Alternative substitutions of N332 in HIV-1AD8 gp120 differentially affect envelope glycoprotein function and viral sensitivity to broadly neutralizing antibodies targeting the V3-glycan.

The envelope glycoprotein (Env) trimer on the surface of human immunodeficiency virus type I (HIV-1) mediates viral entry into host CD4+ T cells and is the sole target of neutralizing antibodies. Broadly neutralizing antibodies (bnAbs) that target gp120 V3-glycan of HIV-1 Env trimer are potent and block the entry of diverse HIV-1 strains. Most V3-glycan bnAbs interact, to a different extent, with a glycan attached to N332, but Asn at this position is not absolutely conserved or required for HIV-1 entry based on the prevalence of N332 in different circulating HIV-1 strains from diverse clades. Here, we studied the effects of amino acid changes at position 332 of HIV-1AD8 Envs on HIV-1 sensitivity to antibodies, cold exposure, and soluble CD4. We further investigated how these changes affect Env function and HIV-1 infectivity in vitro. Our results suggest robust tolerability of HIV-1AD8 Env N332 to changes, with specific changes that resulted in extended exposure of gp120 V3 loop, which is typically concealed in most primary HIV-1 isolates. Viral evolution leading to Asn at position 332 of HIVAD8 Envs is supported by the selection advantage of high levels of cell-cell fusion, transmission, and infectivity with high levels of cell surface expression and slightly higher gp120 shedding than most N332 variants. Thus, tolerance of HIV-1AD8 Envs to different amino acids at position 332 provides increased flexibility to respond to changing conditions/environments and evade the immune system. Modeling studies of the distance between N332 glycan and specific bnAbs were in agreement with N332 glycan dependency on bnAb neutralization. Overall, our studies provide insights into the contribution of specific amino acids at position 332 to Env antigenicity, stability on ice, and conformational states. IMPORTANCE: Glycan attached to amino acid asparagine at position 332 of HIV-1 envelope glycoproteins is a main target of a subset of broadly neutralizing antibodies that block HIV-1 infection. Here, we defined the contribution of different amino acids at this position to Env antigenicity, stability on ice, and conformational states.

Alternative substitutions of N332 in HIV-1AD8 gp120 differentially affect envelope glycoprotein function and viral sensitivity to broadly neutralizing antibodies targeting the V3-glycan.

The envelope glycoprotein (Env) trimer on the surface of human immunodeficiency virus type I (HIV-1) mediates viral entry into host CD4+ T cells and is the sole target of neutralizing antibodies. Broadly neutralizing antibodies (bnAbs) that target gp120 V3-glycan of HIV-1 Env trimer are potent and block the entry of diverse HIV-1 strains. Most V3-glycan bnAbs interact, to a different extent, with a glycan attached to N332 but Asn at this position is not absolutely conserved or required for HIV-1 entry based on prevalence of N332 in different circulating HIV-1 strains from diverse clades. Here, we studied the effects of amino acid changes at position 332 of HIV-1AD8 Envs on HIV-1 sensitivity to antibodies, cold exposure, and soluble CD4. We further investigated how these changes affect Env function and HIV-1 infectivity in vitro. Our results suggest robust tolerability of HIV-1AD8 Env N332 to changes with specific changes that resulted in extended exposure of gp120 V3 loop, which is typically concealed in most primary HIV-1 isolates. Viral evolution leading to Asn at position 332 of HIVAD8 Envs is supported by the selection advantage of high levels of cell-cell fusion, transmission, and infectivity even though cell surface expression levels are lower than most N332 variants. Thus, tolerance of HIV-1AD8 Envs to different amino acids at position 332 provides increased flexibility to respond to changing conditions/environments and to evade the immune system. Modeling studies of the distance between N332 glycan and specific bnAbs was in agreement with N332 glycan dependency on bnAb neutralization. Overall, our studies provide insights into the contribution of specific amino acids at position 332 to Env antigenicity, stability on ice, and conformational states.

Conformational flexibility of HIV-1 envelope glycoproteins modulates transmitted / founder sensitivity to broadly neutralizing antibodies.

HIV-1 envelope glycoproteins (Envs) mediate viral entry and are the sole target of neutralizing antibodies. Envs of most primary HIV-1 strains exist in a closed conformation and occasionally sample more open states. Thus, current knowledge guides immunogen design to mimic the closed Env conformation as the preferred target for eliciting broadly neutralizing antibodies (bnAbs) to block HIV-1 entry. Here we show that Env-preferred conformations of 6 out of 13 (46%) transmitted/founder (T/F) strains tested are incompletely closed. As a result, entry of these T/Fs into target cells is sensitive to antibodies that recognize internal epitopes exposed on open Env conformations. A cryo-electron microscopy structure of unliganded, incompletely closed T/F Envs (1059-SOSIP) at 3.6 Å resolution exhibits an asymmetric configuration of Env protomers with increased sampling of states with incompletely closed trimer apex. Double electron-electron resonance spectroscopy provided further evidence for enriched occupancy of more open Env conformations. Consistent with conformational flexibility, 1059 Envs were associated with resistance to most bnAbs that exhibit reduced potency against functional Env intermediates. To follow the fate of incompletely closed Env in patients, we reconstructed de novo the post-transmission evolutionary pathway of a second T/F Env (CH040), which is sensitive to the V3-targeting antibody 19b and highly resistant to most bnAbs. Evolved viruses exhibited increased resistance to cold, soluble CD4 and 19b, all of which correlate with closing of the adapted Env trimer. Lastly, we show a correlation between efficient neutralization of multiple Env conformations and increased antiviral breadth of CD4-binding site (CD4bs) bnAbs. In particular, N6 bnAb, which uniquely recognizes different Env conformations, efficiently neutralizes 50% of the HIV-1 strains that were resistant to VRC01 and transmitted during the first-in-humans antibody-mediated prevention trial (HVTN 704). VRC01-resistant Envs are incompletely closed based on their sensitivity to cold and on partial sensitivity to antibodies targeting internal, typically occluded, epitopes. Most VRC01-resistant Envs retain the VRC01 epitope according to VRC01 binding to their gp120 subunit at concentrations that have no significant effect on virus entry, and they exhibit cross resistance to other CD4bs bnAbs that poorly recognize functional Env intermediates. Our findings refine current knowledge of Env conformational states and provide guidance for developing new strategies for bnAb immunotherapy and Env-based immunogen design.

Low-carbohydrate diets and men's cortisol and testosterone: Systematic review and meta-analysis.

Background: Low-carbohydrate diets may have endocrine effects, although individual studies are conflicting. Therefore, a review was conducted on the effects of low- versus high-carbohydrate diets on men's testosterone and cortisol. Methods: The review was registered on PROSPERO (CRD42021255957). The inclusion criteria were: intervention study, healthy adult males, and low-carbohydrate diet: ≤35% carbohydrate. Eight databases were searched from conception to May 2021. Cochrane's risk of bias tool was used for quality assessment. Random-effects, meta-analyses using standardized mean differences and 95% confidence intervals, were performed with Review Manager. Subgroup analyses were conducted for diet duration, protein intake, and exercise duration. Results: Twenty-seven studies were included, with a total of 309 participants. Short-term (<3 weeks), low- versus high-carbohydrate diets moderately increased resting cortisol (0.41 [0.16, 0.66], p < 0.01). Whereas, long-term (≥3 weeks), low-carbohydrate diets had no consistent effect on resting cortisol. Low- versus high-carbohydrate diets resulted in much higher post-exercise cortisol, after long-duration exercise (≥20 min): 0 h (0.78 [0.47, 1.1], p < 0.01), 1 h (0.81 [0.31, 1.31], p < 0.01), and 2 h (0.82 [0.33, 1.3], p < 0.01). Moderate-protein (<35%), low-carbohydrate diets had no consistent effect on resting total testosterone, however high-protein (≥35%), low-carbohydrate diets greatly decreased resting (-1.08 [-1.67, -0.48], p < 0.01) and post-exercise total testosterone (-1.01 [-2, -0.01] p = 0.05). Conclusions: Resting and post-exercise cortisol increase during the first 3 weeks of a low-carbohydrate diet. Afterwards, resting cortisol appears to return to baseline, whilst post-exercise cortisol remains elevated. High-protein diets cause a large decrease in resting total testosterone (∼5.23 nmol/L).

Smokers Increasingly Motivated and Able to Quit as Smoking Prevalence Falls: Umbrella and Systematic Review of Evidence Relevant to the "Hardening Hypothesis," Considering Transcendence of Manufactured Doubt.

INTRODUCTION: The "hardening hypothesis" proposes that as the prevalence of smoking in a population declines, there will be a "hardening" of the remaining smoker population. This review examines the evidence regarding smokers' motivation, dependence, and quitting behavior as smoking prevalence declines, to assess whether population "hardening" (decreasing propensity to quit) or "softening" (the converse) is occurring. METHODS: MEDLINE, PsychINFO, Scopus, Web of Science, and Cochrane Library were searched to July 2019, using terms related to smoking and hardening, for reviews and large, population-based repeat cross-sectional studies. There were additional searches of reference lists and citations of key research articles. Two reviewers screened half the titles and abstracts each, and two reviewers screened full texts independently using tested criteria. Four reviewers independently and systematically extracted data from eligible publications, with one reviewer per study, checked by another reviewer. RESULTS: Of 265 titles identified, three reviews and ten repeat cross-sectional studies were included. Reviews concluded that hardening has not occurred among the general smoking population over time. Among repeated cross-sectional studies, five examined motivation, nine examined dependence, five examined hardcore smoking, and two examined quit outcomes. All but one study found a lack of hardening. Most found softening within the smoking population, consistent across hardening indicators, definitions, countries (and tobacco control environments), and time periods examined. CONCLUSIONS: Tobacco control reduces smoking prevalence and fosters a smoking population more amenable to evidence-based interventions. Based on the weight of the available evidence, the "hardening hypothesis" should be rejected and the reality of softening accepted. IMPLICATIONS: This umbrella review and systematic review provides a critical consideration of evidence from epidemiology and psychology and other fields regarding the "hardening hypothesis"-a persistent myth undermining tobacco control. It reaches the conclusion that the sum-total of the worldwide evidence indicates either "softening" of the smoking population, or a lack of hardening. Hence, tobacco control reduces smoking prevalence and fosters a smoking population more amenable to evidence-based interventions. The review indicates that the time has come to take active steps to combat the myth of hardening and to replace it with the reality of "softening."

Framework for the public health assessment of electronic cigarettes.

BACKGROUND: Electronic cigarettes (e-cigarettes) are relatively new products with substantial public health impacts. Evidence on their effects is diverse and emerging rapidly, presenting challenges to high-quality policymaking and decision-making. This paper addresses these challenges by developing and presenting a framework for the public health assessment of e-cigarettes, using the Australian context as an example. METHODS: Framework development involved stakeholder engagement, development of guiding principles, and consideration of existing relevant frameworks and the evidence requirements of current policy options, identified in published and grey literature. RESULTS: Guiding principles include the need for the framework to: be evidence based; include consideration of the likely balance of benefits and risks of e-cigarettes, uncertainty and safety; support equity; support the ongoing application of evidence to high-quality policy and practice; and consider potential competing interests. The framework draws upon: health technology assessment; health impact assessment; environmental health risk assessment; healthcare recommendations evidence evaluation; consumer goods regulation; medicine and chemical scheduling; tobacco product evaluation; previous reviews and the precautionary principle. Final framework components are: (1) characterisation of products under consideration; (2) definition of populations of interest; (3) characterisation of tobacco smoking, control and impacts on health and well-being; (4) review of evidence on patterns of e-cigarette use; (5) review of evidence on e-cigarette use and health outcomes; (6) assessment of likely risks, benefits and safety; (7) identification and assessment of policy options to optimise health outcomes. CONCLUSIONS: Structured and ongoing public health assessment of e-cigarette use is likely to support health through enhancing evidence-based decision-making.

The ketogenic diet: its impact on human gut microbiota and potential consequent health outcomes: a systematic literature review.

Aim: This systematic review examined the diet's impact on the human gut microbiota to identify potential consequent health outcomes. Background: The extreme macronutrient profile of the ketogenic diet (KD) instigates compositional shifts in the gut's microbial community. Methods: In this systematic literature review, an evidence-based and methodical approach was undertaken, which involved systematic searches of the Medical Literature Analysis and Retrieval System Online (MEDLINE), PubMed and Cumulative Index to Nursing and Allied Health Literature (CINAHL) databases, generating a total of 263 relevant research papers. Following the application of inclusion and exclusion criteria, eight papers were deemed suitable for inclusion. These papers were critically appraised using a checklist tool adapted from the National Institute of Care and Excellence (NICE). The findings were analysed using a simplified thematic analysis. Results: The results provide strong evidence for a persistent reduction in Bifidobacterium abundance following KD adherence. A reduced abundance of key Firmicutes butyrate-producing bacteria was found to be a likely impact, although two studies with extended intervention periods indicate this may be time-limited. Studies investigating short-chain fatty acids (SCFA's) indicate KD reduces total faecal SCFA's, acetate, and butyrate. Conclusion: Changes to microbial communities resulting from KD adherence are potentially detrimental to colonic health. The persistent reduction in Bifidobacterium abundance was concerning, with obesity, type-2 diabetes, and depression highlighted as potential consequent risks. For nutrition and healthcare professionals, the findings emphasize the importance of considering KDs microbial effects and resulting health implications at an individual level.

Intra- and extra-cellular environments contribute to the fate of HIV-1 infection.

HIV-1 entry into host cells leads to one of the following three alternative fates: (1) HIV-1 elimination by restriction factors, (2) establishment of HIV-1 latency, or (3) active viral replication in target cells. Here, we report the development of an improved system for monitoring HIV-1 fate at single-cell and population levels and show the diverse applications of this system to study specific aspects of HIV-1 fate in different cell types and under different environments. An analysis of the transcriptome of infected, primary CD4+ T cells that support alternative fates of HIV-1 identifies differential gene expression signatures in these cells. Small molecules are able to selectively target cells that support viral replication with no significant effect on viral latency. In addition, HIV-1 fate varies in different tissues following infection of humanized mice in vivo. Altogether, these studies indicate that intra- and extra-cellular environments contribute to the fate of HIV-1 infection.

Nutrition, the digestive system and immunity in COVID-19 infection.

The current review aimed to synthesize the literature on the complex relationship between food consumption and nutritional status as well as the digestive system in order to examine the relationship between immunity and potential responses to COVID-19 infection. The goal is to help inform the many healthcare professionals working with COVID-19 patients. A literature search was performed on PubMed, Scopus, and EMBASE databases. Hand searches were also undertaken using Google and reference lists to identify recent evidence. Studies were critically appraised, and the findings were analyzed by narrative synthesis. Nutritional status can impact immunity in several ways, including affecting susceptibility to infection, severity of disease, and recovery time, and is therefore a significant consideration in the management of COVID-19. COVID-19 can also impact digestive function, which can further impact nutritional status. The role of Vitamin D deficiency in vulnerability to severe respiratory infections, including COVID-19, has been recognized, and it may have a role in treatment where deficiency is indicated. Healthcare professionals should be aware that obesity may be accompanied by micronutrient malnutrition including vitamin D deficiency and alterations in the microbiome and inflammatory responses, which can further impact immunity and disease severity. Multidisciplinary team-work is recommended in the management of patients with COVID-19, and approaches should include a consideration of nutritional status (both macronutrients and micronutrients), body weight, and gastrointestinal signs and symptom.

Slow Receptor Binding of the Noncytopathic HIV-2UC1 Envs Is Balanced by Long-Lived Activation State and Efficient Fusion Activity.

Many HIV strains downregulate the levels of CD4 receptor on the surface of infected cells to prevent superinfection. In contrast, the rare HIV-2UC1 strain is noncytopathic and has no effect on CD4 expression in infected cells but still replicates as efficiently as more cytopathic strains in peripheral blood mononuclear cells (PBMCs). Here, we show that HIV-2UC1 Env interactions with the CD4 receptor exhibit slow association kinetics, whereas the dissociation kinetics is within the range of cytopathic strains. Despite the resulting 10- to 100-fold decrease in binding affinity, HIV-2UC1 Envs exhibit long-lived activation state and efficient fusion activity. These observations suggest that HIV-2UC1 Envs evolved to balance low affinity with an improved and readily triggerable molecular machinery to mediate entry. Resistance to cold exposure, similar to many primary HIV-1 isolates, and to sCD4 neutralization suggests that HIV-2UC1 Envs preferentially sample a closed Env conformation. Our data provide insights into the mechanism of HIV entry.

Muddying the waters: A political ecology of mosquito-borne disease in coastal Ecuador.

The mosquito-borne arboviral diseases dengue, chikungunya, and Zika are major public health burdens in Latin America. To analyze the socio-environmental dynamics of these diseases, we apply a political ecology of health and disease framework that is attentive to local etiological frameworks, structural sociopolitical conditions, processes of identity construction, and the contested, politicized nature of public health work. We use multiple qualitative methods to analyze perceptions and interactions with the local environment in relation to mosquito-borne disease across three small communities in Manabí Province, Ecuador. We find that participants' perceptions and practices are complex and multilayered: subjects possess a mixed theory of causation, where these diseases are caused not only by mosquitoes, but also by people's interactions with a changing environment; most environmental management to control vector mosquitoes is carried out informally by women as part of domestic routines; and contrary to public health messaging that stresses the importance of individual agency, participants prefer some of the most invasive techniques for mosquito control (i.e. fumigation with insecticides). However, individual agency in disease control is constrained by poor water infrastructure and lack of public health coordination. Our approach advocates for recognition of local knowledges and sociopolitical constraints in the development of public health messages and interventions.

Ready, Cell, Go! Guided Movement Exercises in an Introductory Cell Biology Course.

Cells and dance are each dynamic manifestations of energy, shape, time, and space. Here we present a novel application of movement learning in cell biology education. "Ready, Cell, Go!" is a set of movement exercises for introductory cell biology students designed to teach concepts of fluidity, crowding, and chaos. These aspects of cells are difficult to glean from two-dimensional illustrations in textbooks or animations where necessary simplification abstracts processes from their full cellular context. Forty-four undergraduate biology students were guided to move using three sets of cues in a dance studio setting where each exercise aimed to experientially highlight and deepen understanding of a different aspect of cellular structure and function. Students described their experiences and personal learning outcomes in written reflections. The movement-based exercises we describe provided a means of discovery, inquiry, and interest for introductory cell biology students and serve as a template to teach other central concepts in cell biology.