Adaptation of a transmitted/founder simian-human immunodeficiency virus for enhanced replication in rhesus macaques.

Transmitted/founder (TF) simian-human immunodeficiency viruses (SHIVs) express HIV-1 envelopes modified at position 375 to efficiently infect rhesus macaques while preserving authentic HIV-1 Env biology. SHIV.C.CH505 is an extensively characterized virus encoding the TF HIV-1 Env CH505 mutated at position 375 shown to recapitulate key features of HIV-1 immunobiology, including CCR5-tropism, a tier 2 neutralization profile, reproducible early viral kinetics, and authentic immune responses. SHIV.C.CH505 is used frequently in nonhuman primate studies of HIV, but viral loads after months of infection are variable and typically lower than those in people living with HIV. We hypothesized that additional mutations besides Δ375 might further enhance virus fitness without compromising essential components of CH505 Env biology. From sequence analysis of SHIV.C.CH505-infected macaques across multiple experiments, we identified a signature of envelope mutations associated with higher viremia. We then used short-term in vivo mutational selection and competition to identify a minimally adapted SHIV.C.CH505 with just five amino acid changes that substantially improve virus replication fitness in macaques. Next, we validated the performance of the adapted SHIV in vitro and in vivo and identified the mechanistic contributions of selected mutations. In vitro, the adapted SHIV shows improved virus entry, enhanced replication on primary rhesus cells, and preserved neutralization profiles. In vivo, the minimally adapted virus rapidly outcompetes the parental SHIV with an estimated growth advantage of 0.14 days-1 and persists through suppressive antiretroviral therapy to rebound at treatment interruption. Here, we report the successful generation of a well-characterized, minimally adapted virus, termed SHIV.C.CH505.v2, with enhanced replication fitness and preserved native Env properties that can serve as a new reagent for NHP studies of HIV-1 transmission, pathogenesis, and cure.

The inhibin/activin signalling pathway in human gonadal and adrenal cancers.

The biological function of the inhibin-α subunit (INHA) in gonadal tumorigenesis is different in humans compared with mouse. The INHA subunit is up-regulated in most human ovarian and testicular cancers but knock-out studies in mice showed the INHA subunit is a tumour suppressor with gonadal and adrenal specificity. The INHA subunit is a component of the inhibin/activin signalling pathway, which includes activin receptors ActRIIA/IIB and intracellular Smads-2/3. To resolve the incongruity in function in humans versus mouse, we re-evaluated the inhibin/activin pathway in human gonadal and adrenal cancers using contemporary protein and mRNA expression data for multiple pathway components rather than INHA alone. We used an INHA antibody raised against the N-terminal domain to compare immunoreactivity with the more commonly used antibody raised against the C-terminal domain. This study also described, for the first time, a comprehensive protein expression profile of activin-ßC in reproductive and adrenal cancers, and its effect on a human granulosa cell line, providing evidence for a role in ovarian, testis and adrenal tumour biology. Our data show reduced INHA expression at both protein and mRNA levels, and increased activin signalling in human testicular, ovarian and malignant versus benign forms of adrenal cancer. We also found that activin-C acts as an activin-A antagonist by binding to activin receptor subunits IIA and IIB and modulating the canonical Smad pathway. In conclusion, analysis of the inhibin/activin signalling pathway helps to explain discrepancies arising from studies of only one hormone or subunit and suggests that altered expression of the inhibin and activin subunits is associated with reproductive and adrenal cancer biology.

Activin-ß(c) reduces reproductive tumour progression and abolishes cancer-associated cachexia in inhibin-deficient mice.

Activins are involved in the regulation of a diverse range of physiological processes including development, reproduction, and fertility, and have been implicated in the progression of cancers. Bioactivity is regulated by the inhibin α-subunit and by an activin-binding protein, follistatin. The activin-ß(C) subunit was not considered functionally significant in this regard due to an absence of phenotype in knockout mice. However, activin-ß(C) forms heterodimers with activin-ß(A) and activin-C antagonizes activin-A in vitro. Thus, it is proposed that overexpression, rather than loss of activin-ß(C) , regulates activin-A bioactivity. In order to prove biological efficacy, inhibin α-subunit knockout mice (α-KO) were crossed with mice overexpressing activin-ß(C) (ActC++). Deletion of inhibin leads to Sertoli and granulosa cell tumours, increased activin-A, and cancer-associated cachexia. Therefore, cachexia and reproductive tumour development should be modulated in α-KO/ActC++ mice, where excessive activin-A is the underlying cause. Accordingly, a reduction in activin-A, no significant weight loss, and reduced incidence of reproductive tumours were evident in α-KO/ActC++ mice. Overexpression of activin-ß(C) antagonized the activin signalling cascade; thus, the tumourigenic effects of activin-A were abrogated. This study provides proof of the biological relevance of activin-ß(C) . Being a regulator of activin-A, it is able to abolish cachexia and modulate reproductive tumour development in α-KO mice.

A rapid, specific, extraction-less, and cost-effective RT-LAMP test for the detection of SARS-CoV-2 in clinical specimens.

In 2019 a newly identified coronavirus, designated as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread rapidly from the epicenter in Wuhan (China) to more than 150 countries around the world, causing the Coronavirus disease 2019 (COVID-19) pandemic. In this study, we describe an extraction-less method based on reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) intended for the rapid qualitative detection of nucleic acid from SARS-CoV-2 in upper respiratory specimens, including oropharyngeal and nasopharyngeal swabs, anterior nasal and mid-turbinate nasal swabs, nasopharyngeal washes/aspirates or nasal aspirates as well as bronchoalveolar lavage (BAL) from individuals suspected of COVID-19 by their healthcare provider. The assay's performance was evaluated and compared to an RT quantitative PCR-based assay (FDA-approved). With high sensitivity, specificity, and bypassing the need for RNA extraction, the RT-LAMP Rapid Detection assay is a valuable and fast test for an accurate and rapid RNA detection of the SARS-CoV-2 virus and potentially other pathogens. Additionally, the versatility of this test allows its application in virtually every laboratory setting and remote location where access to expensive laboratory equipment is a limiting factor for testing during pandemic crises.

Regional localization of activin-ßA, activin-ßC, follistatin, proliferation, and apoptosis in adult and developing mouse prostate ducts.

Activins and inhibins, members of the TGF-ß superfamily, are growth and differentiation factors involved in the regulation of several biological processes, including reproduction, development, and fertility. Previous studies have shown that the activin-ßA subunit plays a pivotal role in prostate development. Activin-A inhibits branching morphogenesis in the developing prostate, and its expression is associated with increased apoptosis in the adult prostate. Follistatin, a structurally unrelated protein to activins, is an antagonist of activin-A. A balance between endogenous activin-A and follistatin is required to maintain prostatic branching morphogenesis. Deregulation of this balance leads to branching inhibition or excessive branching and increased maturation of the stroma surrounding the differentiating epithelial ducts. Recent work identified another member of the TGF-ß superfamily, the activin-ßC subunit, as a novel antagonist of activin-A. Over-expression of activin-C (ßC-ßC) alters prostate homeostasis, by interfering with the activin-A signaling. The current study characterized the spatiotemporal localization of activin-A, activin-C and follistatin in the adult and developing mouse prostate using immunohistochemical analysis. Results showed activin-C and follistatin are differentially expressed during prostate development and suggested that the antagonistic property of follistatin is secondary to the action of activin-C. In conclusion, the present study provides evidence to support a role of activin-C in prostate development and provides new insights in the spatiotemporal localization of activins and their antagonists during mouse prostate development.

Activin-ßC modulates cachexia by repressing the ubiquitin-proteasome and autophagic degradation pathways.

BACKGROUND: Cancer-associated cachexia and muscle wasting are considered key determinants of cancer-related death and reduction in the quality of life of cancer patients. A crucial link has been established between activin signaling and skeletal muscle atrophy-hypertrophy. We previously showed that activin-ßC, a novel activin-A antagonist, is a tumor modulator that abolishes the cancer-associated cachexia in a mouse genetic model of gonadal tumorigenesis, in which the normal balance of inhibin/activin signalling is disrupted by a targeted mutation in the Inha gene (inhibin α-KO mouse). This study aimed to identify the molecular mechanism by which activin-ßC increases survival and abolishes cancer-associated cachexia in α-KO mice. We hypothesized that overexpression of activin-ßC modulates the cachexia phenotype by antagonizing the activin signaling pathway and repressing muscle wasting via the ubiquitin-proteasome and the autophagic-lysosomal degradation pathways. METHODS: Male and female ActC++, α-KO, and α-KO/ActC++ mice and WT littermate controls were studied. Western blot analysis for the specific E3 ubiquitin ligases, atrogin-1 and MuRF1, markers of the autophagic-lysosomal pathway, Beclin-1, p62, and LC3A/B, effectors Smad-2, Smad-3 and myostatin was performed in the gastrocnemius of age-matched mice. Histopathology of the gastrocnemius and survival analysis were also conducted in animals from the same breeding cohort. Serum levels of activin-A, inflammatory cytokines, hormonal profile, and bone density were also assessed. RESULTS: Increased levels of atrogin-1, MuRF-1, Beclin-1, p62, LC3A/B-I, Smad-2 and serum levels of activin-A were noted in the α-KO mice. These mice developed gonadal cancers followed by severe weight loss, and reduced survival. Overexpression of activin- ßC antagonized the activin signaling cascade, attenuating the ubiquitin-proteasome and the autophagic-lysosomal degradation pathways, and reduced serum levels of activin-A. α-KO/ActC++ mice displayed a less aggressive cachectic phenotype, reduced tumor weight, and prolonged survival. CONCLUSION: Our findings show for the first time a specific effect of activin-ßC on muscle wasting and transcription factors involved in muscle protein degradation. The study indicates that activin-ßC may be a novel therapy to abrogate cancer-associated weight loss and prolong survival.

Re-evaluating the role of activin-ßC in cancer biology.

Transforming growth factor-ß (TGF-ß) superfamily signaling pathway and its ligands are essential regulators of cellular processes such as proliferation, differentiation, migration, and survival. Alteration of this pathway results in uncontrolled proliferation and cancer progression. This review focuses on a specific member of the TGF-ß superfamily: activin-ßC. After its initial discovery, activin-ßC has been considered non-biologically relevant. Therefore, for years several experimental designs have ignored the potential contribution of this molecule to the final biological outcome. Here we focus on recent advances in the activin field, with a particular emphasis on activin-ßC, its antagonistic mechanism, and the physiological relevance of activin-ßC actions in reproductive and cancer biology. Covering a novel and previously unexplored function of activin-ßC on cancer associated weight loss and muscle metabolism, this review suggests an imminent need to re-evaluate the function of activin-ßC in biological systems and advances the understanding of how activin-ßC antagonizes the activin signaling pathway. Thus, challenging activin biologists to consider the impact of activin-ßC when interpreting their work.

Activin-ßC modulates gonadal, but not adrenal tumorigenesis in the inhibin deficient mice.

Activins and inhibins are involved in the regulation of several biological processes, including reproduction, development and fertility. Deregulation of the inhibin/activin signaling pathway has been implicated in the progression of reproductive and adrenal cancers. Deletion of the inhibin α-subunit results in up-regulation of the circulating levels of activins and this leads to the development of sex-cord stromal tumors followed by a cancer associated-cachexia in mice. When gonadectomy is performed, development of adrenocortical carcinomas is observed. We previously showed that overexpression of activin-ßC modulates the development of sex-cord stromal tumors and reduces cancer-cachexia in the inhibin-deficient mice by antagonizing the activin signaling pathway. The adrenal cortex and gonads share in common a large subset of genes, consistent with their common embryonic lineage. Additionally, it has been shown that adrenocortical carcinomas adopt an altered cellular identity resembling the ovary. Therefore, a study to assess the impact of overexpression of activin-ßC on the onset of adrenocortical carcinoma in gonadectomized inhibin-deficient mice was warranted. Within the current study we evaluated markers of apoptosis, proliferation, tumor burden, survival analysis and serum levels of activin-A in gonadectomized mice versus sham operated controls. Results showed that overexpression of activin-ßC modulated the development of reproductive tumors but had no effect on adrenal tumorigenesis. Our data reinforces the importance of activin-ßC in reproductive biology and suggest that activin-ßC is a tumor modulator with gonadal specificity.

The therapeutic potential of blocking the activin signalling pathway.

Members of the transforming growth factor ß (TGF-ß) family regulate fundamental physiological process, such as cell growth, differentiation and apoptosis. As a result, defects in this pathway have been linked to uncontrolled proliferation and cancer progression. Here we explore the signal transduction mechanism of TGF-ß focusing on therapeutic intervention in human diseases. Like TGF-ß, another member of the TGF-ß superfamily, activin has been proven to play an important role in maintenance of tissue homeostasis and dysregulation leads to disease. Several studies showed elevated levels of activin are responsible for the development of gonadal tumours and a cachexia-like weight loss syndrome. Discussing the recent advances in approaches developed to antagonise the activin pathway and the encouraging results obtained in animal models, this review presents a therapeutic rationale for targeting the activin pathway in conditions such as cachexia, neuromuscular and/or musculoskeletal disorders.