing dependence on medications, in both states of illness and health, unfulfilled expectations); and preserving graft survival (maintaining the ability to participate in life, avoiding rejection, enacting a social responsibility of giving back). Transplant recipients take medications to preserve graft function, but dependence on medications jeopardizes their sense of normality. Interventions supporting the adaptation to medicine-taking and addressing treatment burdens may improve patient satisfaction and capacities to take medications for improved outcomes.

Rare/cryptic Aspergillus species infections and importance of antifungal susceptibility testing.

BACKGROUND: The number of patients infected with Aspergillus rose dramatically in recent years. However, studies on the clinical spectrum and antifungal susceptibilities of non-classical (non-fumigatus, non-flavus, non-niger and non-terreus) pathogenic Aspergillus species are very limited. OBJECTIVES: We examined the clinical spectrum and antifungal susceptibilities of 34 non-duplicated, non-classical Aspergillus isolates collected from Hong Kong, Shenzhen and Shanghai. METHODS: The Aspergillus isolates were identified by internal transcribed spacer, partial BenA and partial CaM sequencing and phylogenetic analyses. Susceptibility testing against eight antifungals was performed following the European Committee for Antimicrobial Susceptibility Testing's methodology. RESULTS: The 34 Aspergillus isolates were identified as 14 different rare/cryptic species of four sections (Flavi [n = 8], Nidulantes [n = 8], Nigri [n = 17] and Restricti [n = 1]). Except for one patient whose clinical history could not be retrieved, 72.7% of the remaining patients had underlying conditions predisposing them to Aspergillus infections. The most common diseases were pulmonary infections (n = 15), followed by skin/nail infections (n = 6), chronic otitis externa and/or media (n = 5), wound infections (n = 2) and mastoiditis/radionecrosis (n = 1), while three were colonisations. Five patients succumbed due to the infections during the admission, and another two died 5 years later because of chronic pulmonary aspergillosis. Antifungal susceptibility testing showed that they possessed different susceptibility profiles compared to the classical Aspergillus species. The majority of isolates characterised were sensitive or wild-type to amphotericin B. The minimum effective concentrations for all the three echinocandins were also low. CONCLUSION: Susceptibility testing should be performed for infections due to these rare/cryptic Aspergillus species to guide proper patient management.

A Rapid, Simple, Inexpensive, and Mobile Colorimetric Assay COVID-19-LAMP for Mass On-Site Screening of COVID-19.

To control the COVID-19 pandemic and prevent its resurgence in areas preparing for a return of economic activities, a method for a rapid, simple, and inexpensive point-of-care diagnosis and mass screening is urgently needed. We developed and evaluated a one-step colorimetric reverse-transcriptional loop-mediated isothermal amplification assay (COVID-19-LAMP) for detection of SARS-CoV-2, using SARS-CoV-2 isolate and respiratory samples from patients with COVID-19 (n = 223) and other respiratory virus infections (n = 143). The assay involves simple equipment and techniques and low cost, without the need for expensive qPCR machines, and the result, indicated by color change, is easily interpreted by naked eyes. COVID-19-LAMP can detect SARS-CoV-2 RNA with detection limit of 42 copies/reaction. Of 223 respiratory samples positive for SARS-CoV-2 by qRT-PCR, 212 and 219 were positive by COVID-19-LAMP at 60 and 90 min (sensitivities of 95.07% and 98.21%) respectively, with the highest sensitivities among nasopharyngeal swabs (96.88% and 98.96%), compared to sputum/deep throat saliva samples (94.03% and 97.02%), and throat swab samples (93.33% and 98.33%). None of the 143 samples with other respiratory viruses were positive by COVID-19-LAMP, showing 100% specificity. Samples with higher viral load showed shorter detection time, some as early as 30 min. This inexpensive, highly sensitive and specific COVID-19-LAMP assay can be useful for rapid deployment as mobile diagnostic units to resource-limiting areas for point-of-care diagnosis, and for unlimited high-throughput mass screening at borders to reduce cross-regional transmission.

Stimuli-Responsive, Pentapeptide, Nanofiber Hydrogel for Tissue Engineering.

Short peptides are uniquely versatile building blocks for self-assembly. Supramolecular peptide assemblies can be used to construct functional hydrogel biomaterials-an attractive approach for neural tissue engineering. Here, we report a new class of short, five-residue peptides that form hydrogels with nanofiber structures. Using rheology and spectroscopy, we describe how sequence variations, pH, and peptide concentration alter the mechanical properties of our pentapeptide hydrogels. We find that this class of seven unmodified peptides forms robust hydrogels from 0.2-20 kPa at low weight percent (less than 3 wt %) in cell culture media and undergoes shear-thinning and rapid self-healing. The peptides self-assemble into long fibrils with sequence-dependent fibrillar morphologies. These fibrils exhibit a unique twisted ribbon shape, as visualized by transmission electron microscopy (TEM) and Cryo-EM imaging, with diameters in the low tens of nanometers and periodicities similar to amyloid fibrils. Experimental gelation behavior corroborates our molecular dynamics simulations, which demonstrate peptide assembly behavior, an increase in ß-sheet content, and patterns of variation in solvent accessibility. Our rapidly assembling pentapeptides for injectable delivery (RAPID) hydrogels are syringe-injectable and support cytocompatible encapsulation of oligodendrocyte progenitor cells (OPCs), as well as their proliferation and three-dimensional process extension. Furthermore, RAPID gels protect OPCs from mechanical membrane disruption and acute loss of viability when ejected from a syringe needle, highlighting the protective capability of the hydrogel as potential cell carriers for transplantation therapies. The tunable mechanical and structural properties of these supramolecular assemblies are shown to be permissive to cell expansion and remodeling, making this hydrogel system suitable as an injectable material for cell delivery and tissue engineering applications.

Clinical characteristics, rapid identification, molecular epidemiology and antifungal susceptibilities of Talaromyces marneffei infections in Shenzhen, China.

Although case series of talaromycosis have been reported in China, their detailed clinical and microbiological characteristics have never been systematically profiled. In this study, we report the clinical characteristics, molecular epidemiology, rapid identification and antifungal susceptibilities of talaromycosis in The University of Hong Kong-Shenzhen Hospital in Shenzhen. Seven cases of talaromycosis were observed since commencement of hospital service in 2012. Three patients were local Shenzhen residents, whereas the other four were immigrants from other parts of China. Two patients were HIV-negative, but with underlying diseases requiring immunosuppressive therapy. Two of the seven patients succumbed. All the seven isolates were successfully identified as T. marneffei by MALDI-TOF MS using Bruker database expanded with in-house generated T. marneffei mass spectra. MLST showed that the seven strains belonged to six different, novel sequences types. Phylogenetic analyses of the concatenated five-locus sequence revealed that the seven strains were scattered amongst other T. marneffei strains. The MICs of itraconazole, isavuconazole, posaconazole and voriconazole against the seven clinical isolates were low but MICs of anidulafungin were high. Underlying diseases other than HIV infection are increasingly important risk factors of talaromycosis. MALDI-TOF MS is useful for rapid identification. Highly diverse T. marneffei sequence types were observed.

Temperature-Dependent Complex Coacervation of Engineered Elastin-like Polypeptide and Hyaluronic Acid Polyelectrolytes.

Coacervates have enormous potential due to their diverse functional properties supporting a wide number of applications in personal care products, pharmaceuticals, and food processing. Normally, separation of coacervate phases is induced by changes in pH, ionic strength, and/or polyelectrolyte concentration. This study investigates the microphase separation and coacervate complex formation of two natural polyelectrolytes, elastin-like polypeptides (ELPs) and hyaluronic acid (HA), as simple models for biological coacervates. These complex coacervates are formed over a wide range of stoichiometric molar charge ratios without the presence of salt or changes in pH and are primarily induced by changes in temperature. Unlike pure ELP solutions, the ELP/HA coacervates result in well-formed spherical particles after the temperature-induced phase transition. We also note that the formation of these complex coacervates is reversible with low hysteresis. We have demonstrated via fluorescent imaging and dynamic light scattering that high positive/negative charge ratios at elevated temperatures produced 400-600 nm particles with relatively low polydispersity indices (PDIs) of ∼0.1. Furthermore, dynamic light scattering, fluorescence microscopy, and optical microscopy revealed that the ratio of the two polyions strongly influenced the size and structure of these ELP/HA complex coacervates. Finally, we showed that the ELP/HA coacervates were able to sequester the hydrophobic fluorescent molecule pyrene, highlighting their potential for use as delivery vehicles for hydrophobic payloads.

Tailoring the Supramolecular Structure of Guanidinylated Pullulan toward Enhanced Genetic Photodynamic Therapy.

In the progress of designing a gene carrier system, what is urgently needed is a balance of excellent safety and satisfactory efficiency. Herein, a straightforward and versatile synthesis of a cationic guanidine-decorated dendronized pullulan (OGG3P) for efficient genetic photodynamic therapy was proposed. OGG3P was able to block the mobility of DNA from a weight ratio of 2. However, G3P lacking guanidine residues could not block DNA migration until at a weight ratio of 15, revealing guanidination could facilitate DNA condensation via specific guanidinium-phosphate interactions. A zeta potential plateau (∼+23 mV) of OGG3P complexes indicated the nonionic hydrophilic hydroxyl groups in pullulan might neutralize the excessive detrimental cationic charges. There was no obvious cytotoxicity and hemolysis, but also enhancement of transfection efficiency with regard to OGG3P in comparison with that of native G3P in Hela and HEK293T cells. More importantly, we found that the uptake efficiency in Hela cells between OGG3P and G3P complexes was not markedly different. However, guanidination caused changes in uptake pathway and led to macropinocytosis pathway, which may be a crucial reason for improved transfection efficiency. After introducing a therapeutic pKillerRed-mem plasmid, OGG3P complexes achieved significantly enhanced KillerRed protein expression and ROS production under irradiation. ROS-induced cancer cells proliferation suppression was also confirmed. This study highlights the guanidine-decorated dendronized pullulan could emerge as a reliable nonviral gene carrier to specifically deliver therapeutic genes.

Ignatzschineria cameli sp. nov., isolated from necrotic foot tissue of dromedaries (Camelus dromedarius) and associated maggots (Wohlfahrtia species) in Dubai.

Five bacterial strains, UAE-HKU57T, UAE-HKU58, UAE-HKU59, UAE-HKU60 and UAE-HKU61, were isolated in Dubai, UAE, from necrotic foot tissue samples of four dromedaries (Camelus dromedarius) and associated maggots (Wohrlfartia species). They were non-sporulating, Gram-negative, non-motile bacilli. They grew well under aerobic conditions at 37 °C, but not anaerobically. The pH range for growth was pH 7.0-9.0 (optimum, pH 7.5-8.0) and the strains could tolerate NaCl concentrations (w/v) up to 2 % (optimum, 0.5 %). They were catalase- and cytochrome oxidase-positive, but caseinase-, gelatinase- and urease-negative. Their phenotypic characters were distinguishable from other closely related species. Phylogenetic analyses of the almost-complete 16S rRNA gene and partial 23S rRNA gene, gyrB, groEL and recA sequences revealed that the five isolates were most closely related to undescribed Ignatzschineria strain F8392 and Ignatzschineria indica, but in most phylogenies clustered separately from these close relatives. Average nucleotide identity analysis showed that genomes of the five isolates (2.47-2.52 Mb, G+C content 41.71-41.86 mol%) were 98.00-99.97% similar to each other, but ≤87.18 % similar to other Ignatzschineriaspecies/strains. Low DNA relatedness between the five isolates to other Ignatzschineriaspecies/strains was also supported by Genome-to-Genome Distance Calculator analysis. The chemotaxonomic traits of the five strains were highly similar. They were non-susceptible (intermediate or resistant) to tetracycline and resistant to trimethoprim/sulphamethoxazole. The name Ignatzschineria cameli sp. nov. is proposed to accommodate these five strains, with strain UAE-HKU57T (=CCOS1165T=NBRC 113042T) as the type strain.

Serodiagnosis of aspergillosis in falcons (Falco spp.) by an Afmp1p-based enzyme-linked immunosorbent assay.

Aspergillosis in falcons may be associated with high mortality and difficulties in clinical and laboratory diagnosis. We previously cloned an immunogenic protein, Afmp1p, in Aspergillus fumigatus and showed that anti-Afmp1p antibodies were present in human patients with A. fumigatus infections. In this study, we hypothesise that a similar Afmp1p-based enzyme-linked immunosorbent assay (ELISA) could be applied to serodiagnose falcon aspergillosis. A specific polyclonal antibody was first generated to detect falcon serum IgY. Horseradish peroxidase-conjugate of this antibody was then used to measure anti-Afmp1p antibodies in sera collected from falcons experimentally infected with A. fumigatus, and the performance of the Afmp1p-based ELISA was evaluated using sera from healthy falcons and falcons with documented A. fumigatus infections. All four experimentally infected falcons developed culture- and histology-proven invasive aspergillosis. Anti-Afmp1p antibodies were detected in their sera. For the Afmp1p-based ELISA, the mean ± SD OD450 nm using sera from 129 healthy falcons was 0.186 ± 0.073. Receiver operating characteristics curve analysis showed an absorbance cut-off value of 0.407. One negative serum gave an absorbance outside the normal range, giving a specificity of 99.2%. For the 12 sera from falcons with confirmed aspergillosis, nine gave absorbance values ≥ cut-off, giving a sensitivity of 75%. The Afmp1p-based ELISA is useful for serodiagnosis of falcons with aspergillosis.

Lactobacillus paracasei modulates LPS-induced inflammatory cytokine release by monocyte-macrophages via the up-regulation of negative regulators of NF-kappaB signaling in a TLR2-dependent manner.

The application of the probiotic lactobacillus is suggested in the treatment of some inflammatory diseases of intestines due to its potential ability to attenuate inflammation. However, the mechanism is not completely understood. In PBMCs, Lactobacillus paracasei (L. Paracasei) down-regulated the LPS-induced production of TNF-α and IL-6. Using a macrophage-like differentiated THP-1 cell line induced by PMA, we investigated the effect of L. paracasei on the production of pro-inflammatory cytokines by monocyte-macrophages. Treatment of the differentiated THP-1 cells with L. paracasei either concurrently with or before LPS challenge attenuated the LPS-induced secretion of TNF-α and IL-1ß. This effect was due to a decrease in IκB phosphorylation and NF-κB nuclear translocation. Furthermore, treatment of the differentiated THP-1 cells with L. paracasei induced the expression of negative regulators of the NF-κB signaling pathway, including the deubiquitinating enzyme A20, suppressor of cytokine signaling (SOCS) 1, SOCS3, and IL-1 receptor-associated kinase (IRAK) 3. Pretreatment with an IRAK4 inhibitor suppressed the L. paracasei-induced expression of these negative regulators and further increased the LPS-mediated expressions of TNF-α and IL-1ß. Moreover, treatment with an antibody against Toll-like receptor (TLR) 2 reversed the effect of L. paracasei on inducing negative regulators and inhibiting TNF-α and IL-1ß productions. Our findings suggest that L. paracasei inhibits the production of pro-inflammatory cytokines by monocyte-macrophages via the induction of negative regulators of the NF-κB signaling pathway in a TLR2-IRAK4-dependent manner.

Toward a Designable Extracellular Matrix: Molecular Dynamics Simulations of an Engineered Laminin-Mimetic, Elastin-Like Fusion Protein.

Native extracellular matrices (ECMs) exhibit networks of molecular interactions between specific matrix proteins and other tissue components. Guided by these naturally self-assembling supramolecular systems, we have designed a matrix-derived protein chimera that contains a laminin globular-like (LG) domain fused to an elastin-like polypeptide (ELP). This bipartite design offers a flexible protein engineering platform: (i) laminin is a key multifunctional component of the ECM in human brains and other neural tissues, making it an ideal bioactive component of our fusion, and (ii) ELPs, known to be well-tolerated in vivo, provide a self-assembly scaffold with tunable physicochemical (viscoelastic, thermoresponsive) properties. Experimental characterization of novel proteins is resource-intensive, and examining many conceivable designs would be a formidable challenge in the laboratory. Computational approaches offer a way forward: molecular dynamics (MD) simulations can be used to analyze the structural/physical behavior of candidate LG-ELP fusion proteins, particularly in terms of conformational properties salient to our design goals, such as assembly propensity in a temperature range spanning the inverse temperature transition. As a first step in examining the physical characteristics of a model LG-ELP fusion protein, including its temperature-dependent structural behavior, we simulated the protein over a range of physiologically relevant temperatures (290-320 K). We find that the ELP region, built upon the archetypal (VPGXG)5 scaffold, is quite flexible and has a propensity for ß-rich secondary structures near physiological (310-315 K) temperatures. Our trajectories indicate that the temperature-dependent burial of hydrophobic patches in the ELP region, coupled to the local water structure dynamics and mediated by intramolecular contacts between aliphatic side chains, correlates with the temperature-dependent structural transitions in known ELP polymers. Because of the link between compaction of ELP segments into ß-rich structures and differential solvation properties of this region, we posit that future variation of ELP sequence and composition can be used to systematically alter the phase transition profiles and, thus, the general functionality of our LG-ELP fusion protein system.

Adaptation of HIV-1 to human leukocyte antigen class I.

The rapid and extensive spread of the human immunodeficiency virus (HIV) epidemic provides a rare opportunity to witness host-pathogen co-evolution involving humans. A focal point is the interaction between genes encoding human leukocyte antigen (HLA) and those encoding HIV proteins. HLA molecules present fragments (epitopes) of HIV proteins on the surface of infected cells to enable immune recognition and killing by CD8(+) T cells; particular HLA molecules, such as HLA-B*57, HLA-B*27 and HLA-B*51, are more likely to mediate successful control of HIV infection. Mutation within these epitopes can allow viral escape from CD8(+) T-cell recognition. Here we analysed viral sequences and HLA alleles from >2,800 subjects, drawn from 9 distinct study cohorts spanning 5 continents. Initial analysis of the HLA-B*51-restricted epitope, TAFTIPSI (reverse transcriptase residues 128-135), showed a strong correlation between the frequency of the escape mutation I135X and HLA-B*51 prevalence in the 9 study cohorts (P = 0.0001). Extending these analyses to incorporate other well-defined CD8(+) T-cell epitopes, including those restricted by HLA-B*57 and HLA-B*27, showed that the frequency of these epitope variants (n = 14) was consistently correlated with the prevalence of the restricting HLA allele in the different cohorts (together, P < 0.0001), demonstrating strong evidence of HIV adaptation to HLA at a population level. This process of viral adaptation may dismantle the well-established HLA associations with control of HIV infection that are linked to the availability of key epitopes, and highlights the challenge for a vaccine to keep pace with the changing immunological landscape presented by HIV.

Enantioseparation of Racemic Flurbiprofen by Aqueous Two-Phase Extraction With Binary Chiral Selectors of L-dioctyl Tartrate and L-tryptophan.

A novel method for chiral separation of flurbiprofen enantiomers was developed using aqueous two-phase extraction (ATPE) coupled with biphasic recognition chiral extraction (BRCE). An aqueous two-phase system (ATPS) was used as an extracting solvent which was composed of ethanol (35.0% w/w) and ammonium sulfate (18.0% w/w). The chiral selectors in ATPS for BRCE consideration were L-dioctyl tartrate and L-tryptophan, which were screened from amino acids, ß-cyclodextrin derivatives, and L-tartrate esters. Factors such as the amounts of L-dioctyl tartrate and L-tryptophan, pH, flurbiprofen concentration, and the operation temperature were investigated in terms of chiral separation of flurbiprofen enantiomers. The optimum conditions were as follows: L-dioctyl tartrate, 80 mg; L-tryptophan, 40 mg; pH, 4.0; flurbiprofen concentration, 0.10 mmol/L; and temperature, 25 °C. The maximum separation factor α for flurbiprofen enantiomers could reach 2.34. The mechanism of chiral separation of flurbiprofen enantiomers is discussed and studied. The results showed that synergistic extraction has been established by L-dioctyl tartrate and L-tryptophan, which enantioselectively recognized R- and S-enantiomers in top and bottom phases, respectively. Compared to conventional liquid-liquid extraction, ATPE coupled with BRCE possessed higher separation efficiency and enantioselectivity without the use of any other organic solvents. The proposed method is a potential and powerful alternative to conventional extraction for separation of various enantiomers.

Self-assembly of pH-sensitive fluorinated peptide dendron functionalized dextran nanoparticles for on-demand intracellular drug delivery.

In this study, the amphiphilic fluorinated peptide dendrons functionalized dextran (FPD-HZN-Dex) via an acid-sensitive hydrazone linkage was successfully designed and prepared for the first time. We demonstrated a spontaneous self-assembly of amphiphilic FPD-HZN-Dex into the well-defined nanoparticles with the core-shell architecture in aqueous media, which is attributed to the efficient amphiphilic functionalization of dextran by the hydrophobic fluorinated peptide dendrons. The spherical morphology, uniform particle size and good storage stability of the prepared FPD-HZN-Dex nanoparticles were characterized by dynamic light scattering and transmission electron microscopy, respectively. In vitro drug release studies showed a controlled and pH dependent hydrophobic drug release profile. The cell viability assays show excellent biocompatibility of the FPD-HZN-Dex nanoparticles for both normal cells and tumor cells. Moreover, the FPD-HZN-Dex self-assembled systems based on pH-sensitive hydrazone linkage also can serve as stimulus bioresponsive carriers for on-demand intracellular drug delivery. These self-assembled nanoparticles exhibit a stimulus-induced response to endo/lysosome pH (pH 5.0) that causes their disassembly over time, enabling controlled release of encapsulated DOX. This work has unveiled a unique non-covalent interaction useful for engineering amphiphilic dendrons or dendrimers self-assembled systems.

The Interaction Mechanism of Intramuscular Gene Delivery Materials with Cell Membranes.

It has been confirmed that skeletal muscle cells have the capability to receive foreign plasmid DNA (pDNA) and express functional proteins. This provides a promisingly applicable strategy for safe, convenient, and economical gene therapy. However, intramuscular pDNA delivery efficiency was not high enough for most therapeutic purposes. Some non-viral biomaterials, especially several amphiphilic triblock copolymers, have been shown to significantly improve intramuscular gene delivery efficiency, but the detailed process and mechanism are still not well understood. In this study, the molecular dynamics simulation method was applied to investigate the structure and energy changes of the material molecules, the cell membrane, and the DNA molecules at the atomic and molecular levels. From the results, the interaction process and mechanism of the material molecules with the cell membrane were revealed, and more importantly, the simulation results almost completely matched the previous experimental results. This study may help us design and optimize better intramuscular gene delivery materials for clinical applications.

Understanding how transmembrane domains regulate interactions between human BST-2 and the SARS-CoV-2 accessory protein ORF7a.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID, replicates at intracellular membranes. Bone marrow stromal antigen 2 (BST-2; tetherin) is an antiviral response protein that inhibits transport of viral particles after budding within infected cells. RNA viruses such as SARS-CoV-2 use various strategies to disable BST-2, including use of transmembrane 'accessory' proteins that interfere with BST-2 oligomerization. ORF7a is a small, transmembrane protein present in SARS-CoV-2 shown previously to alter BST-2 glycosylation and function. In this study, we investigated the structural basis for BST-2 ORF7a interactions, with a particular focus on transmembrane and juxtamembrane interactions. Our results indicate that transmembrane domains play an important role in BST-2 ORF7a interactions and mutations to the transmembrane domain of BST-2 can alter these interactions, particularly single-nucleotide polymorphisms in BST-2 that result in mutations such as I28S. Using molecular dynamics simulations, we identified specific interfaces and interactions between BST-2 and ORF7a to develop a structural basis for the transmembrane interactions. Differences in glycosylation are observed for BST-2 transmembrane mutants interacting with ORF7a, consistent with the idea that transmembrane domains play a key role in their heterooligomerization. Overall, our results indicate that ORF7a transmembrane domain interactions play a key role along with extracellular and juxtamembrane domains in modulating BST-2 function.