Halal cultivated meat: an untapped opportunity.

The global Halal food market is forecast to reach US$1.67 trillion by 2025, growing to meet the dietary demands of a rapidly increasing Muslim population, set to comprise 30% of the global population by mid-century. Meat consumption levels are increasing in many Muslim countries, with important implications for health and environmental sustainability. Alt protein products are currently being manufactured and positioned as one possible solution to reduce the environmental impact of meat consumption, yet, little is currently known about the Halal status of these products, nor the extent to which they appeal to Muslim consumers in emerging markets in Asia and Africa. Here, we explore key considerations regarding the acceptability of alt protein products for Muslim consumers, explore Halal certification requirements in the context of cultivated meat, and examine some unique beliefs within the Islamic faith that may support, as well as impede, widespread adoption of alt protein among the 2.8 billion Muslims of the future.

Global Biological Threats: Novel Tools and Multi-Disciplinary Approaches to Sustainable Development.

The Covid-19 pandemic has raised awareness of future biological threats, how we can prepare and develop mitigation strategies. Technology has allowed us to quickly identify the pathogen, map its evolution in real time and develop scores of vaccines within months. This review looks at disease threats from a perspective of human development, and the futuristic technologies that may help in the fight. Most importantly, cooperation across political and ideological boundaries would be needed in a highly inter-connected world. A new disease emerging anywhere is a threat everywhere.

Update: proposed reference sequences for subtypes of hepatitis E virus (species Orthohepevirus A).

In this recommendation, we update our 2016 table of reference sequences of subtypes of hepatitis E virus (HEV; species Orthohepevirus A, family Hepeviridae) for which complete genome sequences are available (Smith et al., 2016). This takes into account subsequent publications describing novel viruses and additional proposals for subtype names; there are now eight genotypes and 36 subtypes. Although it remains difficult to define strict criteria for distinguishing between virus subtypes, and is not within the remit of the International Committee on Taxonomy of Viruses (ICTV), the use of agreed reference sequences will bring clarity and stability to researchers, epidemiologists and clinicians working with HEV.

India Research Management Initiative (IRMI) - an initiative for building research capacity in India.

Research and innovation are growing in India with significant investments being made towards institutions, researchers and research infrastructure. Although still under 1% of GDP, funding for science and technology in India has increased each year for over two decades. There is also increasing realization that public funding for research should be supplemented with that from industry and philanthropy. Like their counterparts worldwide, Indian researchers require access to professional research management support at their institutions to fully leverage emerging scientific opportunities and collaborations. However, there are currently significant  gaps in the research management support available to these researchers and this has implications for research in India. The India Research Management Initiative (IRMI) was launched by the Wellcome Trust/DBT (Department of Biotechnology, Government of India) India Alliance (hereafter India Alliance) in February 2018 to narrow these gaps. A 12-month pilot phase has enabled conversations across multiple stakeholders. In this Open Letter, we share some insights from the IRMI pilot phase, which could aid systemic development and scaling up of research management as a professional support service across India. We anticipate these will stimulate dialogue and guide future policy and interventions towards building robust research and innovation ecosystems in India.

Hepatitis E virus in bottlenose dolphins Tursiops truncatus.

Hepatitis E virus (HEV) infects several animal species that act as zoonotic reservoirs for viral transmission. Solid and liquid residues from infected animals could lead to HEV contamination of food and surface waters. Evidence of human HEV infection through ingestion of seafood (shellfish, mussels) has been reported. Dolphins generally feed on fish and squid but are able to adapt to an environment and consume whatever prey is available. Clinical signs of infected dolphins include lethargy, inappetence, behavioral aberrations and increased serum alanine aminotransferase (ALT). The dolphins examined in this study were maintained at the National Aquarium, Havana, Cuba. A total of 31 dolphins were evaluated for HEV markers. Sera were collected and screened for total immunoglobin (Ig) anti-HEV. Sera and liver homogenate were tested for HEV RNA by nested RT-PCR using primers targeting the open reading frame 1. Phylogenetic analysis was performed using partial nucleotide sequences at the amplified RNA-dependent RNA polymerase gene. Total anti-HEV Ig was detected in 32.2% (10 of 31), and 16.1% (5 of 31) of these dolphins were positive by both serology and HEV RNA testing. Nucleotide sequence analyses revealed that HEV strains identified in dolphins were genotype 3. This virus may represent an environmental contamination of food or wastewater as a source of HEV exposure and infection. Our findings provide evidence that HEV is associated with liver disorders in cetaceans and that it is advisable to screen for exposure of this virus in captive dolphins, particularly animals with elevated serum ALT or compromised liver function test results of undetermined cause.

Proposed reference sequences for hepatitis E virus subtypes.

The nomenclature of hepatitis E virus (HEV) subtypes is inconsistent and makes comparison of different studies problematic. We have provided a table of proposed complete genome reference sequences for each subtype. The criteria for subtype assignment vary between different genotypes and methodologies, and so a conservative pragmatic approach has been favoured. Updates to this table will be posted on the International Committee on Taxonomy of Viruses website (http://talk.ictvonline.org/r.ashx?C). The use of common reference sequences will facilitate communication between researchers and help clarify the epidemiology of this important human pathogen. This subtyping procedure might be adopted for other taxa of the genus Orthohepevirus.

Changes in gene expression in liver tissue from patients with fulminant hepatitis E.

AIM: To study host gene expression and number of immune cells in liver tissues from patients with fulminant hepatitis E (FH-E). METHODS: Microarray-based expression profiling was done using Illumina Human WG-6_v3_BeadChip arrays on post-mortem liver tissue from 5 patients with FH-E, and compared with similar tissue from 6 patients with fulminant hepatitis B (FH-B; disease controls) and normal liver tissue from 6 persons. Differential expression was defined as ≥ 2.0-fold change with Benjamini-Hochberg false discovery rate below 0.05 using t-test in liver tissue from FH-B and FH-E, than healthy liver tissue. For some genes that showed differential expression in FH-E, microarray data were validated using quantitative reverse transcription PCR. Differentially expressed gene lists were then subjected to "Gene Ontology" analysis for biological processes, and pathway analysis using BioCarta database on the DAVID server. In addition, tissue sections were stained for CD4(+), CD8(+) and CD56(+) cells using indirect immunohistochemistry; cells staining positive for each of these markers were counted and compared between groups. RESULTS: Compared to normal livers, those from patients with FH-E and FH-B showed differential expression of 3377 entities (up-regulated 1703, downregulated 1674) and 2572 entities (up 1164, down 1408), respectively. This included 2142 (up 896, down 1246) entities that were common between the two sets; most of these belonged to metabolic, hemostatic and complement pathways, which are active in normal livers. Gene expression data from livers of patients with FH-E but not those of FH-B showed activation of several immune response pathways, particularly those involving cytotoxic T cells. The fold-change values of mRNA for selected genes in livers from FH-E than in normal liver tissue determined using quantitative reverse transcription PCR showed excellent concordance with microarray analysis. At immunohistochemistry, CD8(+) T cells showed an increase in liver biopsies from both FH-E [median 53.4 per arbitrary unit area (range 31.2-99.9)] and FH-B [median 49.3 (19.3-51.0); P = 0.005] compared to control liver tissue [median 6.9 (3.1-14.9)]. CONCLUSION: FH-E patients show CD8(+) T cell infiltration and increased gene expression of cytotoxic T cell pathways in liver, suggesting a possible pathogenetic role for these cells.

Mycobacterium tuberculosis and Human Immunodeficiency Virus Type 1 Cooperatively Modulate Macrophage Apoptosis via Toll Like Receptor 2 and Calcium Homeostasis.

The emergence of drug resistant strains of Mycobacterium tuberculosis (M. tuberculosis) together with reports of co-infections with the human immunodeficiency virus (HIV) has renewed interest to better understand the intricate mechanisms prevalent during co-infections. In this study we report a synergistic effect of M. tuberculosis and HIV-1, and their antigens Rv3416 and Nef, respectively, in inhibiting apoptosis of macrophages. This inhibition involves the TLR2 pathway and second messengers that play complementing and contrasting roles in regulating apoptosis. Interestingly, the route of calcium influx into cells differentially regulates apoptosis during antigenic co-stimulation. While calcium released from intracellular stores was anti-apoptotic, calcium influx from the external milieu was pro-apoptotic. Further, molecular sensors of intracellular calcium release aid in antigen mediated inhibition of apoptosis. A cross-regulation between oxidative burst and differential routing of calcium influx governed apoptosis. Interestingly, the HIV-1 Nef supported anti-apoptotic responses in macrophages whereas Vpu had no significant effect. These results point to a synergistic liaison between M. tuberculosis and HIV-1 in regulating macrophage apoptosis.

Transcriptomic Analysis of mRNAs in Human Monocytic Cells Expressing the HIV-1 Nef Protein and Their Exosomes.

The Nef protein of human immunodeficiency virus (HIV) promotes viral replication and progression to AIDS. Besides its well-studied effects on intracellular signaling, Nef also functions through its secretion in exosomes, which are nanovesicles containing proteins, microRNAs, and mRNAs and are important for intercellular communication. Nef expression enhances exosome secretion and these exosomes can enter uninfected CD4 T cells leading to apoptotic death. We have recently reported the first miRNome analysis of exosomes secreted from Nef-expressing U937monocytic cells. Here we show genome-wide transcriptome analysis of Nef-expressing U937 cells and their exosomes. We identified four key mRNAs preferentially retained in Nef-expressing cells; these code for MECP2, HMOX1, AARSD1, and ATF2 and are important for chromatin modification and gene expression. Interestingly, their target miRNAs are exported out in exosomes. We also identified three key mRNAs selectively secreted in exosomes from Nef-expressing U937 cells and their corresponding miRNAs being preferentially retained in cells. These are AATK, SLC27A1, and CDKAL and are important in apoptosis and fatty acid transport. Thus, our study identifies selectively expressed mRNAs in Nef-expressing U937 cells and their exosomes and supports a new mode on intercellular regulation by the HIV-1 Nef protein.

The lncRNA NRON modulates HIV-1 replication in a NFAT-dependent manner and is differentially regulated by early and late viral proteins.

A majority of the human genome is transcribed into noncoding RNAs, of which the functions of long noncoding RNAs (lncRNAs) are poorly understood. Many host proteins and RNAs have been characterized for their roles in HIV/AIDS pathogenesis, but there is only one lncRNA, NEAT1, which is shown to affect the HIV-1 life cycle. We profiled 90 disease-related lncRNAs and found NRON (noncoding repressor of Nuclear Factor of Activated T cells [NFAT]) to be one of several lncRNAs whose expression was significantly altered following HIV-1 infection. The regulation of NRON expression during the HIV-1 life cycle was complex; its levels were reduced by the early viral accessory protein Nef and increased by the late protein Vpu. Consequently, Nef and Vpu also modulated activity of the transcription factor NFAT. The knockdown of NRON enhanced HIV-1 replication through increased activity of NFAT and the viral LTR. Using siRNA-mediated NFAT knockdown, we show the effects of NRON on HIV-1 replication to be mediated by NFAT, and the viral Nef and Vpu proteins to modulate NFAT activity through their effects on NRON. These findings add the lncRNA, NRON to the vast repertoire of host factors utilized by HIV for infection and persistence.

Hepatitis E virus enters liver cells through a dynamin-2, clathrin and membrane cholesterol-dependent pathway.

The hepatitis E virus (HEV) causes large outbreaks and sporadic cases of acute viral hepatitis in developing countries. In the developed world, HEV occurrence has increased as a result of zoonotic transmission from swine. The cellular aspects of HEV infection, especially the determinants of entry, are poorly understood. In the absence of a robust in vitro culture system for HEV, it is not possible to produce high titre infectious virus that can be labeled for tracking its internalization. We have therefore used an Escherichia coli expressed HEV-like particle (HEV-LP) to study HEV entry. Following internalization, the HEV-LP initially trafficks to Rab5-positive compartments en route to acidic lysosomal compartments where it is degraded. Using pharmacological inhibitors, dominant negative and constitutively active mutants, and siRNA-mediated perturbations, we show that HEV entry requires dynamin-2, clathrin, membrane cholesterol and actin, but is independent of factors associated with macropinocytosis. The HEV-LP results were further validated through infection of liver cells with virus from the stool of an infected patient. The comparative analysis also showed involvement of the phosphatidylinositol-3-kinase/Akt pathway in an early post-entry step of viral replication. This report provides a detailed description of endocytic processes associated with HEV infection.

Measuring glutathione redox potential of HIV-1-infected macrophages.

Redox signaling plays a crucial role in the pathogenesis of human immunodeficiency virus type-1 (HIV-1). The majority of HIV redox research relies on measuring redox stress using invasive technologies, which are unreliable and do not provide information about the contributions of subcellular compartments. A major technological leap emerges from the development of genetically encoded redox-sensitive green fluorescent proteins (roGFPs), which provide sensitive and compartment-specific insights into redox homeostasis. Here, we exploited a roGFP-based specific bioprobe of glutathione redox potential (E(GSH); Grx1-roGFP2) and measured subcellular changes in E(GSH) during various phases of HIV-1 infection using U1 monocytic cells (latently infected U937 cells with HIV-1). We show that although U937 and U1 cells demonstrate significantly reduced cytosolic and mitochondrial E(GSH) (approximately -310 mV), active viral replication induces substantial oxidative stress (E(GSH) more than -240 mV). Furthermore, exposure to a physiologically relevant oxidant, hydrogen peroxide (H2O2), induces significant deviations in subcellular E(GSH) between U937 and U1, which distinctly modulates susceptibility to apoptosis. Using Grx1-roGFP2, we demonstrate that a marginal increase of about ∼25 mV in E(GSH) is sufficient to switch HIV-1 from latency to reactivation, raising the possibility of purging HIV-1 by redox modulators without triggering detrimental changes in cellular physiology. Importantly, we show that bioactive lipids synthesized by clinical drug-resistant isolates of Mycobacterium tuberculosis reactivate HIV-1 through modulation of intracellular E(GSH). Finally, the expression analysis of U1 and patient peripheral blood mononuclear cells demonstrated a major recalibration of cellular redox homeostatic pathways during persistence and active replication of HIV.

The microRNA miR-29a is associated with human immunodeficiency virus latency.

BACKGROUND: Latent reservoirs of HIV-1 provide a major challenge to its cure. There are increasing reports of interplay between HIV-1 replication and host miRNAs. Several host miRNAs, which potentially target the nef-3'LTR region of HIV-1 RNA, including miR-29a, are proposed to promote latency. FINDINGS: We used two established cellular models of HIV-1 latency - the U1 monocytic and J1.1 CD4+ T cell lines to show an inverse relationship between HIV-1 replication and miR-29a levels, which was mediated by the HIV-1 Nef protein. Using a miR-29a responsive luciferase reporter plasmid, an expression plasmid and an anti-miR29a LNA, we further demonstrate increased miR-29a levels during latency and reduced levels following active HIV replication. Finally, we show that miR-29a levels in the PBMCs and plasma of HIV infected persons also correlate inversely with latency and active viral replication. CONCLUSIONS: The levels of miR-29a correlate inversely with active HIV-1 replication in cell culture models and in HIV infected persons. This links miR-29a to viral latency and suggests another approach to activate and destroy latent HIV-1 reservoirs.

Atomistic detailed mechanism and weak cation-conducting activity of HIV-1 Vpu revealed by free energy calculations.

The viral protein U (Vpu) encoded by HIV-1 has been shown to assist in the detachment of virion particles from infected cells. Vpu forms cation-specific ion channels in host cells, and has been proposed as a potential drug target. An understanding of the mechanism of ion transport through Vpu is desirable, but remains limited because of the unavailability of an experimental structure of the channel. Using a structure of the pentameric form of Vpu--modeled and validated based on available experimental data--umbrella sampling molecular dynamics simulations (cumulative simulation time of more than 0.4 µs) were employed to elucidate the energetics and the molecular mechanism of ion transport in Vpu. Free energy profiles corresponding to the permeation of Na+ and K+ were found to be similar to each other indicating lack of ion selection, consistent with previous experimental studies. The Ser23 residue is shown to enhance ion transport via two mechanisms: creating a weak binding site, and increasing the effective hydrophilic length of the channel, both of which have previously been hypothesized in experiments. A two-dimensional free energy landscape has been computed to model multiple ion permeation, based on which a mechanism for ion conduction is proposed. It is shown that only one ion can pass through the channel at a time. This, along with a stretch of hydrophobic residues in the transmembrane domain of Vpu, explains the slow kinetics of ion conduction. The results are consistent with previous conductance studies that showed Vpu to be a weakly conducting ion channel.

Consensus proposals for classification of the family Hepeviridae.

The family Hepeviridae consists of positive-stranded RNA viruses that infect a wide range of mammalian species, as well as chickens and trout. A subset of these viruses infects humans and can cause a self-limiting acute hepatitis that may become chronic in immunosuppressed individuals. Current published descriptions of the taxonomical divisions within the family Hepeviridae are contradictory in relation to the assignment of species and genotypes. Through analysis of existing sequence information, we propose a taxonomic scheme in which the family is divided into the genera Orthohepevirus (all mammalian and avian hepatitis E virus (HEV) isolates) and Piscihepevirus (cutthroat trout virus). Species within the genus Orthohepevirus are designated Orthohepevirus A (isolates from human, pig, wild boar, deer, mongoose, rabbit and camel), Orthohepevirus B (isolates from chicken), Orthohepevirus C (isolates from rat, greater bandicoot, Asian musk shrew, ferret and mink) and Orthohepevirus D (isolates from bat). Proposals are also made for the designation of genotypes within the human and rat HEVs. This hierarchical system is congruent with hepevirus phylogeny, and the three classification levels (genus, species and genotype) are consistent with, and reflect discontinuities in the ranges of pairwise distances between amino acid sequences. Adoption of this system would include the avoidance of host names in taxonomic identifiers and provide a logical framework for the assignment of novel variants.