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ABSTRACT Rational engineering of gas-fermenting bacteria for high yields of bioproducts is vital for a sustainable bioeconomy. It will allow the microbial chassis to renewably valorize natural resources from carbon oxides, hydrogen, and/or lignocellulosic feedstocks more efficiently. To date, rational design of gas-fermenting bacteria such as changing the expression levels of individual enzymes to obtain the desired pathway flux is challenging, because pathway design must follow a verifiable metabolic blueprint indicating where interventions should be executed. Based on recent advances in constraint-based thermodynamic and kinetic models, we identify key enzymes in the gas-fermenting acetogen Clostridium ljungdahlii that correlate with the production of isopropanol. To this extent, we integrated a metabolic model in comparison with proteomics measurements and quantified the uncertainty for a variety of pathway targets needed to improve the bioproduction of isopropanol. Based on in silico thermodynamic optimization, minimal protein requirement analysis, and ensemble modeling-based robustness analysis, we identified the top two significant flux control sites, i.e., acetoacetyl-coenzyme A (CoA) transferase (AACT) and acetoacetate decarboxylase (AADC), overexpression of which could lead to increased isopropanol production. Our predictions directed iterative pathway construction, which enabled a 2.8-fold increase in isopropanol production compared to the initial version. The engineered strain was further tested under gas-fermenting mixotrophic conditions, where more than 4 g/L isopropanol was produced when CO, CO2, and fructose were provided as the substrates. In a bioreactor environment sparging with CO, CO2, and H2 only, the strain produced 2.4 g/L isopropanol. Our work highlighted that the gas-fermenting chasses can be fine-tuned for high-yield bioproduction by directed and elaborative pathway engineering. IMPORTANCE Highly efficient bioproduction from gaseous substrates (e.g., hydrogen and carbon oxides) will require systematic optimization of the host microbes. To date, the rational redesign of gas-fermenting bacteria is still in its infancy, due in part to the lack of quantitative and precise metabolic knowledge that can direct strain engineering. Here, we provide a case study by engineering isopropanol production in gas-fermenting Clostridium ljungdahlii. We demonstrate that a modeling approach based on the thermodynamic and kinetic analysis at the pathway level can provide actionable insights into strain engineering for optimal bioproduction. This approach may pave the way for iterative microbe redesign for the conversion of renewable gaseous feedstocks.

3-Hydroxybutyrate (3HB) is a product of interest as it is a precursor to the commercially produced bioplastic polyhydroxybutyrate. It can also serve as a platform for fine chemicals, medicines, and biofuels, making it a value-added product and feedstock. Acetogens non-photosynthetically fix CO2 into acetyl-CoA and have been previously engineered to convert acetyl-CoA into 3HB. However, as acetogen metabolism is poorly understood, those engineering efforts have had varying levels of success. 3HB, using acetyl-CoA as a precursor, can be synthesized by a variety of different pathways. Here we systematically compare various pathways to produce 3HB in acetogens and discover a native (S)-3-hydroxybutyryl-CoA dehydrogenase, hbd2, responsible for endogenous 3HB production. In conjunction with the heterologous thiolase atoB and CoA transferase ctfAB, hbd2 overexpression improves yields of 3HB on both sugar and syngas (CO/H2/CO2), outperforming the other tested pathways. These results uncovered a previously unknown 3HB production pathway, inform data from prior metabolic engineering efforts, and have implications for future physiological and biotechnological anaerobic research.

The sustainable production of chemicals from non-petrochemical sources is one of the greatest challenges of our time. CO2 release from industrial activity is not environmentally friendly yet provides an inexpensive feedstock for chemical production. One means of addressing this problem is using acetogenic bacteria to produce chemicals from CO2, waste streams, or renewable resources. Acetogens are attractive hosts for chemical production for many reasons: they can utilize a variety of feedstocks that are renewable or currently waste streams, can capture waste carbon sources and covert them to products, and can produce a variety of chemicals with greater carbon efficiency over traditional fermentation technologies. Here we investigated the metabolism of Clostridium ljungdahlii, a model acetogen, to probe carbon and electron partitioning and understand what mechanisms drive product formation in this organism. We utilized CRISPR/Cas9 and an inducible riboswitch to target enzymes involved in fermentation product formation. We focused on the genes encoding phosphotransacetylase (pta), aldehyde ferredoxin oxidoreductases (aor1 and aor2), and bifunctional alcohol/aldehyde dehydrogenases (adhE1 and adhE2) and performed growth studies under a variety of conditions to probe the role of those enzymes in the metabolism. Finally, we demonstrated a switch from acetogenic to ethanologenic metabolism by these manipulations, providing an engineered bacterium with greater application potential in biorefinery industry.

Volumetric muscle defect, caused by trauma or combat injuries, is a major health concern leading to severe morbidity. It is characterized by partial or full thickness loss of muscle and its bio-scaffold, resulting in extensive fibrosis and scar formation. Therefore, the ideal therapeutic option is to use stem cells combined with bio-scaffolds to restore muscle. For this purpose, muscle-derived stem cells (MDSCs) are a great candidate due to their unique multi-lineage differentiation potential.In this study, we evaluated the regeneration potential of MDSCs for muscle loss repair using a novel in situ fibrin gel casting. Muscle defect was created by a partial thickness wedge resection in the tibialis anterior (TA) muscles of NSG mice which created an average of 25% mass loss. If untreated, this defect leads to severe muscle fibrosis. Next, MDSCs were delivered using a novel in situ fibrin gel casting method.Our results demonstrated MDSCs are able to engraft and form new myofibers in the defect when casted along with fibrin gel. LacZ labeled MDSCs were able to differentiate efficiently into new myofibers and significantly increase muscle mass. This was also accompanied by significant reduction of fibrotic tissue in the engrafted muscles. Furthermore, transplanted cells also contributed to new vessel formation and satellite cell seeding.These results confirmed the therapeutic potential of MDSCs and feasibility of direct in situ casting of fibrin/MDSC mixture to repair muscle mass defects. Keywords: Skeletal muscle, Volumetric muscle loss, Fibrin gel, Muscle-derived stem cells (MDSCs), Muscle repair, Bio-scaffold

Recently, a new type of limb-girdle muscular dystrophy (LGMD type 2Z) has been identified due to a missense mutation in POGLUT1 (protein O-glucosyltransferase-Rumi), an enzyme capable of adding glucose to a distinct serine residue of epidermal growth factor-like repeats containing a C-X-S-X-(P/A)-C consensus sequence such as Notch receptors. Affected patients demonstrate reduced Notch signaling, decreased muscle stem cell pool and hypoglycosylation of α-dystroglycan, leading to LGMD phenotype. Here we report the generation and characterization of an iPSC line (CSCRMi001-A) from a LGMD-2Z patient with missense mutation in POGLUT1 which can be used for in vitro disease modeling.

Stable isotope based metabolic flux analysis is currently the unique methodology that allows the experimental study of the integrated responses of metabolic networks. This method primarily relies on isotope labeling and modeling, which could be a challenge in both experimental and computational biology. In particular, the algorithm implementation for isotope simulation is a critical step, limiting extensive usage of this powerful approach. Here, we introduce EMUlator a Python-based isotope simulator which is developed on Elementary Metabolite Unit (EMU) algorithm, an efficient and powerful algorithm for isotope modeling. We propose a novel adjacency matrix method to implement EMU modeling and exemplify it stepwise. This method is intuitively straightforward and can be conveniently mastered for various customized purposes. We apply this arithmetic pipeline to understand the phosphoketolase flux in the metabolic network of an industrial microbe Clostridium acetobutylicum. The resulting design enables a high-throughput and non-invasive approach for estimating phosphoketolase flux in vivo. Our computational insights allow the systematic design and prediction of isotope-based metabolic models and yield a comprehensive understanding of their limitations and potentials.

Summary: Myogenic differentiation of human pluripotent stem cells (hPSCs) has been done by gene overexpression or directed differentiation. However, viral integration, long-term culture, and the presence of unwanted cells are the main obstacles. By using CRISPR/Cas9n, a double-reporter human embryonic stem cell (hESC) line was generated for PAX7/MYF5, allowing prospective readout. This strategy allowed pathway screen to define efficient myogenic induction in hPSCs. Next, surface marker screen allowed identification of CD10 and CD24 for purification of myogenic progenitors and exclusion of non-myogenic cells. CD10 expression was also identified on human satellite cells and skeletal muscle progenitors. In vitro and in vivo studies using transgene and/or reporter-free hPSCs further validated myogenic potential of the cells by formation of new fibers expressing human dystrophin as well as donor-derived satellite cells in NSG-mdx4Cv mice. This study provides biological insights for myogenic differentiation of hPSCs using a double-reporter cell resource and defines an improved myogenic differentiation and purification strategy. : Wu et al. demonstrate that, by generation of a double-reporter human embryonic stem cell line for skeletal muscle lineage, cells can be screened for governing pathways and surface markers, defining myogenic induction and purification protocol and evaluation of their regenerative potential using in vivo studies in a mouse model for muscular dystrophy.

Cellulolytic bacteria have the potential to perform lignocellulose hydrolysis and fermentation simultaneously. The metabolic pathways of these bacteria, therefore, require more comprehensive and quantitative understanding. Using isotope tracer, gas chromatography-mass spectrometry, and metabolic flux modeling, we decipher the metabolic network of Clostridium thermocellum, a model cellulolytic bacterium which represents as an attractive platform for conversion of lignocellulose to dedicated products. We uncover that the Embden–Meyerhof–Parnas (EMP) pathway is the predominant glycolytic route whereas the Entner–Doudoroff (ED) pathway and oxidative pentose phosphate pathway are inactive. We also observe that C. thermocellum's TCA cycle is initiated by both Si- and Re-citrate synthase, and it is disconnected between 2-oxoglutarate and oxaloacetate in the oxidative direction; C. thermocellum uses a citramalate shunt to synthesize isoleucine; and both the one-carbon pathway and the malate shunt are highly active in this bacterium. To gain a quantitative understanding, we further formulate a fluxome map to quantify the metabolic fluxes through central metabolic pathways. This work represents the first global in vivo investigation of the principal carbon metabolism of C. thermocellum. Our results elucidate the unique structure of metabolic network in this cellulolytic bacterium and demonstrate the capability of isotope-assisted metabolite studies in understanding microbial metabolism of industrial interests.

Abstract Methotrexate (MTX) doses on days +1, +3, +6, and +11 after match unrelated donor allogeneic stem cell transplant (MUD HSCT) is a common graft‐versus‐host disease (GVHD) prophylaxis regimen. However, the overlapping toxicity of MTX with conditioning chemotherapy sometimes warrants the omission of the fourth dose of MTX. Prior single‐institution studies showed conflicting results comparing the outcomes of patients who received three versus four doses of MTX, but to our knowledge, the effect of concomitant antithymocyte globulin (ATG) has not been reported. Charts of patients who underwent MUD HSCT between 2009 and 2023 were reviewed. Patients received rabbit ATG (Thymoglobulin), given at 0.5 mg/kg on day −3, 2 mg/kg on day −2, and 2.5 mg/kg on day −1. MTX is given at 15 mg/m2 on day +1 and 10 mg/m2 on days +3, +6, and +11. Severe mucositis was the most common indication for day +11 MTX omission (82%). We identified 292 patients (116 in 3 dose cohort and 176 in 4 dose cohort). Median follow‐up was 23 months (range 1–151). Patients in the 4 doses cohort were more frequently male (68% vs. 50%, p

Plant-parasitic nematodes constrain global food security. During parasitism, they secrete effectors into the host plant from two types of pharyngeal gland cells. These effectors elicit profound changes in host biology to suppress immunity and establish a unique feeding organ from which the nematode draws nutrition. Despite the importance of effectors in nematode parasitism, there has been no comprehensive identification and characterisation of the effector repertoire of any plant-parasitic nematode. To address this, we advance techniques for gland cell isolation and transcriptional analysis to define a stringent annotation of putative effectors for the cyst nematode Heterodera schachtii at three key life-stages. We define 717 effector gene loci: 269 "known" high-confidence homologs of plant-parasitic nematode effectors, and 448 "novel" effectors with high gland cell expression. In doing so we define the most comprehensive "effectorome" of a plant-parasitic nematode to date. Using this effector definition, we provide the first systems-level understanding of the origin, deployment and evolution of a plant-parasitic nematode effectorome. The robust identification of the effector repertoire of a plant-parasitic nematode will underpin our understanding of nematode pathology, and hence, inform strategies for crop protection.

Abstract Most patients with advanced ovarian cancer (AOC) ultimately relapse after platinum-based chemotherapy. Combining bevacizumab, olaparib, and durvalumab likely drives synergistic activity. This open-label phase 2 study (NCT04015739) aimed to assess activity and safety of this triple combination in female patients with relapsed high-grade AOC following prior platinum-based therapy. Patients were treated with olaparib (300 mg orally, twice daily), the bevacizumab biosimilar FKB238 (15 mg/kg intravenously, once-every-3-weeks), and durvalumab (1.12 g intravenously, once-every-3-weeks) in nine French centers. The primary endpoint was the non-progression rate at 3 months for platinum-resistant relapse or 6 months for platinum-sensitive relapse per RECIST 1.1 and irRECIST. Secondary endpoints were CA-125 decline with CA-125 ELIMination rate constant K (KELIM-B) per CA-125 longitudinal kinetics over 100 days, progression free survival and overall survival, tumor response, and safety. Non-progression rates were 69.8% (90%CI 55.9%-80.0%) at 3 months for platinum-resistant relapse patients (N = 41), meeting the prespecified endpoint, and 43.8% (90%CI 29.0%-57.4%) at 6 months for platinum-sensitive relapse (N = 33), not meeting the prespecified endpoint. Median progression-free survival was 4.1 months (95%CI 3.5–5.9) and 4.9 months (95%CI 2.9–7.0) respectively. Favorable KELIM-B was associated with better survival. No toxic deaths or major safety signals were observed. Here we show that further investigation of this triple combination may be considered in AOC patients with platinum-resistant relapse.

Objective The aim of this study was to determine the effectiveness of a mechanical thrombectomy device in treating iliofemoral deep vein thrombosis (DVT) without thrombolysis and factors associated with post-thrombectomy occlusion.

Tree establishment following severe or stand-replacing disturbance is critical for achieving U.S. climate change mitigation goals and for maintaining the co-benefits of intact forest ecosystems. In many contexts, natural post-fire tree regeneration is sufficient to maintain forest cover and associated ecosystem services, but increasingly the pattern and scale of disturbance exceeds ecological thresholds and active reforestation may be warranted. Our capacity to plant trees, however, is not keeping pace with reforestation needs. This shortfall is uniquely apparent in the western U.S., where wildfire size and severity have increased in recent decades and long-term divestment in the reforestation supply chain has limited our ability to respond to existing needs. Here we present an analysis of key facets of both the supply and demand side of reforestation in the western U.S. and address six questions: (1) What is the current backlog of potential reforestation needs driven by high-severity wildfire?; (2) How will increasing wildfire activity through the end of the century affect potential reforestation needs?; (3) What is our capacity to meet current and future reforestation needs?; (4) How can we scale the reforestation supply chain to meet current and future demands?; (5) What approaches to reforestation can promote forest resilience to climate change and wildfire?; and (6) Where are opportunities emerging from recent policy initiatives, innovative public-private partnerships, and natural capital markets for scaling reforestation? Between 1984 and 2000, annual tree planting capacity met post-fire needs but cumulatively over the last two decades (2000 to 2021) it has fallen short of fire-driven needs by an estimated 1.5 million ha (ca. 3.8 million ac). We anticipate this gap will increase 2 to 3 fold by 2050. Scaling up reforestation efforts to close this gap will require increased investment across all facets of the reforestation supply chain, public-private partnerships, and novel approaches to reforestation that increase the resilience of western forests to drought and wildfire. We highlight emerging opportunities from recent policy initiatives and conservation finance for expanding reforestation efforts.

Primary gastric Burkitt’s lymphoma is an aggressive non-Hodgkin’s lymphoma that has been rarely reported in the literature. The majority of primary gastric lymphomas are diffuse large B-cell lymphomas and mucosa-associated lymphoid tissue (MALT) lymphomas. Patients with primary gastric Burkitt’s lymphoma can present with abdominal pain, hematemesis, melena, perforation, and obstruction. Diagnosis is made with a combination of clinical, radiological, and pathological findings. Treatment data are limited due to the limited cases reported. We present a case of a 47-year-old female who presented with diffuse abdominal pain, melena, and coffee-ground emesis that was diagnosed with primary gastric Burkitt’s lymphoma following biopsies taken from a gastric ulcerated mass found on upper endoscopy.

Short-rotation crop (SRC) systems with woody species have been planted in Costa Rica. However, information about different tree species and spacing is limited. The objective of the present study was to examine biomass production and the physical, energy, and chemical properties of feedstock of four tropical tree species (Cordia alliodora, Dipteryx panamensis, Gmelina arborea, and Tectona grandis) in 34-month-old plants planted at four spacings (0.5x1.0 m, 1.0x1.0 m, 1.0x2.0 m, and 2.0x2.0 m). The highest mortality rate (50%) was found in G. arborea; however, diameter, height, basal area, and biomass production in G. arborea plantations were higher than T. grandis, C. alliodora, and D. panamensis. Spacing effects on diameter, height, basal area, and biomass production were observed in 10-month-olds. Wide spacing presented the highest values in diameter and height, but the highest biomass production was found in the narrow spacing. Also, biomass distribution was different in D. panamensis in relation to other species. Specify gravity, density, and moisture content of biomass showed high variation between species and spacing, but the energy and chemical properties of biomass showed few differences. These results suggest that these four species were uniform in terms of their energy and chemical properties, regardless of the spacing used. Finally, three species (G. arborea, C. alliodora, and T. grandis) presented important potential for use in SRC systems. G. arborea was the species with the highest production but a high mortality rate.

Abstract Combination chemotherapy is crucial for successfully treating cancer. However, the enormous number of possible drug combinations means discovering safe and effective combinations remains a significant challenge. To improve this process, we conduct large-scale targeted CRISPR knockout screens in drug-treated cells, creating a genetic map of druggable genes that sensitize cells to commonly used chemotherapeutics. We prioritize neuroblastoma, the most common extracranial pediatric solid tumor, where ~50% of high-risk patients do not survive. Our screen examines all druggable gene knockouts in 18 cell lines (10 neuroblastoma, 8 others) treated with 8 widely used drugs, resulting in 94,320 unique combination-cell line perturbations, which is comparable to the largest existing drug combination screens. Using dense drug-drug rescreening, we find that the top CRISPR-nominated drug combinations are more synergistic than standard-of-care combinations, suggesting existing combinations could be improved. As proof of principle, we discover that inhibition of PRKDC, a component of the non-homologous end-joining pathway, sensitizes high-risk neuroblastoma cells to the standard-of-care drug doxorubicin in vitro and in vivo using patient-derived xenograft (PDX) models. Our findings provide a valuable resource and demonstrate the feasibility of using targeted CRISPR knockout to discover combinations with common chemotherapeutics, a methodology with application across all cancers.

Summary: Background: Although immunomodulators have established benefit against the new coronavirus disease (COVID-19) in general, it is uncertain whether such agents improve outcomes without increasing the risk of secondary infections in the specific subgroup of previously immunocompromised patients. We assessed the effect of immunomodulators on outcomes of immunocompromised patients hospitalized for COVID-19. Methods: The protocol was prospectively registered with PROSPERO (CRD42022335397). MEDLINE, Cochrane Central Register of Controlled Trials and references of relevant articles were searched up to 01-06-2022. Authors of potentially eligible randomized controlled trials were contacted to provide data on immunocompromised patients randomized to immunomodulators vs control (i.e., placebo or standard-of-care). Findings: Eleven randomized controlled trials involving 397 immunocompromised patients hospitalized for COVID-19 were included. Ten trials had low risk of bias. There was no difference between immunocompromised patients randomized to immunomodulators vs control regarding mortality [30/182 (16.5%) vs 41/215 (19.1%); RR 0.93, 95% CI 0.61–1.41; p = 0.74], secondary infections (RR 1.00, 95% CI 0.64–1.58; p = 0.99) and change in World Health Organization ordinal scale from baseline to day 15 (weighed mean difference 0.27, 95% CI -0.09–0.63; p = 0.15). In subgroup analyses including only patients with hematologic malignancy, only trials with low risk of bias, only trials administering IL-6 inhibitors, or only trials administering immunosuppressants, there was no difference between comparators regarding mortality. Interpretation: Immunomodulators, compared to control, were not associated with harmful or beneficial outcomes, including mortality, secondary infections, and change in ordinal scale, when administered to immunocompromised patients hospitalized for COVID-19. Funding: Hellenic Foundation for Research and Innovation.

Implication Statement Assessment of clinical teachers is a requirement by family medicine residency programs in Canada. This facilitates feedback to teachers and ensures the curriculum is delivered in an efficient and safe way. To protect resident confidentiality, preceptors often receive their teaching evaluations months to years later. Teachers have requested shorter feedback loops, greater numbers, and more frequent assessments to improve their skills. The preceptor field note (PFN) is a tool that allows learners to evaluate teachers during a single encounter providing more frequent and immediate feedback. This study documents teachers’ and residents’ initial impressions of the first iteration of the PFN.

Coronary atherosclerosis remains a leading cause of morbidity and mortality worldwide. The underlying pathophysiology includes a complex interplay of endothelial dysfunction, lipid accumulation and inflammatory pathways. Multiple structural and inflammatory features of the atherosclerotic lesions have become targets to identify high-risk lesions. Various intracoronary imaging devices have been developed to assess the morphological, biocompositional and molecular profile of the intracoronary atheromata. These techniques guide interventional and therapeutical management and allow the identification and stratification of atherosclerotic lesions. We sought to provide an overview of the inflammatory pathobiology of atherosclerosis, distinct high-risk plaque features and the ability to visualize this process with contemporary intracoronary imaging techniques.

Service-learning integrates community service with academic study. Students apply skills and knowledge in practical situations and reflect on their experiences. Our 3000-level service-learning college course, Leadership Development in Agricultural Sciences, involved student-led development and execution of a one-day event, “AgCamp,” held on a university campus for high school students. The objective of this study was to evaluate if an intervention (i.e., a service-learning project) changed undergraduate students’ perceptions of their ability to collaborate, ability to lead, and overall perceptions of the usefulness of service-learning. An electronic mixed methods questionnaire-based survey was developed, tested for validity and reliability, and distributed to college students enrolled in Leadership Development in Agricultural Sciences as a pre- and post-test at the beginning and end of two semesters. Data were analyzed in SPSS 26.0 using measures of central tendency and paired sample t-tests. Undergraduate students’ (n = 74) had positive perceptions of service-learning entering the semester, all measures of which improved as a result of their experiences in the course. In comparison with traditional courses, students felt service-learning courses have more societal benefits and should be increasingly offered. Participating in the service-learning project significantly increased students’ comfort working with people from different cultures or backgrounds and empowered them in different ways, including with decision-making. Ultimately, our data indicate that students enjoyed service-learning and received soft skill development through the course. We recommend service-learning be increasingly integrated into post-secondary curriculum.

Background: Standard views in two-dimensional echocardiography are well established but the qualities of acquired images are highly dependent on operator skills and are assessed subjectively. This study was aimed at providing an objective assessment pipeline for echocardiogram image quality by defining a new set of domain-specific quality indicators. Consequently, image quality assessment can thus be automated to enhance clinical measurements, interpretation, and real-time optimization. Methods: We developed deep neural networks for the automated assessment of echocardiographic frames that were randomly sampled from 11,262 adult patients. The private echocardiography dataset consists of 33,784 frames, previously acquired between 2010 and 2020. Unlike non-medical images where full-reference metrics can be applied for image quality, echocardiogram's data are highly heterogeneous and requires blind-reference (IQA) metrics. Therefore, deep learning approaches were used to extract the spatiotemporal features and the image's quality indicators were evaluated against the mean absolute error. Our quality indicators encapsulate both anatomical and pathological elements to provide multivariate assessment scores for anatomical visibility, clarity, depth-gain and foreshortedness. Results: The model performance accuracy yielded 94.4%, 96.8%, 96.2%, 97.4% for anatomical visibility, clarity, depth-gain and foreshortedness, respectively. The mean model error of 0.375±0.0052 with computational speed of 2.52 ms per frame (real-time performance) was achieved. Conclusion: The novel approach offers new insight to the objective assessment of transthoracic echocardiogram image quality and clinical quantification in A4C and PLAX views. It also lays stronger foundations for the operator's guidance system which can leverage the learning curve for the acquisition of optimum quality images during the transthoracic examination.

Abstract Background Several investigations on the microbial diversity and functional properties of the International Space Station (ISS) environment were carried out to understand the influence of spaceflight conditions on the microbial population. However, metagenome-assembled genomes (MAGs) of ISS samples are yet to be generated and subjected to various genomic analyses, including phylogenetic affiliation, predicted functional pathways, antimicrobial resistance, and virulence characteristics. Results In total, 46 MAGs were assembled from 21 ISS environmental metagenomes, in which metaSPAdes yielded 20 MAGs and metaWRAP generated 26 MAGs. Among 46 MAGs retrieved, 18 bacterial species were identified, including one novel genus/species combination (Kalamiella piersonii) and one novel bacterial species (Methylobacterium ajmalii). In addition, four bins exhibited fungal genomes; this is the first-time fungal genomes were assembled from ISS metagenomes. Phylogenetic analyses of five bacterial species showed ISS-specific evolution. The genes pertaining to cell membranes, such as transmembrane transport, cell wall organization, and regulation of cell shape, were enriched. Variations in the antimicrobial-resistant (AMR) and virulence genes of the selected 20 MAGs were characterized to predict the ecology and evolution of biosafety level (BSL) 2 microorganisms in space. Since microbial virulence increases in microgravity, AMR gene sequences of MAGs were compared with genomes of respective ISS isolates and corresponding type strains. Among these 20 MAGs characterized, AMR genes were more prevalent in the Enterobacter bugandensis MAG, which has been predominantly isolated from clinical samples. MAGs were further used to analyze if genes involved in AMR and biofilm formation of viable microbes in ISS have variation due to generational evolution in microgravity and radiation pressure. Conclusions Comparative analyses of MAGs and whole-genome sequences of related ISS isolates and their type strains were characterized to understand the variation related to the microbial evolution under microgravity. The Pantoea/Kalamiella strains have the maximum single-nucleotide polymorphisms found within the ISS strains examined. This may suggest that Pantoea/Kalamiella strains are much more subjective to microgravity changes. The reconstructed genomes will enable researchers to study the evolution of genomes under microgravity and low-dose irradiation compared to the evolution of microbes here on Earth. Video Abstract

In the current era, digital games have been adopted as a part of teaching and learning tools in education and learning. Digital games can motivate users, attract interest in playing games, and learn what is offered. Object-oriented programming (OOP) is one of today's most important programming paradigms because it can help solve team problems in large systems. The conventional teaching system is considered insufficient for educating students in programming courses. Other studies have also shown that students have difficulty learning OOP. In this research, the Fisher-Yates Shuffle Algorithm is used for randomizing the question in educational game form. The results were obtained using the GUESS-18 modelling to get the user acceptance of the educational game OOP learning media using the Fisher- Yates algorithm with a user acceptance rate of 88.45%, meaning the user is “very satisfied” with the game being played. Based on this level of acceptance, it can be concluded that the 2D Platformer game for OOP learning media with the Fisher-Yates algorithm can be an alternative OOP learning media for users.

Background The utility of long-read genome sequencing platforms has been shown in many fields including whole genome assembly, metagenomics, and amplicon sequencing. Less clear is the applicability of long reads to reference-guided human genomics, which is the foundation of genomic medicine. Here, we benchmark available platform-agnostic alignment tools on datasets from nanopore and single-molecule real-time platforms to understand their suitability in producing a genome representation. Results For this study, we leveraged publicly-available data from sample NA12878 generated on Oxford Nanopore and sample NA24385 on Pacific Biosciences platforms. We employed state of the art sequence alignment tools including GraphMap2, long-read aligner (LRA), Minimap2, CoNvex Gap-cost alignMents for Long Reads (NGMLR), and Winnowmap2. Minimap2 and Winnowmap2 were computationally lightweight enough for use at scale, while GraphMap2 was not. NGMLR took a long time and required many resources, but produced alignments each time. LRA was fast, but only worked on Pacific Biosciences data. Each tool widely disagreed on which reads to leave unaligned, affecting the end genome coverage and the number of discoverable breakpoints. No alignment tool independently resolved all large structural variants (1,001–100,000 base pairs) present in the Database of Genome Variants (DGV) for sample NA12878 or the truthset for NA24385. Conclusions These results suggest a combined approach is needed for LRS alignments for human genomics. Specifically, leveraging alignments from three tools will be more effective in generating a complete picture of genomic variability. It should be best practice to use an analysis pipeline that generates alignments with both Minimap2 and Winnowmap2 as they are lightweight and yield different views of the genome. Depending on the question at hand, the data available, and the time constraints, NGMLR and LRA are good options for a third tool. If computational resources and time are not a factor for a given case or experiment, NGMLR will provide another view, and another chance to resolve a case. LRA, while fast, did not work on the nanopore data for our cluster, but PacBio results were promising in that those computations completed faster than Minimap2. Due to its significant burden on computational resources and slow run time, Graphmap2 is not an ideal tool for exploration of a whole human genome generated on a long-read sequencing platform.

Summary: Melanoma is the deadliest form of skin cancer due to its propensity to metastasize. It arises from melanocytes, which are attached to keratinocytes within the basal epidermis. Here, we hypothesize that, in addition to melanocyte-intrinsic modifications, dysregulation of keratinocyte functions could initiate early-stage melanoma cell invasion. We identified the lysolipid sphingosine 1-phosphate (S1P) as a tumor paracrine signal from melanoma cells that modifies the keratinocyte transcriptome and reduces their adhesive properties, leading to tumor invasion. Mechanistically, tumor cell-derived S1P reduced E-cadherin expression in keratinocytes via S1P receptor dependent Snail and Slug activation. All of these effects were blocked by S1P2/3 antagonists. Importantly, we showed that epidermal E-cadherin expression was inversely correlated with the expression of the S1P-producing enzyme in neighboring tumors and the Breslow thickness in patients with early-stage melanoma. These findings support the notion that E-cadherin loss in the epidermis initiates the metastatic cascade in melanoma.

Objectives and importance of the study: Most older Aboriginal peoples live in urban locations. Many of these people were displaced by the policies and practices that produced the Stolen Generations. As a result, access to ‘Country’ and cultural landscapes that are minimally impacted by urbanisation can be limited for older Aboriginal peoples, restricting the health and wellbeing benefits these environments promote. Study type: Qualitative study. Methods: Our study worked collaboratively with Aboriginal traditional cultural knowledge holders to observe and analyse how participation in a ‘cultural camp’ on a Yuwaalaraay sacred site in New South Wales (NSW), Australia, impacted wellbeing and connection to place among older Aboriginal people who were survivors or descendants of the Stolen Generations. Results: Eight participants (three women; five men) attended the cultural camp and took part in the yarning circle. Thematic analysis of a yarning circle uncovered memories of traumatic experiences of institutionalisation, including abuse and loss of Country, community, and culture. Experiences of the cultural camp generated a sense of reconnection, cultural pride, wellbeing and place attachment. The sensory experience of Country emphasised a sense of belonging and healing. Conclusions: Our findings reflect the importance of sensory-led experiences on Country for older urban Aboriginal peoples and reinforce previous evidence on the ‘therapeutic’ aspects of culture and natural landscapes minimally impacted by colonisation. Policies and resources supporting grassroots initiatives such as Aboriginal cultural camps are needed to ensure accessibility for older Aboriginal peoples living in urban places.

Introduction Liposomes have been used as carriers for vaccine adjuvants and antigens due to their inherent biocompatibility and versatility as delivery vehicles. Two vial admixture of protein antigens with liposome-formulated immunostimulatory adjuvants has become a broadly used clinical vaccine preparation approach. Compared to freely soluble antigens, liposome-associated forms can enhance antigen delivery to antigen-presenting cells and co-deliver antigens with adjuvants, leading to improved vaccine efficacy. Areas covered Several antigen-capture strategies for liposomal vaccines have been developed for proteins, peptides, and nucleic acids. Specific antigen delivery methodologies are discussed, including electrostatic adsorption, encapsulation inside the liposome aqueous core, and covalent and non-covalent antigen capture. Expert opinion Several commercial vaccines include active lipid components, highlighting an increasingly prominent role of liposomes and lipid nanoparticles in vaccine development. Utilizing liposomes to associate antigens offers potential advantages, including antigen and adjuvant dose-sparing, co-delivery of antigen and adjuvant to immune cells, and enhanced immunogenicity. Antigen capture by liposomes has demonstrated feasibility in clinical testing. New antigen-capture techniques have been developed and appear to be of interest for vaccine development.

As Lawrence wrote in Sea and Sardinia (1921) “How glad to be on a ship! What a golden hour for the heart of man! Ah if one could sail for ever, on a small quiet, lonely ship, from land to land and isle to isle, and saunter through the spaces of this lovely world, always through the spaces of this lovely world.” (SS 47.27 – 31). He wrote those words as a record of his response to the sea on a steamer travelling from Sicily to Sardinia in January 1921. They are a good example of how conflicted he was about his use of technology. This essay will outline Lawrence’s principal journeys by sea, a significant feature of and expense in his life, how they were necessary to enable him to undertake his “savage pilgrimage” and what Lawrence wrote on board ship, providing examples of the people he met on board ship, and how they and his experience of the sea as opposed to his experiences on land helped or featured in some of his best work.

Introduction: Necrotizing fasciitis is a rare but serious complication that can occur in children with chickenpox, even in immunocompetent children. Prompt diagnosis and immediate surgical treatment are mandatory to avoid potentially fatal complications. Cases presentation: The first patient was an 8-year-old female who presented with a 3-day history of varicella infection. Clinical examination revealed general varicella-related exanthema with necrotizing fasciitis in the left groin. In addition to immediate parenteral antibacterial treatment, emergency surgical exploration was done within 3 hours of arrival to the emergency department. Wide surgical debridement was the initial treatment. Primary closure was achieved using a rectus abdominis myocutaneous (RAM) pedicled flap. The postoperative course was uneventful. The second patient was a 6-year-old male with a history of renal disease who presented with a 3-day history of varicella infection to our emergency department. The patient was in a poor general condition and was initially treated with antibiotics for presumed urosepsis. After approximately 2 hours, he developed clear signs of necrotizing fasciitis in the right hemiscrotum. Surgical debridement was immediately performed, but the patient eventually succumbed due to uncontrolled sepsis. Conclusion: Necrotizing fasciitis is a potential complication of patients with chickenpox. Emergent surgical debridement is mandatory.

Intelligent transport systems (ITS) are pivotal in the development of sustainable and green urban living. ITS is data-driven and enabled by the profusion of sensors ranging from pneumatic tubes to smart cameras which are used to detect and categorise passing vehicles. Simple sensors, such as pneumatic tubes, are successfully deployed for counting passing vehicles but are not useful for vehicle tracking or re-identification. Smart cameras, on the other hand, collect comprehensive information but suffer from occlusion, patchy coverage, and compromised vision in adverse weather and visibility. This work explores a novel ITS data source based on an optical fibre which acts as an uninterrupted length of virtual sensors using a distributed acoustic sensor (DAS) system. Based on real DAS data collected in the field, we first present a study of latent DAS features that uniquely identify a given vehicle, otherwise referred to as the vehicle signature. We formulate a classification problem that examines incoming DAS data to extract vehicle signatures and identify the different types of vehicle. To this end, we implement different classification methods and present a comparative performance analysis that reveals novel insights into the potential role of DAS for ITS applications. This work is a pilot study of DAS for vehicle classification that is driven by real DAS data and validated by promising results where a vehicle’s type is correctly identified with 94% accuracy and the size of a vehicle with 95% accuracy.

Abstract A novel method for the extraction of form factors of unstable particles on the lattice is proposed. The approach is based on the study of two-particle scattering in a static, spatially periodic external field by using a generalization of the Lüscher method in the presence of such a field. It is shown that the resonance form factor is given by the derivative of the resonance pole position in the complex plane with respect to the coupling constant to the external field. Unlike the standard approach, this proposal does not suffer from problems caused by the presence of the triangle diagram.

Fifth Generation Mobile Network (5G) is a heterogeneous network in nature, made up of multiple systems and supported by different technologies. It will be supported by network services such as device-to-device (D2D) communications. This will enable the new use cases to provide access to other services within the network and from third-party service providers (SPs). End-users with their user equipment (UE) will be able to access services ubiquitously from multiple SPs that might share infrastructure and security management, whereby implementing security from one domain to another will be a challenge. This highlights a need for a new and effective security approach to address the security of such a complex system. This article proposes a network service security (NSS) modular framework for 5G and beyond that consists of different security levels of the network. It reviews the security issues of D2D communications in 5G, and it is used to address security issues that affect the users and SPs in an integrated and heterogeneous network such as the 5G enabled D2D communications network. The conceptual framework consists of a physical layer, network access, service and D2D security levels. Finally, it recommends security mechanisms to address the security issues at each level of the 5G-enabled D2D communications network.

Circulating tumour DNA (ctDNA) is a promising biomarker that may better identify stage II colon cancer (CC) patients who will benefit from adjuvant chemotherapy (AC) compared to standard clinicopathological parameters. The DYNAMIC study demonstrated that ctDNA-informed treatment decreased AC utilisation without compromising recurrence free survival, but medical oncologists’ willingness to utilise ctDNA results to inform AC decision is unknown. Medical oncologists from Australia, Canada and New Zealand were presented with clinical vignettes for stage II CC comprised of two variables with three levels each (age: ≤50, 52–69, ≥70 years; and clinicopathological risk of recurrence: low, intermediate, high) and were queried about ctDNA testing and treatment recommendations based on results. Sixty-four colorectal oncologists completed at least one vignette (all vignettes, n = 59). The majority of oncologist were Australian (70%; Canada: n = 13; New Zealand: n = 6) and had over 10 years of clinical experience (n = 41; 64%). The proportion of oncologists requesting ctDNA testing exceeded 80% for all vignettes, except for age ≥ 70 and low-risk disease (63%). Following a positive ctDNA result, the proportion of oncologists recommending AC (p < 0.01) and recommending oxaliplatin-based doublet (p < 0.01) increased in all vignettes. Following a negative result, the proportion recommending AC decreased in all intermediate and high-risk vignettes (p < 0.01).

The autonomic nervous systems densely innervate the pancreas, but its contribution to pancreatic ductal adenocarcinoma (PDAC) progression is not fully understood. Here, the authors characterize the pattern of sympathetic innervation by 3D imaging in a murine model of PDAC and show that sympathectomy aggravates cancer progression.

We report a case of metastatic pancreatic neuroendocrine carcinoma associated with paraneoplastic Cushing’s syndrome, successively treated with five lines of treatment (platin-etoposide, LV5FU2-dacarbazine, FOLFIRINOX, pembrolizumab, and paclitaxel) and anti-secretory treatment. Circulating-free DNA (cfDNA) was analysed at each morphological evaluation starting from the second-line treatment. cfDNA changes were well correlated with the disease course, and cfDNA may be used as a predictive marker and/or as an early marker of response. In addition, the absolute count of atypical cells was elevated upon disease progression.

Abstract Background Lymphopenia is a hallmark of severe coronavirus disease 19 (COVID-19). Similar alterations have been described in bacterial sepsis and therapeutic strategies targeting T cell function such as recombinant human interleukin 7 (rhIL-7) have been proposed in this clinical context. As COVID-19 is a viral sepsis, the objectives of this study were to characterize T lymphocyte response over time in severe COVID-19 patients and to assess the effect of ex vivo administration of rhIL-7. Results Peripheral blood mononuclear cells from COVID-19 patients hospitalized in intensive care unit (ICU) were collected at admission and after 20 days. Transcriptomic profile was evaluated through NanoString technology. Inhibitory immune checkpoints expressions were determined by flow cytometry. T lymphocyte proliferation and IFN-γ production were evaluated after ex vivo stimulation in the presence or not of rhIL-7. COVID-19 ICU patients were markedly lymphopenic at admission. Mononuclear cells presented with inhibited transcriptomic profile prevalently with impaired T cell activation pathways. CD4 + and CD8 + T cells presented with over-expression of co-inhibitory molecules PD-1, PD-L1, CTLA-4 and TIM-3. CD4 + and CD8 + T cell proliferation and IFN-γ production were markedly altered in samples collected at ICU admission. These alterations, characteristic of a T cell exhaustion state, were more pronounced at ICU admission and alleviated over time. Treatment with rhIL-7 ex vivo significantly improved both T cell proliferation and IFN-γ production in cells from COVID-19 patients. Conclusions Severe COVID-19 patients present with features of profound T cell exhaustion upon ICU admission which can be reversed ex vivo by rhIL-7. These results reinforce our understanding of severe COVID-19 pathophysiology and opens novel therapeutic avenues to treat such critically ill patients based of immunomodulation approaches. Defining the appropriate timing for initiating such immune-adjuvant therapy in clinical setting and the pertinent markers for a careful selection of patients are now warranted to confirm the ex vivo results described so far. Trial registration ClinicalTrials.gov identifier: NCT04392401 Registered 18 May 2020, http:// clinicaltrials.gov/ct2/show/NCT04392401.

ABSTRACT: Objectives: To describe the characteristics of nosocomial cases of Ebola virus disease (EVD) in the Democratic Republic of the Congo between July 2018 and May 2020 in order to inform future interventions. Methods: Nosocomial cases of EVD were identified during outbreak response surveillance, and a retrospective analysis of cases was conducted according to demographic characteristics and type of health facility (HF). Results: Of 3481 cases of EVD, 579 (16.6%) were nosocomial. Of these, 332 cases occurred in women (57.3%). Patients and visitors accounted for 419 cases (72.4%), of which 79 (18.9%) were aged 6–≤18 years and 108 (25.8%) were aged ≤5 years. Health workers (HWs) accounted for the remaining 160 (27.6%) nosocomial cases. The case fatality rate (CFR) for HWs (66/160, 41.3%) was significantly lower than the CFR for patients and visitors (292/419, 69.7%) (P39 beds) had the highest prevalence of nosocomial EVD (148/579, 25.6%). Among HFs with at least one case of nosocomial infection, 50.0% (98/196) were privately owned. Conclusions: Nurses and traditional healers should be targeted for infection prevention and control training, and supportive supervision should be provided to HFs to mitigate EVD transmission.

Organizational intelligence (IO) has raised an interest from academia and the organizational environment. From the literature, studies of the IO are evidenced that reveal the benefits of its management in the organizational field. From the approach to a definition of the IO and the conception of a management model, the present work aims to develop a methodology to evaluate the IO, identifying precisely and in detail the dimensional elements that constitute it. The measurement of this variable in organizations is relevant for control and management processes, identification of improvements and closing gaps, among others, to maintain compliance with the current demanding business environment

CX-5461 recently progressed through phase I clinical trial as a first-inhuman inhibitor of RNA-POL I. Here, the authors demonstrate that CX-5461 synergizes with topoisomerase I inhibitors to inhibit neuroblastoma cells and that its primary target in this disease is topoisomerase II beta and not RNA-POL I.

In “Accumulated Mail” (1925) Lawrence wrote that “it’s no fun, writing unpopular books” (RDP 239). But he was keen to see his books sold. When Lady Chatterley’s Lover (1928) was widely pirated he sought to beat the pirates at their own game by arranging for the so-called “Paris popular edition” of Lady Chatterley’s Lover to be published there in 1929 with an initial print run of 3000 copies. It was quickly followed by second and third printings of the same quantity. Lawrence’s wish is confirmed by Frieda Lawrence in her “open letter” prefixed to the Odyssey Press edition of the book published in 1933, where she stated that Lawrence “wanted to reach the people he voiced and from whom he came.” This paper will explore the intended audience of the Florence first edition, bearing in mind the print run of only 1000 copies. It will then look at Lawrence’s view of the piracies, with some illustrations of those, and then consider whether the Paris popular edition marked a rethink by Lawrence of who the intended audience was or whether it was a natural progression from the Florence first edition. Finally, comparisons will be made with other editions of Lawrence’s books that were considered to be “popular” editions.

The end user’s Quality of Experience (QoE) will be improved while accessing services in Fifth Generation Mobile Network (5G), supported by enhanced security and privacy. The security guarantees offered by the Authentication and Key Agreement (AKA) protocols will be depended upon by end users and network operators. The AKA protocols have been standardized for 5G networks, and the Extensible Authentication Protocol (EAP)-AKA’ protocol is one of the main authentication mechanisms that has been specified for User Equipment (UE) and network mutual authentication. This article models the EAP-AKA’ protocol and conducts an extensive formal verification of the EAP-AKA’ protocol as defined in the 5G security standard to determine whether the protocol is verifiably secure for 5G. It provides a security evaluation of the EAP-AKA’ protocol based on the current 5G specifications using ProVerif, a security protocol proof verifier. It also presents security properties that support the security verification, as well as quantitative properties that are used to assess the protocol’s performance. Finally, it compares the EAP-AKA’ and 5G-AKA protocols’ security and performance results.

Microsatellite instability (MSI) is a molecular signature of mismatch repair deficiency (dMMR), a predictive marker of immune checkpoint inhibitor therapy response. Despite its recognized pan-cancer value, most methods only support detection of this signature in colorectal cancer. In addition to the tissue-specific differences that impact the sensitivity of MSI detection in other tissues, the performance of most methods is also affected by patient ethnicity, tumor content, and other sample-specific properties. These limitations are particularly important when only tumor samples are available and restrict the performance and adoption of MSI testing. Here we introduce MSIdetect, a novel solution for NGS-based MSI detection. MSIdetect models the impact of indel burden and tumor content on read coverage at a set of homopolymer regions that we found are minimally impacted by sample-specific factors. We validated MSIdetect in 139 Formalin-Fixed Paraffin-Embedded (FFPE) clinical samples from colorectal and endometrial cancer as well as other more challenging tumor types, such as glioma or sebaceous adenoma or carcinoma. Based on analysis of these samples, MSIdetect displays 100% specificity and 96.3% sensitivity. Limit of detection analysis supports that MSIdetect is sensitive even in samples with relatively low tumor content and limited microsatellite instability. Finally, the results obtained using MSIdetect in tumor-only data correlate well (R=0.988) with what is obtained using tumor-normal matched pairs, demonstrating that the solution addresses the challenges posed by MSI detection from tumor-only data. The accuracy of MSI detection by MSIdetect in different cancer types coupled with the flexibility afforded by NGS-based testing will support the adoption of MSI testing in the clinical setting and increase the number of patients identified that are likely to benefit from immune checkpoint inhibitor therapy.