Mitochondrial sulfide promotes life span and health span through distinct mechanisms in developing versus adult treated Caenorhabditis elegans.

Living longer without simultaneously extending years spent in good health ("health span") is an increasing societal burden, demanding new therapeutic strategies. Hydrogen sulfide (H2S) can correct disease-related mitochondrial metabolic deficiencies, and supraphysiological H2S concentrations can pro health span. However, the efficacy and mechanisms of mitochondrion-targeted sulfide delivery molecules (mtH2S) administered across the adult life course are unknown. Using a Caenorhabditis elegans aging model, we compared untargeted H2S (NaGYY4137, 100 µM and 100 nM) and mtH2S (AP39, 100 nM) donor effects on life span, neuromuscular health span, and mitochondrial integrity. H2S donors were administered from birth or in young/middle-aged animals (day 0, 2, or 4 postadulthood). RNAi pharmacogenetic interventions and transcriptomics/network analysis explored molecular events governing mtH2S donor-mediated health span. Developmentally administered mtH2S (100 nM) improved life/health span vs. equivalent untargeted H2S doses. mtH2S preserved aging mitochondrial structure, content (citrate synthase activity) and neuromuscular strength. Knockdown of H2S metabolism enzymes and FoxO/daf-16 prevented the positive health span effects of mtH2S, whereas DCAF11/wdr-23 - Nrf2/skn-1 oxidative stress protection pathways were dispensable. Health span, but not life span, increased with all adult-onset mtH2S treatments. Adult mtH2S treatment also rejuvenated aging transcriptomes by minimizing expression declines of mitochondria and cytoskeletal components, and peroxisome metabolism hub components, under mechanistic control by the elt-6/elt-3 transcription factor circuit. H2S health span extension likely acts at the mitochondrial level, the mechanisms of which dissociate from life span across adult vs. developmental treatment timings. The small mtH2S doses required for health span extension, combined with efficacy in adult animals, suggest mtH2S is a potential healthy aging therapeutic.

Mitochondrial hydrogen sulfide supplementation improves health in the C. elegans Duchenne muscular dystrophy model.

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder characterized by progressive muscle degeneration and weakness due to mutations in the dystrophin gene. The symptoms of DMD share similarities with those of accelerated aging. Recently, hydrogen sulfide (H2S) supplementation has been suggested to modulate the effects of age-related decline in muscle function, and metabolic H2S deficiencies have been implicated in affecting muscle mass in conditions such as phenylketonuria. We therefore evaluated the use of sodium GYY4137 (NaGYY), a H2S-releasing molecule, as a possible approach for DMD treatment. Using the dys-1(eg33) Caenorhabditis elegans DMD model, we found that NaGYY treatment (100 µM) improved movement, strength, gait, and muscle mitochondrial structure, similar to the gold-standard therapeutic treatment, prednisone (370 µM). The health improvements of either treatment required the action of the kinase JNK-1, the transcription factor SKN-1, and the NAD-dependent deacetylase SIR-2.1. The transcription factor DAF-16 was required for the health benefits of NaGYY treatment, but not prednisone treatment. AP39 (100 pM), a mitochondria-targeted H2S compound, also improved movement and strength in the dys-1(eg33) model, further implying that these improvements are mitochondria-based. Additionally, we found a decline in total sulfide and H2S-producing enzymes in dystrophin/utrophin knockout mice. Overall, our results suggest that H2S deficit may contribute to DMD pathology, and rectifying/overcoming the deficit with H2S delivery compounds has potential as a therapeutic approach to DMD treatment.

Vaxign2: the second generation of the first Web-based vaccine design program using reverse vaccinology and machine learning.

Vaccination is one of the most significant inventions in medicine. Reverse vaccinology (RV) is a state-of-the-art technique to predict vaccine candidates from pathogen's genome(s). To promote vaccine development, we updated Vaxign2, the first web-based vaccine design program using reverse vaccinology with machine learning. Vaxign2 is a comprehensive web server for rational vaccine design, consisting of predictive and computational workflow components. The predictive part includes the original Vaxign filtering-based method and a new machine learning-based method, Vaxign-ML. The benchmarking results using a validation dataset showed that Vaxign-ML had superior prediction performance compared to other RV tools. Besides the prediction component, Vaxign2 implemented various post-prediction analyses to significantly enhance users' capability to refine the prediction results based on different vaccine design rationales and considerably reduce user time to analyze the Vaxign/Vaxign-ML prediction results. Users provide proteome sequences as input data, select candidates based on Vaxign outputs and Vaxign-ML scores, and perform post-prediction analysis. Vaxign2 also includes precomputed results from approximately 1 million proteins in 398 proteomes of 36 pathogens. As a demonstration, Vaxign2 was used to effectively analyse SARS-CoV-2, the coronavirus causing COVID-19. The comprehensive framework of Vaxign2 can support better and more rational vaccine design. Vaxign2 is publicly accessible at http://www.violinet.org/vaxign2.

Identification of a chemical fingerprint linking the undeclared 2017 release of 106Ru to advanced nuclear fuel reprocessing.

The undeclared release and subsequent detection of ruthenium-106 (106Ru) across Europe from late September to early October of 2017 prompted an international effort to ascertain the circumstances of the event. While dispersion modeling, corroborated by ground deposition measurements, has narrowed possible locations of origin, there has been a lack of direct empirical evidence to address the nature of the release. This is due to the absence of radiological and chemical signatures in the sample matrices, considering that such signatures encode the history and circumstances of the radioactive contaminant. In limiting cases such as this, we herein introduce the use of selected chemical transformations to elucidate the chemical nature of a radioactive contaminant as part of a nuclear forensic investigation. Using established ruthenium polypyridyl chemistry, we have shown that a small percentage (1.2 ± 0.4%) of the radioactive 106Ru contaminant exists in a polychlorinated Ru(III) form, partly or entirely as ß-106RuCl3, while 20% is both insoluble and chemically inert, consistent with the occurrence of RuO2, the thermodynamic endpoint of the volatile RuO4 Together, these findings present a clear signature for nuclear fuel reprocessing activity, specifically the reductive trapping of the volatile and highly reactive RuO4, as the origin of the release. Considering that the previously established 103Ru:106Ru ratio indicates that the spent fuel was unusually young with respect to typical reprocessing protocol, it is likely that this exothermic trapping process proved to be a tipping point for an already turbulent mixture, leading to an abrupt and uncontrolled release.

Guide Swap enables genome-scale pooled CRISPR-Cas9 screening in human primary cells.

CRISPR-Cas9 screening allows genome-wide interrogation of gene function. Currently, to achieve the high and uniform Cas9 expression desirable for screening, one needs to engineer stable and clonal Cas9-expressing cells-an approach that is not applicable in human primary cells. Guide Swap permits genome-scale pooled CRISPR-Cas9 screening in human primary cells by exploiting the unexpected finding that editing by lentivirally delivered, targeted guide RNAs (gRNAs) occurs efficiently when Cas9 is introduced in complex with nontargeting gRNA. We validated Guide Swap in depletion and enrichment screens in CD4+ T cells. Next, we implemented Guide Swap in a model of ex vivo hematopoiesis, and identified known and previously unknown regulators of CD34+ hematopoietic stem and progenitor cell (HSPC) expansion. We anticipate that this platform will be broadly applicable to other challenging cell types, and thus will enable discovery in previously inaccessible but biologically relevant human primary cell systems.

Continuous Droplet-Actuating Platforms via an Electric Field Gradient: Electrowetting and Liquid Dielectrophoresis.

This work develops a technology for actuating droplets of any size without the requirement for high voltages or active control systems, which are typically found in competitive systems. The droplet actuation relies on two microelectrodes separated by a variable gap distance to generate an electrostatic gradient. The physical mechanism for the droplet motion is a combination of liquid dielectrophoresis and electrowetting. Investigating the system behavior as a function of the driving frequency identified the relative contribution of these two mechanisms and the optimum operating conditions. A fixed signal frequency of 0.5 kHz actuated various liquids and contaminants. Droplet actuation was demonstrated on several platforms, including linear, radial-symmetric, and bilateral-symmetric droplet motion. The electrode designs are scalable and can be fabricated on a flexible and optically transparent substrate: these key advancements will enable consumer applications that were previously inaccessible. A self-cleaning platform was also tested under laboratory conditions and on the road. This technology has significant potential in microfluidics and self-cleaning platforms, for example, in the automotive sector to clean body parts, camera covers, and sensors.

Structural properties of cationic surfactant-fatty alcohol bilayers: insights from dissipative particle dynamics.

Bilayers, self-assembled by cationic surfactants and fatty alcohols in water, are the basic units of lamellar gel networks - creamy formulations extensively used in cosmetics and pharmaceutics. Mesoscopic modelling and study of the bilayers formed by single- or double-tail cationic surfactants (CTAC or DHDAC), and fatty alcohols (FAs) in the lamellar fluid and gel phases were employed. Fatty alcohols with alkyl tail equal to or greater than the surfactant alkyl tail, i.e., C16FA or C18FA and C22FA, were considered. A model formulation was explored with the FA concentration greater than that of the surfactant and the structure of the fluid and gel bilayers in tensionless state characterised via the density profiles across the bilayers, orientational order parameters of the surfactant and FA chains, intrinsic analysis of the bilayer interfaces, and bending rigidity. The intrinsic analysis allows identification and quantification of the coexistence of the interdigitated and non-interdigitated phases present within the gel bilayers. The FA chains were found to conform the primary scaffolding of the bilayers while the surfactant chains tessellate bilayer monolayers from their water-hydrophobic interface. Further, the overlap of the FA chains from the apposed monolayers of the fluid bilayers rises with increasing FA length. Finally, the prevalence of the non-interdigitated phase over the interdigitated phase within the gel bilayers becomes enhanced upon the FA length increase with a preference of the surfactant chains to reside in the non-interdigitated phase rather than the interdigitated phase.

Mitochondrial dysfunction causes Ca2+ overload and ECM degradation-mediated muscle damage in C. elegans.

Mitochondrial dysfunction impairs muscle health and causes subsequent muscle wasting. This study explores the role of mitochondrial dysfunction as an intramuscular signal for the extracellular matrix (ECM)-based proteolysis and, consequentially, muscle cell dystrophy. We found that inhibition of the mitochondrial electron transport chain causes paralysis as well as muscle structural damage in the nematode Caenorhabditis elegans. This was associated with a significant decline in collagen content. Both paralysis and muscle damage could be rescued with collagen IV overexpression, matrix metalloproteinase (MMP), and Furin inhibitors in Antimycin A-treated animal as well as in the C. elegans Duchenne muscular dystrophy model. Additionally, muscle cytosolic calcium increased in the Antimycin A-treated worms, and its down-regulation rescued the muscle damage, suggesting that calcium overload acts as one of the early triggers and activates Furin and MMPs for collagen degradation. In conclusion, we have established ECM degradation as an important pathway of muscle damage.-Sudevan, S., Takiura, M., Kubota, Y., Higashitani, N., Cooke, M., Ellwood, R. A., Etheridge, T., Szewczyk, N. J., Higashitani, A. Mitochondrial dysfunction causes Ca2+ overload and ECM degradation-mediated muscle damage in C. elegans.

An Injectable Hyaluronan-Methylcellulose (HAMC) Hydrogel Combined with Wharton's Jelly-Derived Mesenchymal Stromal Cells (WJ-MSCs) Promotes Degenerative Disc Repair.

Intervertebral disc (IVD) degeneration is one of the predominant causes of chronic low back pain (LBP), which is a leading cause of disability worldwide. Despite substantial progress in cell therapy for the treatment of IVD degeneration, significant challenges remain for clinical application. Here, we investigated the effectiveness of hyaluronan-methylcellulose (HAMC) hydrogels loaded with Wharton's Jelly-derived mesenchymal stromal cell (WJ-MSCs) in vitro and in a rat coccygeal IVD degeneration model. Following induction of injury-induced IVD degeneration, female Sprague-Dawley rats were randomized into four groups to undergo a single intradiscal injection of the following: (1) phosphate buffered saline (PBS) vehicle, (2) HAMC, (3) WJ-MSCs (2 × 104 cells), and (4) WJ-MSCs-loaded HAMC (WJ-MSCs/HAMC) (n = 10/each group). Coccygeal discs were removed following sacrifice 6 weeks after implantation for radiologic and histologic analysis. We confirmed previous findings that encapsulation in HAMC increases the viability of WJ-MSCs for disc repair. The HAMC gel maintained significant cell viability in vitro. In addition, combined implantation of WJ-MSCs and HAMC significantly promoted degenerative disc repair compared to WJ-MSCs alone, presumably by improving nucleus pulposus cells viability and decreasing extracellular matrix degradation. Our results suggest that WJ-MSCs-loaded HAMC promotes IVD repair more effectively than cell injection alone and supports the potential clinical use of HAMC for cell delivery to arrest IVD degeneration or to promote IVD regeneration.

Comparing Two Independent Satellite-Based Algorithms for Detecting and Tracking Ash Clouds by Using SEVIRI Sensor.

The Eyjafjallajökull (Iceland) volcanic eruption of April-May 2010 caused unprecedented air-traffic disruption in Northern Europe, revealing some important weaknesses of current operational ash-monitoring and forecasting systems and encouraging the improvement of methods and procedures for supporting the activities of Volcanic Ash Advisory Centers (VAACs) better. In this work, we compare two established satellite-based algorithms for ash detection, namely RSTASH and the operational London VAAC method, both exploiting sensor data of the spinning enhanced visible and infrared imager (SEVIRI). We analyze similarities and differences in the identification of ash clouds during the different phases of the Eyjafjallajökull eruption. The work reveals, in some cases, a certain complementary behavior of the two techniques, whose combination might improve the identification of ash-affected areas in specific conditions. This is indicated by the quantitative comparison of the merged SEVIRI ash product, achieved integrating outputs of the RSTASH and London VAAC methods, with independent atmospheric infrared sounder (AIRS) DDA (dust-detection algorithm) observations.

In Vitro and In Vivo Antifungal Profile of a Novel and Long-Acting Inhaled Azole, PC945, on Aspergillus fumigatus Infection.

The profile of PC945, a novel triazole antifungal designed for administration via inhalation, was assessed in a range of in vitro and in vivo studies. PC945 was characterized as a potent, tightly binding inhibitor of Aspergillus fumigatus sterol 14α-demethylase (CYP51A and CYP51B) activity (50% inhibitory concentrations [IC50s], 0.23 µM and 0.22 µM, respectively) with characteristic type II azole binding spectra. Against 96 clinically isolated A. fumigatus strains, the MIC values of PC945 ranged from 0.032 to >8 µg/ml, while those of voriconazole ranged from 0.064 to 4 µg/ml. Spectrophotometric analysis of the effects of PC945 against itraconazole-susceptible and -resistant A. fumigatus growth yielded IC50 (determined based on optical density [OD]) values of 0.0012 to 0.034 µg/ml, whereas voriconazole (0.019 to >1 µg/ml) was less effective than PC945. PC945 was effective against a broad spectrum of pathogenic fungi (with MICs ranging from 0.0078 to 2 µg/ml), including Aspergillus terreus, Trichophyton rubrum, Candida albicans, Candida glabrata, Candida krusei, Cryptococcus gattii, Cryptococcus neoformans, and Rhizopus oryzae (1 or 2 isolates each). In addition, when A. fumigatus hyphae or human bronchial cells were treated with PC945 and then washed, PC945 was found to be absorbed quickly into both target and nontarget cells and to produce persistent antifungal effects. Among temporarily neutropenic immunocompromised mice infected with A. fumigatus intranasally, 50% of the animals survived until day 7 when treated intranasally with PC945 at 0.56 µg/mouse, while posaconazole showed similar effects (44%) at 14 µg/mouse. This profile affirms that topical treatment with PC945 should provide potent antifungal activity in the lung.

A conserved salt bridge in the G loop of multiple protein kinases is important for catalysis and for in vivo Lyn function.

The glycine-rich G loop controls ATP binding and phosphate transfer in protein kinases. Here we show that the functions of Src family and Abl protein tyrosine kinases require an electrostatic interaction between oppositely charged amino acids within their G loops that is conserved in multiple other phylogenetically distinct protein kinases, from plants to humans. By limiting G loop flexibility, it controls ATP binding, catalysis, and inhibition by ATP-competitive compounds such as Imatinib. In WeeB mice, mutational disruption of the interaction results in expression of a Lyn protein with reduced catalytic activity, and in perturbed B cell receptor signaling. Like Lyn(-/-) mice, WeeB mice show profound defects in B cell development and function and succumb to autoimmune glomerulonephritis. This demonstrates the physiological importance of the conserved G loop salt bridge and at the same time distinguishes the in vivo requirement for the Lyn kinase activity from other potential functions of the protein.

An image-based screen identifies a small molecule regulator of megakaryopoiesis.

Molecules that control the lineage commitment of hematopoietic stem cells (HSCs) may allow the expansion of enriched progenitor populations for both research and therapeutic uses. In an effort to better understand and control the differentiation of HSCs to megakaryocytes, we carried out an image-based screen of a library of 50,000 heterocycles using primary human CD34(+) cells. A class of naphthyridinone derivatives was identified that induces the differentiation of common myeloid progenitors (CMP) to megakaryocytes. Kinase profiling and subsequent functional assays revealed that these compounds act through inhibition of platelet-derived growth factor receptor (PDGFR) signaling in CMPs. Such molecules may ultimately have clinical utility in the treatment of thrombocytopenia.

Uncovering regulatory pathways that affect hematopoietic stem cell function using 'genetical genomics'.

We combined large-scale mRNA expression analysis and gene mapping to identify genes and loci that control hematopoietic stem cell (HSC) function. We measured mRNA expression levels in purified HSCs isolated from a panel of densely genotyped recombinant inbred mouse strains. We mapped quantitative trait loci (QTLs) associated with variation in expression of thousands of transcripts. By comparing the physical transcript position with the location of the controlling QTL, we identified polymorphic cis-acting stem cell genes. We also identified multiple trans-acting control loci that modify expression of large numbers of genes. These groups of coregulated transcripts identify pathways that specify variation in stem cells. We illustrate this concept with the identification of candidate genes involved with HSC turnover. We compared expression QTLs in HSCs and brain from the same mice and identified both shared and tissue-specific QTLs. Our data are accessible through WebQTL, a web-based interface that allows custom genetic linkage analysis and identification of coregulated transcripts.

Interleukin-15 rescues tolerant CD8+ T cells for use in adoptive immunotherapy of established tumors.

CD8+ T cells can mediate eradication of established tumors, and strategies to amplify tumor-reactive T-cell numbers by immunization or ex vivo expansion followed by adoptive transfer are currently being explored in individuals with cancer. Generating effective CD8+ T cell-mediated responses to tumors is often impeded by T-cell tolerance to relevant tumor antigens, as most of these antigens are also expressed in normal tissues. We examined whether such tolerant T cells could be rescued and functionally restored for use in therapy of established tumors. We used a transgenic T-cell receptor (TCR) mouse model in which peripheral CD8+ T cells specific for a candidate tumor antigen also expressed in liver are tolerant, failing to proliferate or secrete interleukin (IL)-2 in response to antigen. Molecular and cellular analysis showed that these tolerant T cells expressed the IL-15 receptor alpha chain, and could be induced to proliferate in vitro in response to exogenous IL-15. Such proliferation abrogated tolerance and the rescued cells became effective in treating leukemia. Therefore, high-affinity CD8+ T cells are not necessarily deleted by encounter with self-antigen in the periphery, and can potentially be rescued and expanded for use in tumor immunotherapy.

Catalytic asymmetric synthesis of allylic alcohols and derivatives and their applications in organic synthesis.

Allylic alcohols represent an important and highly versatile class of chiral building blocks for organic synthesis. This Review summarizes the plethora of methods developed for the catalytic asymmetric synthesis of enantioenriched allylic alcohols. These include: dynamic kinetic resolution (DKR/DKAT), nucleophilic 1,2-addition to carbonyl groups, allylic substitution, oxidation of C-H bonds, the addition of O nucleophiles to π systems, reduction of unsaturated carbonyl compounds, and an alternative route from enantioenriched propargylic alcohols. Furthermore, these catalytic asymmetric processes are exemplified by their applications in the syntheses of complex molecules such as natural products and potential therapeutic agents.

Barriers to and Facilitators of Participation in Health and Social Care Governance: Categories and Cross-Cutting Themes from a Survey of SHAPES Project Partners.

Good governance-aligned with human rights and rights-based care, participation, inclusion, and person-centredness-of digital care systems is integral to their ability to meet their objectives. To gain insight into existing governance structures and processes and participation experiences across Europe and lay foundations for the SHAPES Project's network governance (a healthy and active ageing Innovation Action consortium), our objectives included: 1) expand the list of known stakeholders, 2) explore how the range of stakeholders participate in health and social care governance, 3) develop an inventory of barriers and facilitators. Using an empirical, survey method, we consulted SHAPES Project partner organisations, with respondents invited to suggest specific participation barriers and facilitators. 16 organisations responded. Numerous additional stakeholders were identified. Circa 150 unique barriers and facilitators were reported, rationalised into 20 superordinate categories. Six cross-cutting themes were assembled: dimensionality and flux; power; opportunity and environments; interest, motivation, and choice; valuing governance participation, and duality. This work allows consideration of a wide range of stakeholders for the SHAPES collaborative governance model and future research, and for system design with the benefit of a detailed inventory of barriers and facilitators, and thematic contextualisation. Participation is modifiable and we suggest intervention targets and mechanisms.

Mechanisms for the Participatory Governance of Technology-Mediated Health and Social Care.

Increasingly, health and social care providers are adopting technology-mediated processes to optimise the delivery of care and to influence policy- and decision-makers. However, fragmentation persists in and between health and social care, impeding the provision of rounded person-centred care. Health and care delivery for an ageing population involves many diverse stakeholders with a range of motivations and agendas. The creation of a functional and sustainable network may promote the achievement of a well-functioning and integrated health and care sector. This work-in-progress paper outlines the evolution of an optimal governance model for the SHAPES network.

SHAPES Marketplace: Transparency, Trust and Fair Competition in the Healthy Ageing Market.

Access to digital health and care solutions and services that promote healthy ageing, independent living, and ageing in place is limited due to significant market barriers and challenges. The SHAPES project addresses the challenge of ageing populations by developing a sociotechnical ecosystem comprising a variety of health and care digital solutions, tools and services to enable and facilitate active, independent, and healthy ageing at home. Within the SHAPES project, the SHAPES Marketplace serves as a one-stop-shop for digital solutions and services designed for the Silver Economy that target the smart and healthy ageing and independent living markets. Delivering a dynamic catalogue of health and care digital solutions and services, the Marketplace promotes a transparent expansion of a trusted market offer on digital solutions and services for healthy ageing and independent living on a pan-European scale, thereby preventing vendor lock-in and enhancing the agile and fair competitiveness of the health and care industry, particularly in Europe. This paper introduces the SHAPES Marketplace and considers its function as a market driver to raise awareness on the benefits and impact of health and care digital solutions and services, as well as to shape the healthy ageing market, upholding the Systems-Market for Assistive and Related Technologies (SMART) Thinking Matrix to stimulate transparency, trust and fair competition.

VIGET: A web portal for study of vaccine-induced host responses based on Reactome pathways and ImmPort data.

Host responses to vaccines are complex but important to investigate. To facilitate the study, we have developed a tool called Vaccine Induced Gene Expression Analysis Tool (VIGET), with the aim to provide an interactive online tool for users to efficiently and robustly analyze the host immune response gene expression data collected in the ImmPort/GEO databases. VIGET allows users to select vaccines, choose ImmPort studies, set up analysis models by choosing confounding variables and two groups of samples having different vaccination times, and then perform differential expression analysis to select genes for pathway enrichment analysis and functional interaction network construction using the Reactome's web services. VIGET provides features for users to compare results from two analyses, facilitating comparative response analysis across different demographic groups. VIGET uses the Vaccine Ontology (VO) to classify various types of vaccines such as live or inactivated flu vaccines, yellow fever vaccines, etc. To showcase the utilities of VIGET, we conducted a longitudinal analysis of immune responses to yellow fever vaccines and found an intriguing complex activity response pattern of pathways in the immune system annotated in Reactome, demonstrating that VIGET is a valuable web portal that supports effective vaccine response studies using Reactome pathways and ImmPort data.