Human iPSC Modeling Reveals Mutation-Specific Responses to Gene Therapy in a Genotypically Diverse Dominant Maculopathy.

Dominantly inherited disorders are not typically considered to be therapeutic candidates for gene augmentation. Here, we utilized induced pluripotent stem cell-derived retinal pigment epithelium (iPSC-RPE) to test the potential of gene augmentation to treat Best disease, a dominant macular dystrophy caused by over 200 missense mutations in BEST1. Gene augmentation in iPSC-RPE fully restored BEST1 calcium-activated chloride channel activity and improved rhodopsin degradation in an iPSC-RPE model of recessive bestrophinopathy as well as in two models of dominant Best disease caused by different mutations in regions encoding ion-binding domains. A third dominant Best disease iPSC-RPE model did not respond to gene augmentation, but showed normalization of BEST1 channel activity following CRISPR-Cas9 editing of the mutant allele. We then subjected all three dominant Best disease iPSC-RPE models to gene editing, which produced premature stop codons specifically within the mutant BEST1 alleles. Single-cell profiling demonstrated no adverse perturbation of retinal pigment epithelium (RPE) transcriptional programs in any model, although off-target analysis detected a silent genomic alteration in one model. These results suggest that gene augmentation is a viable first-line approach for some individuals with dominant Best disease and that non-responders are candidates for alternate approaches such as gene editing. However, testing gene editing strategies for on-target efficiency and off-target events using personalized iPSC-RPE model systems is warranted. In summary, personalized iPSC-RPE models can be used to select among a growing list of gene therapy options to maximize safety and efficacy while minimizing time and cost. Similar scenarios likely exist for other genotypically diverse channelopathies, expanding the therapeutic landscape for affected individuals.

Multi-omics analysis to characterize molecular adaptation of Entamoeba histolytica during serum stress.

Entamoeba histolytica is responsible for dysentery and extraintestinal disease in humans. To establish successful infection, it must generate adaptive response against stress due to host defense mechanisms. We have developed a robust proteomics workflow by combining miniaturized sample preparation, low flow-rate chromatography, and ultra-high sensitivity mass spectrometry, achieving increased proteome coverage, and further integrated proteomics and RNA-seq data to decipher regulation at translational and transcriptional levels. Label-free quantitative proteomics led to identification of 2344 proteins, an improvement over the maximum number identified in E. histolytica proteomic studies. In serum-starved cells, 127 proteins were differentially abundant and were associated with functions including antioxidant activity, cytoskeleton, translation, catalysis, and transport. The virulence factor, Gal/GalNAc-inhibitable lectin subunits, was significantly altered. Integration of transcriptomic and proteomic data revealed that only 30% genes were coordinately regulated at both transcriptional and translational levels. Some highly expressed transcripts did not change in protein abundance. Conversely, genes with no transcriptional change showed enhanced protein abundance, indicating post-transcriptional regulation. This multi-omics approach enables more refined gene expression analysis to understand the adaptive response of E. histolytica during growth stress.

Quality metrics in academia: time to revisit the rules?

Publication and citation metrics have been used for many years now as apparently objective parameters to evaluate educational institutions as well as individual researchers. A recent report in Science, about the Saveetha Institute of Medical and Technical Sciences (SIMATS), near Chennai, Tamil Nadu [1], highlights concerns about the value and limitations of such metrics in evaluating the importance of research publications, authors, journals and institutions.

Authorship controversies in academic publication: implications for science and the research environment.

The inspiration for this theme issue came from one of the biggest challenges that the journal has faced since its inception.

A basement membrane discovery pipeline uncovers network complexity, regulators, and human disease associations.

Basement membranes (BMs) are ubiquitous extracellular matrices whose composition remains elusive, limiting our understanding of BM regulation and function. By developing a bioinformatic and in vivo discovery pipeline, we define a network of 222 human proteins and their animal orthologs localized to BMs. Network analysis and screening in C. elegans and zebrafish uncovered BM regulators, including ADAMTS, ROBO, and TGFß. More than 100 BM network genes associate with human phenotypes, and by screening 63,039 genomes from families with rare disorders, we found loss-of-function variants in LAMA5, MPZL2, and MATN2 and show that they regulate BM composition and function. This cross-disciplinary study establishes the immense complexity of BMs and their impact on in human health.

Placebo in new Covid-19 vaccine trials: data quality prioritised over participants' rights.

The WHO Ad Hoc Expert Group on the Next Steps for Covid-19 Vaccine Evaluation makes recommendations on the use of placebo controlled trials in ongoing and future Covid-19 vaccine research. These recommendations unequivocally prioritise data quality over participants' rights and safety. Participants in trials of vaccines which have received emergency use listing or authorisation would be refused available vaccines. Placebo-controlled trials that would be impossible to conduct in rich countries would be permitted in poor countries. If these suggestions are implemented, the major beneficiary will be the vaccine industry.

Switch to Autophagy the Key Mechanism for Trabecular Meshwork Death in Severe Glaucoma.

PURPOSE: The key differences in cell death mechanisms in the trabecular meshwork (TM) in adult moderate and severe primary glaucoma remain still unanswered. This study explored key differences in cell death mechanisms in the trabecular meshwork (TM) in adult moderate and severe primary glaucoma. DESIGN: In-vitro laboratory study on surgical specimens and primary cell lines. METHODS: Select cell death-related proteins differentially expressed on mass spectrometric analysis in ex-vivo dissected TM specimens patients with severe adult primary open-angle (POAG) or angle-closure glaucoma (PACG) compared to controls (cadaver donor cornea) were validated for temporal changes in cell death-related gene expression on in-vitro primary human TM cell culture after 48 hours (moderate) or 72 hours (severe) oxidative stress with H2O2 (400-1000 uM concentration). These were compared with histone modifications after oxidative stress in human TM (HTM) culture and peripheral blood of patients with moderate and severe glaucoma. RESULTS: Autophagy-related proteins seemed to be the predominant cell-death mechanism over apoptosis in ex-vivo dissected TM specimens in severe glaucoma. Analyzing HTM cell gene expression at 48 hours and 72 hours of oxidative stress, autophagy genes were up-regulated at 48-72 hours of exposure in contrast to apoptosis-related genes, showing down-regulation at 72 hours. There was associated increased expression of H3K14ac in HTM after 72 hours of oxidative stress and in peripheral blood of severe POAG and PACG. CONCLUSION: A preference of autophagy over apoptosis may underlie stage transition from moderate to severe glaucoma in the trabecular meshwork or peripheral blood, which may be tightly regulated by epigenetic modulators.

Scale-up and coverage of Avahan: a large-scale HIV-prevention programme among female sex workers and men who have sex with men in four Indian states.

OBJECTIVE: Documenting the implementation of a public health programme as per its design is critical to interpretation of results from survey-led outcome and impact evaluation activities, the authors describe the scale-up and coverage of large-scale HIV-prevention services provided to female sex workers (FSWs) and high-risk men who have sex with men (HR-MSM) during the first 5 years of the Avahan programme in India. METHODS: Implementing NGO partner-generated denominator estimates from 70 districts were used to estimate the programme's intended coverage. Routine programme-monitoring data until December 2008 were used to describe the service and commodity availability, service utilisation to generate internal estimates of coverage. Coverage was validated in few districts using data from a cross-sectional survey. RESULTS: In December 2008, the estimated denominators for intended services were about 217,000 FSWs and 80,000 HR-MSM. By January 2007, 79% of eventual total clinics and 75% drop-in centres were established, and 83% of eventual peer educators were active. By month 48, sufficient condoms to cover all estimated FSW commercial sex acts were distributed free. By month 60, 75% of the estimated denominator intended to be covered was met monthly. 86% of FSWs and 67% of HR-MSM ever contacted had used sexually transmitted infections services at least once. Cross-sectional survey generated coverage results suggest that programme-monitoring data provide a proxy to coverage of services. CONCLUSION: Avahan's monitoring data show that Avahan achieved infrastructure scale by year 3 and high contact coverage through peers and with commodities by year 5 of implementation as per the design.

Centring patient autonomy in DNAR decisions.

The Response from the ICMR team of Dr Mani et al (1) to the IJME Editorial (2) on the ICMR DNAR Guidelines (3) provides some answers to the gaps and questions raised, and it is hoped that these will find a place in revised versions of the document. The document Disclaimer said "further revisions" were planned, based on perceptions and experiences of clinicians and the public; an early revision will allow for better acceptance of the Guidelines.

Mycobacterium tuberculosis Reactivates HIV-1 via Exosome-Mediated Resetting of Cellular Redox Potential and Bioenergetics.

The synergy between Mycobacterium tuberculosis and human immunodeficiency virus-1 (HIV-1) interferes with therapy and facilitates the pathogenesis of both human pathogens. Fundamental mechanisms by which M. tuberculosis exacerbates HIV-1 infection are not clear. Here, we show that exosomes secreted by macrophages infected with M. tuberculosis, including drug-resistant clinical strains, reactivated HIV-1 by inducing oxidative stress. Mechanistically, M. tuberculosis-specific exosomes realigned mitochondrial and nonmitochondrial oxygen consumption rates (OCR) and modulated the expression of host genes mediating oxidative stress response, inflammation, and HIV-1 transactivation. Proteomics analyses revealed the enrichment of several host factors (e.g., HIF-1α, galectins, and Hsp90) known to promote HIV-1 reactivation in M. tuberculosis-specific exosomes. Treatment with a known antioxidant-N-acetyl cysteine (NAC)-or with inhibitors of host factors-galectins and Hsp90-attenuated HIV-1 reactivation by M. tuberculosis-specific exosomes. Our findings uncover new paradigms for understanding the redox and bioenergetics bases of HIV-M. tuberculosis coinfection, which will enable the design of effective therapeutic strategies.IMPORTANCE Globally, individuals coinfected with the AIDS virus (HIV-1) and with M. tuberculosis (causative agent of tuberculosis [TB]) pose major obstacles in the clinical management of both diseases. At the heart of this issue is the apparent synergy between the two human pathogens. On the one hand, mechanisms induced by HIV-1 for reactivation of TB in AIDS patients are well characterized. On the other hand, while clinical findings clearly identified TB as a risk factor for HIV-1 reactivation and associated mortality, basic mechanisms by which M. tuberculosis exacerbates HIV-1 replication and infection remain poorly characterized. The significance of our research is in identifying the role of fundamental mechanisms such as redox and energy metabolism in catalyzing HIV-M. tuberculosis synergy. The quantification of redox and respiratory parameters affected by M. tuberculosis in stimulating HIV-1 will greatly enhance our understanding of HIV-M. tuberculosis coinfection, leading to a wider impact on the biomedical research community and creating new translational opportunities.

Confronting the medical devices jungle.

The report by the International Consortium of Investigative Journalists (ICIJ) on the international medical device industry adds to the growing documentation of health scandals in India in recent years. A comprehensive picture emerges of manufacturers selling untested products at usurious rates; criminally negligent doctors and medical establishments; and a regulatory system focused on the industry's growth with little regard for patient safety.

Response of the authors to the Letter "Concerns with regard to an article" published online on April 5, 2019.

Response of the authors to the Letter "Concerns with regard to an article" published online on April 5, 2019 (DOI: 10.20529/IJME.2019.014) on the IJME website.

Consultations on human infection studies in India: Do people's voices really count?

The Controlled Human Infection Model or CHIM, sometimes described as a human challenge study, is a relatively specialised medical research technique. Researchers infect healthy participants with a weakened strain of a pathogen in a controlled setting, in order to learn more about the infection and the disease, or to develop new vaccines for that disease. Unlike in other human clinical trials, where participants face a risk of harm because of, for example, the drug's side effects, healthy participants in CHIM trials are deliberately harmed through infection - contrary to every principle and guideline of medical practice and research.

New Drugs and Clinical Trials Rules, 2019: The market trumps ethics and participant rights.

The enactment of the New Drugs and Clinical Trials Rules, 2019 (hereafter New Rules), on March 19 by the Ministry of Health and Family Welfare (MoHFW), Government of India (1), is the use of power delegated to the political executive by sub-section (1) of section 12 and sub-section (1) of section 33 of the Drugs and Cosmetics Act, 1940. Has this power been used wisely? Whose interests do these rules represent?

LipidII interaction with specific residues of Mycobacterium tuberculosis PknB extracytoplasmic domain governs its optimal activation.

The Mycobacterium tuberculosis kinase PknB is essential for growth and survival of the pathogen in vitro and in vivo. Here we report the results of our efforts to elucidate the mechanism of regulation of PknB activity. The specific residues in the PknB extracytoplasmic domain that are essential for ligand interaction and survival of the bacterium are identified. The extracytoplasmic domain interacts with mDAP-containing LipidII, and this is abolished upon mutation of the ligand-interacting residues. Abrogation of ligand-binding or sequestration of the ligand leads to aberrant localization of PknB. Contrary to the prevailing hypothesis, abrogation of ligand-binding is linked to activation loop hyperphosphorylation, and indiscriminate hyperphosphorylation of PknB substrates as well as other proteins, ultimately causing loss of homeostasis and cell death. We propose that the ligand-kinase interaction directs the appropriate localization of the kinase, coupled to stringently controlled activation of PknB, and consequently the downstream processes thereof.

Public communication about CHIM: What is the role of the media?

The possibility of introducing the Controlled Human Infection Model of research into India is being discussed by some Indian scientists in order to develop biomedical technologies such as vaccines. CHIM studies involve the deliberate introduction of an infectious agent into a healthy person in order to observe the development and progression of the disease, or test potential treatments. This idea will be alarming to the Indian public who will demand the assurance that CHIM is needed and safe. Health communication is viewed by researchers as vital to getting communities on board in public health programmes. The role of the media, however, is to provide information and analysis that represents the public's interests, and enables the public to make informed decisions. The starting point for journalists will be the environment in which CHIM would be conducted in India: poor healthcare, poverty, vulnerabilities of various kinds, ethical violations, weak regulation, and industry's impunity. They will also consider that research agendas may be driven by a focus on technological solutions to complex problems, and promote unnecessary vaccines that subsidise the private sector vaccine industry. When talking to the media about its interest in conducting human challenge trials, the research community will have to be honest about its plans, transparent in its functioning, and also ready to admit the possibility that we should not introduce this technology in India.

Response to proposed research to reverse brain death: more than regulatory failure.

In early May 2016, reports of a bizarre study to attempt a reversal of brain death made the headlines, but there was not even a squeak from the authorities. The "Reanima Project" is a collaboration between an Indian surgeon, Himanshu Bansal, and a US-based biotech company, Bioquark Inc. The research will be conducted by Bansal's own biotech company, Revita Life Sciences, at his Anupam hospital in Rudrapur, a city in Uttarakhand state.

Sero-Prevalence of Rodent Pathogens in India.

Health monitoring is an integral part of laboratory animal quality standards. However, current or past prevalence data as well as regulatory requirements dictate the frequency, type and the expanse of health monitoring. In an effort to understand the prevalence of rodent pathogens in India, a preliminary study was carried out by sero-epidemiology. Sera samples obtained from 26 public and private animal facilities were analyzed for the presence of antibodies against minute virus of mice (MVM), ectromelia virus (ECTV), lymphocytic choriomeningitis virus (LCMV), mouse hepatitis virus (MHV), Sendai virus (SeV), and Mycoplasma pulmonis in mice, and SeV, rat parvo virus (RPV), Kilham's rat virus (KRV) and sialodacryoadenitis virus (SDAV) in rats, by sandwich ELISA. It was observed that MHV was the most prevalent agent followed by Mycoplasma pulmonis and MVM in mice, and SDAV followed by RPV were prevalent in rats. On the other hand, none of the samples were positive for ECTV in mice, or SeV or KRV in rats. Multiple infections were common in both mice and rats. The incidence of MHV and Mycoplasma pulmonis was higher in facilities maintained by public organizations than in vivaria of private organizations, although the difference was not statistically different. On the other hand the prevalence of rodent pathogens was significantly higher in the northern part of India than in the South. These studies form the groundwork for detailed sero-prevalence studies which should further lay the foundations for country-specific guidelines for health monitoring of laboratory animals.