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Abstract Background The extreme environment of the International Space Station (ISS) puts selective pressure on microorganisms unintentionally introduced during its 20+ years of service as a low-orbit science platform and human habitat. Such pressure leads to the development of new features not found in the Earth-bound relatives, which enable them to adapt to unfavorable conditions. Results In this study, we generated the functional annotation of the genomes of five newly identified species of Gram-positive bacteria, four of which are non-spore-forming and one spore-forming, all isolated from the ISS. Using a deep-learning based tool—deepFRI—we were able to functionally annotate close to 100% of protein-coding genes in all studied species, overcoming other annotation tools. Our comparative genomic analysis highlights common characteristics across all five species and specific genetic traits that appear unique to these ISS microorganisms. Proteome analysis mirrored these genomic patterns, revealing similar traits. The collective annotations suggest adaptations to life in space, including the management of hypoosmotic stress related to microgravity via mechanosensitive channel proteins, increased DNA repair activity to counteract heightened radiation exposure, and the presence of mobile genetic elements enhancing metabolism. In addition, our findings suggest the evolution of certain genetic traits indicative of potential pathogenic capabilities, such as small molecule and peptide synthesis and ATP-dependent transporters. These traits, exclusive to the ISS microorganisms, further substantiate previous reports explaining why microbes exposed to space conditions demonstrate enhanced antibiotic resistance and pathogenicity. Conclusion Our findings indicate that the microorganisms isolated from ISS we studied have adapted to life in space. Evidence such as mechanosensitive channel proteins, increased DNA repair activity, as well as metallopeptidases and novel S-layer oxidoreductases suggest a convergent adaptation among these diverse microorganisms, potentially complementing one another within the context of the microbiome. The common genes that facilitate adaptation to the ISS environment may enable bioproduction of essential biomolecules need during future space missions, or serve as potential drug targets, if these microorganisms pose health risks. Video Abstract

Abstract Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that causes severe nosocomial infections in susceptible individuals due to the emergence of multidrug-resistant strains. There are no approved vaccines against P. aeruginosa infections nor candidates in active clinical development, highlighting the need for novel candidates and strategies. Using a cell-blot proteomic approach, we reproducibly identified 49 proteins involved in interactions with human lung epithelial cells across four P. aeruginosa strains. Among these were cell division protein FtsZ and outer membrane protein OpmH. Escherichia coli BL21 cells overexpressing recombinant FtsZ or rOpmH showed a 66- and 15-fold increased ability to attach to 16HBE14o− cells, further supporting their involvement in host cell attachment. Both antigens led to proliferation of NK and CD8+ cytotoxic T cells, significant increases in the production of IFN-γ, IL-17A, TNF and IL-4 in immunised mice and elicited strong antigen-specific serological IgG1 and IgG2c responses. Immunisation with FtsZ significantly reduced bacterial burden in the lungs by 1.9-log CFU and dissemination to spleen by 1.8-log CFU. The protective antigen candidate, FtsZ, would not have been identified by traditional approaches relying on either virulence mechanisms or sequence-based predictions, opening new avenues in the development of an anti-P. aeruginosa vaccine.

DIII-D research is being accelerated by leveraging high performance computing (HPC) and data resources available through the National Energy Research Scientific Computing Center (NERSC) Superfacility initiative. As part of this initiative, a high-resolution, fully automated, whole discharge kinetic equilibrium reconstruction workflow was developed that runs at the NERSC for most DIII-D shots in under 20 min. This has eliminated a long-standing research barrier and opened the door to more sophisticated analyses, including plasma transport and stability. These capabilities would benefit from being automated and executed within the larger Department of Energy Advanced Scientific Computing Research program’s Integrated Research Infrastructure (IRI) framework. The goal of IRI is to empower researchers to meld DOE’s world-class research tools, infrastructure, and user facilities seamlessly and securely in novel ways to radically accelerate discovery and innovation. For transport, we are looking at producing flux matched profiles and also using particle tracing to predict fast ion heat deposition from neutral beam injection before a shot takes place. Our starting point for evaluating plasma stability focuses on the pedestal limits that must be navigated to achieve better confinement. This information is meant to help operators run more effective experiments, so it needs to be available rapidly inside the DIII-D control room. So far this has been achieved by ensuring the data is available with existing tools, but as more novel results are produced new visualization tools must be developed. In addition, all of the high-quality data we have generated has been collected into databases that can unlock even deeper insights. This has already been leveraged for model and code validation studies as well as for developing AI/ML surrogates. The workflows developed for this project are intended to serve as prototypes that can be replicated on other experiments and can be run to provide timely and essential information for ITER, as well as next stage fusion power plants.

Biodegradable polymeric coatings are being explored as a preventive strategy for orthopaedic device-related infection. In this study, titanium surfaces (Ti) were coated with poly-D,L-lactide (PDLLA, (P)), polyethylene-glycol poly-D,L-lactide (PEGylated-PDLLA, (PP20)), or multi-layered PEGylated-PDLLA (M), with or without 1 % silver sulfadiazine. The aim was to evaluate their cytocompatibility, resistance to Staphylococcus aureus biofilm formation, and their potential to enhance the susceptibility of any biofilm formed to antibiotics. Using automated high-content screening confocal microscopy, biofilm formation of a clinical methicillin-resistant Staphylococcus aureus (MRSA) isolate expressing GFP was quantified, along with isogenic mutants that were unable to form polysaccharidic or proteinaceous biofilm matrices. The results showed that PEGylated-PDLLA coatings exhibited significant antibiofilm properties, with M showing the highest effect. This inhibitory effect was stronger in S. aureus biofilms with a matrix composed of proteins compared to those with an exopolysaccharide (PIA) biofilm matrix. Our data suggest that the antibiofilm effect may have been due to (i) inhibition of the initial attachment through microbial surface components recognising adhesive matrix molecules (MSCRAMMs), since PEG reduces protein surface adsorption via surface hydration layer and steric repulsion; and (ii) mechanical disaggregation and dispersal of microcolonies due to the bioresorbable/degradable nature of the polymers, which undergo hydration and hydrolysis over time. The disruption of biofilm morphology by the PDLLA-PEG co-polymers increased S. aureus susceptibility to antibiotics like rifampicin and fusidic acid. Adding 1 % AgSD provided additional early bactericidal effects on both biofilm and planktonic S. aureus. Additionally, the coatings were cytocompatible with immune cells, indicating their potential to enhance bacterial clearance and reduce bacterial colonisation of titanium-based orthopaedic biomaterials.

Abstract Comprehensive studies comparing impacts of building and street levels interventions on air temperature at metropolitan scales are still lacking despite increased urban heat‐related mortality and morbidity. We therefore model the impact of 9 interventions on air temperatures at 2 m during 2 hot days from the summer 2018 in the Greater London Authority area using the WRF BEP‐BEM climate model. We find that on average cool roofs most effectively reduce temperatures (∼−1.2°C), outperforming green roofs (∼0°C), solar panels (∼−0.5°C) and street level vegetation (∼−0.3°C). Application of air conditioning across London (United Kingdom) increases air temperatures by ∼+0.15°C. A practicable deployment of solar panels could cover its related energetic consumption. Current practicable deployments of green roofs and solar panels are ineffective at large scale reduction of temperatures. We provide a detailed decomposition of the surface energy balance to explain changes in air temperature and guide future decision‐making.

Abstract The properties of superheavy elements probe extremes of physics and chemistry. They are synthesised at accelerator laboratories using nuclear fusion, where two atomic nuclei collide, stick together (capture), then with low probability evolve to a compact superheavy nucleus. The fundamental microscopic mechanisms controlling fusion are not fully understood, limiting predictive capability. Even capture, considered to be the simplest stage of fusion, is not matched by models. Here we show that collisions of 40Ca with 208Pb, experience an ‘explosion’ of mass and charge transfers between the nuclei before capture, with unexpectedly high probability and complexity. Ninety different partitions of the protons and neutrons between the projectile-like and target-like nuclei are observed. Since each is expected to have a different probability of fusion, the early stages of collisions may be crucial in superheavy element synthesis. Our interpretation challenges the current view of fusion, explains both the successes and failures of current capture models, and provides a framework for improved models.

Abstract Background Both eating disorder (ED) prevention and weight management interventions often focus on the thin ideal. Yet, many Black and Latina women do not view thinness as their body ideal. This study used focus groups to investigate the influence of race, ethnicity, and culture on appearance ideals and inform the cultural adaptation and integration of two established programs addressing EDs and weight management: the Body Project, and Healthy Weight Interventions. Methods White (n = 10), Black (n = 14), and Latina (n = 6) women participated in racially and ethnically homogenous focus groups. Results Thematic analysis identified several themes, including: (1) diverse beauty standards across groups, (2) lack of acknowledgement of racialized beauty standards in prevention and cultural appropriation, (3) culturally-specific impacts of standards, (4) harm of appearance-related comments, (5) limitations of available resources, (6) stigma/minimization of mental health, (7) barriers to inclusive programming, and (8) facilitators of inclusive programming. Conclusions Results suggested that current programs’ emphasis on thinness limit their relevance for women of color, and perpetuate the misconception that EDs primarily affect White women. Findings highlight the need for culturally responsive prevention.

Abstract With the advent of long-term human habitation in space and on the moon, understanding how the built environment microbiome of space habitats differs from Earth habitats, and how microbes survive, proliferate and spread in space conditions, is becoming more important. The microbial tracking mission series has been monitoring the microbiome of the International Space Station (ISS) for almost a decade. During this mission series, six unique strains of Gram-stain-positive bacteria, including two spore-forming and three non-spore-forming species, were isolated from the environmental surfaces of the ISS. The analysis of their 16S rRNA gene sequences revealed > 99% similarities with previously described bacterial species. To further explore their phylogenetic affiliation, whole genome sequencing was undertaken. For all strains, the gyrB gene exhibited

Abstract Public and Patient Involvement in research is becoming a requirement on most research funding applications; this includes both healthcare and lab-based research. Whilst case studies and practical guides have been developed and are well documented for PPI in healthcare research, there is very little guidance available for PPI in lab-based research. In this piece we discuss our experience of how we have successfully involved patients in our translational cancer research, which is focused on developing personalised treatment for high-grade serous ovarian cancer. We discuss the benefits it has made to both our research and to us as researchers. The patients involved write about their experience, what they enjoyed, and the benefits they felt. Although PPI is quite topical and is being widely discussed, there is hesitancy among researchers, especially those in lab-based research about getting started because of a lack of practical guidance about how to implement it. Here, we have shared our experience, hopefully providing a practical example of how PPI can be incorporated into a lab-based research project.

ABSTRACTA comprehensive microbial surveillance was conducted at NASA’s Mars 2020 spacecraft assembly facility (SAF), where whole-genome sequencing (WGS) of 110 bacterial strains was performed. One isolate, designated 179-BFC-A-HST, exhibited less than 80% average nucleotide identity (ANI) to known species, suggesting a novel organism. This strain demonstrated high-level resistance [minimum inhibitory concentration (MIC) >256 mg/L] to third-generation cephalosporins, including ceftazidime, cefpodoxime, combination ceftazidime/avibactam, and the fourth-generation cephalosporin cefepime. The results of a comparative genomic analysis revealed that 179-BFC-A-HST is most closely related to Virgibacillus halophilus 5B73CT, sharing an ANI of 78.7% and a digital DNA-DNA hybridization (dDDH) value of 23.5%, while their 16S rRNA gene sequences shared 97.7% nucleotide identity. Based on these results and the recent recognition that the genus Virgibacillus is polyphyletic, strain 179-BFC-A-HST is proposed as a novel species of a novel genus, Tigheibacillus jepli gen. nov., sp. nov (type strain 179-BFC-A-HST = DSM 115946T = NRRL B-65666T), and its closest neighbor, V. halophilus, is proposed to be reassigned to this genus as Tigheibacillus halophilus comb. nov. (type strain 5B73CT = DSM 21623T = JCM 21758T = KCTC 13935T). It was also necessary to reclassify its second closest neighbor Virgibacillus soli, as a member of a novel genus Paracerasibacillus, reflecting its phylogenetic position relative to the genus Cerasibacillus, for which we propose Paracerasibacillus soli comb. nov. (type strain CC-YMP-6T = DSM 22952T = CCM 7714T). Within Amphibacillaceae (n = 64), P. soli exhibited 11 antibiotic resistance genes (ARG), while T. jepli encoded for 3, lacking any known β-lactamases, suggesting resistance from variant penicillin-binding proteins, disrupting cephalosporin efficacy. P. soli was highly resistant to azithromycin (MIC >64 mg/L) yet susceptible to cephalosporins and penicillins.IMPORTANCEThe significance of this research extends to understanding microbial survival and adaptation in oligotrophic environments, such as those found in SAF. Whole-genome sequencing of several strains isolated from Mars 2020 mission assembly cleanroom facilities, including the discovery of the novel species Tigheibacillus jepli, highlights the resilience and antimicrobial resistance (AMR) in clinically relevant antibiotic classes of microbes in nutrient-scarce settings. The study also redefines the taxonomic classifications within the Amphibacillaceae family, aligning genetic identities with phylogenetic data. Investigating ARG and virulence factors (VF) across these strains illuminates the microbial capability for resistance under resource-limited conditions while emphasizing the role of human-associated VF in microbial survival, informing sterilization practices and microbial management in similar oligotrophic settings beyond spacecraft assembly cleanrooms such as pharmaceutical and medical industry cleanrooms.

Abstract During the construction and assembly of the Mars 2020 mission components at two different NASA cleanrooms, several fungal strains were isolated. Based on their colony morphology, two strains that showed yeast-like appearance were further characterized for their phylogenetic position. The species-level classification of these two novel strains, using traditional colony and cell morphology methods combined with the phylogenetic reconstructions using multi-locus sequence analysis (MLSA) based on several gene loci (ITS, LSU, SSU, RPB1, RPB2, CYTB and TEF1), and whole genome sequencing (WGS) was carried out. This polyphasic taxonomic approach supported the conclusion that the two basidiomycetous yeasts belong to hitherto undescribed species. The strain FJI-L2-BK-P3T, isolated from the Jet Propulsion Laboratory Spacecraft Assembly Facility, was placed in the Naganishia albida clade (Filobasidiales, Tremellomycetes), but is genetically and physiologically different from other members of the clade. Another yeast strain FKI-L6-BK-PAB1T, isolated from the Kennedy Space Center Payload Hazardous and Servicing Facility, was placed in the genus Cystobasidium (Cystobasidiales, Cystobasidiomycetes) and is distantly related to C. benthicum. Here we propose two novel species with the type strains, Naganishia kalamii sp. nov. (FJI-L2-BK-P3T = NRRL 64466 = DSM 115730) and Cystobasidium onofrii sp. nov. (FKI-L6-BK-PAB1T = NRRL 64426 = DSM 114625). The phylogenetic analyses revealed that single gene phylogenies (ITS or LSU) were not conclusive, and MLSA and WGS-based phylogenies were more advantageous for species discrimination in the two genera. The genomic analysis predicted proteins associated with dehydration and desiccation stress-response and the presence of genes that are directly related to osmotolerance and psychrotolerance in both novel yeasts described. Cells of these two newly-described yeasts were exposed to UV-C radiation and compared with N. onofrii, an extremophilic UV-C resistant cold-adapted Alpine yeast. Both novel species were UV resistant, emphasizing the need for collecting and characterizing extremotolerant microbes, including yeasts, to improve microbial reduction techniques used in NASA planetary protection programs.

The endomembrane system is essential for healthy cell function, with the various compartments carrying out a large number of specific biochemical reactions. To date, almost all of our understanding of the endomembrane system has come from the study of cultured cells growing as monolayers. However, monolayer-grown cells only poorly represent the environment encountered by cells in the human body. As a first step to address this disparity, we have developed a platform that allows us to investigate and quantify changes to the endomembrane system in three-dimensional (3D) cell models, in an automated and highly systematic manner. HeLa Kyoto cells were grown on custom-designed micropatterned 96-well plates to facilitate spheroid assembly in the form of highly uniform arrays. Fully automated high-content confocal imaging and analysis were then carried out, allowing us to measure various spheroid-, cellular- and subcellular-level parameters relating to size and morphology. Using two drugs known to perturb endomembrane function, we demonstrate that cell-based assays can be carried out in these spheroids, and that changes to the Golgi apparatus and endosomes can be quantified from individual cells within the spheroids. We also show that image texture measurements are useful tools to discriminate cellular phenotypes. The automated platform that we show here has the potential to be scaled up, thereby allowing large-scale robust screening to be carried out in 3D cell models.

Abstract Background Monitoring the adaptation of microorganisms to the extreme environment of the International Space Station (ISS) is crucial to understanding microbial evolution and infection prevention. Acinetobacter pittii is an opportunistic nosocomial pathogen, primarily impacting immunocompromised patients, that was recently isolated from two missions aboard the ISS. Results Here, we report how ISS-associated A. pittii (n = 20 genomes) has formed its own genetically and functionally discrete clade distinct from most Earth-bound isolates (n = 291 genomes). The antimicrobial susceptibility testing of ISS strains and two related clinical isolates demonstrated that ISS strains acquired more resistance, specifically with regard to expanded-spectrum cephalosporins, despite no prediction of increased resistance based on genomic analysis of resistance genes. By investigating 402 longitudinal environmental and host-associated ISS metagenomes, we observed that viable A. pittii is increasing in relative abundance and therefore potentially exhibiting succession, being identified in >2X more metagenomic samples in back-to-back missions. ISS strains additionally contain functions that enable them to survive in harsh environments, including the transcriptional regulator LexA. Via a genome-wide association study, we identified a high level of mutational burden in methionine sulfoxide reductase genes relative to the most closely related Earth strains. Conclusions Overall, these results indicated a step forward in understanding how microorganisms might evolve and alter their antibiotic resistance phenotype in extreme, resource-limited, human-built environments. Video Abstract

Hereditary spastic paraplegias (HSPs) are a heterogeneous group of mono-genetic inherited neurological disorders, whose primary manifestation is the disruption of the pyramidal system, observed as a progressive impaired gait and leg spasticity in patients. Despite the large list of genes linked to this group, which exceeds 80 loci, the number of cellular functions which the gene products engage is relatively limited, among which endoplasmic reticulum (ER) morphogenesis appears central. Mutations in genes encoding ER-shaping proteins are the most common cause of HSP, highlighting the importance of correct ER organisation for long motor neuron survival. However, a major bottleneck in the study of ER morphology is the current lack of quantitative methods, with most studies to date reporting, instead, on qualitative changes. Here, we describe and apply a quantitative image-based screen to identify genetic modifiers of ER organisation using a mammalian cell culture system. An analysis reveals significant quantitative changes in tubular ER and dense sheet ER organisation caused by the siRNA-mediated knockdown of HSP-causing genes ATL1 and RTN2. This screen constitutes the first attempt to examine ER distribution in cells in an automated and high-content manner and to detect genes which impact ER organisation.

A single strain from the family Paenibacillaceae was isolated from the wall behind the Waste Hygiene Compartment aboard the International Space Station (ISS) in April 2018, as part of the Microbial Tracking mission series. This strain was identified as a gram-positive, rod-shaped, oxidase-positive, catalase-negative motile bacterium in the genus Cohnella, designated as F6_2S_P_1T. The 16S sequence of the F6_2S_P_1T strain places it in a clade with C. rhizosphaerae and C. ginsengisoli, which were originally isolated from plant tissue or rhizosphere environments. The closest 16S and gyrB matches to strain F6_2S_P_1T are to C. rhizosphaerae with 98.84 and 93.99% sequence similarity, while a core single-copy gene phylogeny from all publicly available Cohnella genomes places it as more closely related to C. ginsengisoli. Average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) values to any described Cohnella species are

The Gemini South telescope is now equipped with a new high-resolution spectrograph called the Gemini High-resolution Optical SpecTrograph (GHOST). This instrument provides high-efficiency, high-resolution spectra covering 347–1060 nm in a single exposure of either one or two targets simultaneously, along with precision radial velocity spectroscopy utilizing an internal calibration source. It can operate at a spectral element resolving power of either 76,000 or 56,000, and can reach a signal-to-noise ratio of ∼5 in a 1 hr exposure on a V ∼ 20.8 mag target in median site seeing and dark skies (per resolution element). GHOST was installed on-site in 2022 June, and we report performance after full integration to queue operations in 2023 November, in addition to scientific results enabled by the integration observing runs. These results demonstrate the ability to observe a wide variety of bright and faint targets with high efficiency and precision. With GHOST, new avenues to explore high-resolution spectroscopy have opened up to the astronomical community. These are described, along with the planned and potential upgrades to the instrument.

Abstract Assessing genuine extracellular vesicle (EV) uptake is crucial for understanding the functional roles of EVs. This study measured the bona fide labelling of EVs utilising two commonly used fluorescent dyes, PKH26 and C5-maleimide-Alexa633. MCF7 EVs tagged with mEmerald-CD81 were isolated from conditioned media by size exclusion chromatography (SEC) and characterised using Nanoparticle Tracking Analysis (NTA), Transmission Electron Microscopy (TEM), MACsPlex immunocapture assay and immunoblots. These fluorescently tagged EVs were subsequently stained with C5-maleimide-Alexa633 or PKH26, according to published protocols. Colocalisation of dual-labelled EVs was assessed by confocal microscopy and quantified using the Rank-Weighted Colocalisation (RWC) algorithm. We observed strikingly poor colocalisation between mEmerald-CD81-tagged EVs and C5-Maleimide-Alexa633 (5.4% ± 1.8) or PKH26 (4.6% ± 1.6), that remained low even when serum was removed from preparations. Our data confirms previous work showing that some dyes form contaminating aggregates. Furthermore, uptake studies showed that maleimide and mEmerald-CD81-tagged EVs can be often located into non-overlapping subcellular locations. By using common methods to isolate and stain EVs we observed that most EVs remained unstained and most dye signal does not appear to be EV associated. Our work shows that there is an urgent need for optimisation and standardisation in how EV researchers use these tools to assess genuine EV signals.

Abstract Universal stress proteins (USPs) are ubiquitously expressed in bacteria, archaea, and eukaryotes and play a lead role in adaptation to environmental conditions. They enable adaptation of bacterial pathogens to the conditions encountered in the human niche, including hypoxia, oxidative stress, osmotic stress, nutrient deficiency, or acid stress, thereby facilitating colonization. We previously reported that all six USP proteins encoded within a low‐oxygen activated (lxa) locus in Burkholderia cenocepacia showed increased abundance during chronic colonization of the cystic fibrosis (CF) lung. However, the role of USPs in chronic cystic fibrosis infection is not well understood. Structural modeling identified surface arginines on one lxa‐encoded USP, USP76, which suggested it mediated interactions with heparan sulfate. Using mutants derived from the B. cenocepacia strain, K56‐2, we show that USP76 is involved in host cell attachment. Pretreatment of lung epithelial cells with heparanase reduced the binding of the wild‐type and complement strains but not the Δusp76 mutant strain, indicating that USP76 is directly or indirectly involved in receptor recognition on the surface of epithelial cells. We also show that USP76 is required for growth and survival in many conditions associated with the CF lung, including acidic conditions and oxidative stress. Moreover, USP76 also has a role in survival in macrophages isolated from people with CF. Overall, while further elucidation of the exact mechanism(s) is required, we can conclude that USP76, which is upregulated during chronic infection, is involved in bacterial survival within CF macrophages, a hallmark of Burkholderia infection.

Milk-derived peptides are known to confer anti-inflammatory effects. We hypothesised that milk-derived cell-penetrating peptides might modulate inflammation in useful ways. Using computational techniques, we identified and synthesised peptides from the milk protein Alpha-S1-casein that were predicted to be cell-penetrating using a machine learning predictor. We modified the interpretation of the prediction results to consider the effects of histidine. Peptides were then selected for testing to determine their cell penetrability and anti-inflammatory effects using HeLa cells and J774.2 mouse macrophage cell lines. The selected peptides all showed cell penetrating behaviour, as judged using confocal microscopy of fluorescently labelled peptides. None of the peptides had an effect on either the NF-κB transcription factor or TNFα and IL-1β secretion. Thus, the identified milk-derived sequences have the ability to be internalised into the cell without affecting cell homeostatic mechanisms such as NF-κB activation. These peptides are worthy of further investigation for other potential bioactivities or as a naturally derived carrier to promote the cellular internalisation of other active peptides.

Abstract Background In mammalian cells the endoplasmic reticulum (ER) comprises a highly complex reticular morphology that is spread throughout the cytoplasm. This organelle is of particular interest to biologists, as its dysfunction is associated with numerous diseases, which often manifest themselves as changes to the structure and organisation of the reticular network. Due to its complex morphology, image analysis methods to quantitatively describe this organelle, and importantly any changes to it, are lacking. Results In this work we detail a methodological approach that utilises automated high-content screening microscopy to capture images of cells fluorescently-labelled for various ER markers, followed by their quantitative analysis. We propose that two key metrics, namely the area of dense ER and the area of polygonal regions in between the reticular elements, together provide a basis for measuring the quantities of rough and smooth ER, respectively. We demonstrate that a number of different pharmacological perturbations to the ER can be quantitatively measured and compared in our automated image analysis pipeline. Furthermore, we show that this method can be implemented in both commercial and open-access image analysis software with comparable results. Conclusions We propose that this method has the potential to be applied in the context of large-scale genetic and chemical perturbations to assess the organisation of the ER in adherent cell cultures.

In mammalian cells, membrane traffic pathways play a critical role in connecting the various compartments of the endomembrane system. Each of these pathways is highly regulated, requiring specific machinery to ensure their fidelity. In the early secretory pathway, transport between the endoplasmic reticulum (ER) and Golgi apparatus is largely regulated via cytoplasmic coat protein complexes that play a role in identifying cargo and forming the transport carriers. The secretory pathway is counterbalanced by the retrograde pathway, which is essential for the recycling of molecules from the Golgi back to the ER. It is believed that there are at least two mechanisms to achieve this - one using the cytoplasmic COPI coat complex, and another, poorly characterised pathway, regulated by the small GTPase Rab6. In this work, we describe a systematic RNA interference screen targeting proteins associated with membrane fusion, in order to identify the machinery responsible for the fusion of Golgi-derived Rab6 carriers at the ER. We not only assess the delivery of Rab6 to the ER, but also one of its cargo molecules, the Shiga-like toxin B-chain. These screens reveal that three proteins, VAMP4, STX5, and SCFD1/SLY1, are all important for the fusion of Rab6 carriers at the ER. Live cell imaging experiments also show that the depletion of SCFD1/SLY1 prevents the membrane fusion event, suggesting that this molecule is an essential regulator of this pathway.

On the basis of their training, medical students are considered “the best case scenario” among university students in knowledge of the human papillomavirus (HPV). We evaluated differences in knowledge of HPV, HPV vaccine, and head and neck cancer (HNC) among medical students. A previously validated questionnaire was completed by 247 medical students at a Midwestern university. Outcomes of interest were knowledge score for HPV and HPV vaccine, and HNC, derived from combining questionnaire items to form HPV knowledge and HNC scores, and analyzed using multivariate linear regression. Mean scores for HPV knowledge were 19.4 out of 26, and 7.2 out of 12 for HNC knowledge. In the final multivariate linear regression model, sex, race, and year of study were independently associated with HPV and HPV vaccine knowledge. Males had significantly lower HPV vaccine knowledge than females (β = −1.53; 95% CI: −2.53, −0.52), as did nonwhite students (β = −1.05; 95% CI: −2.07, −0.03). There was a gradient in HPV vaccine knowledge based on the year of study, highest among fourth year students (β = 6.75; 95% CI: 5.17, 8.33). Results were similar for factors associated with HNC knowledge, except for sex. HNC knowledge similarly increased based on year of study, highest for fourth year students (β = 2.50; 95% CI: 1.72, 3.29). Among medical students, gaps remain in knowledge of HPV, HPV vaccine, and HPV-linked HNC. Male medical students have significantly lower knowledge of HPV. This highlights the need to increase medical student knowledge of HPV and HPV-linked HNC.

We interrogated a students as partners (SaP), co-curricular program that focuses on supporting student learning. To center power and equity in SaP, the program was grounded in social design-based experiment methodology. We considered the manifestation of power and equity beyond higher education, to that of broader socio-political contexts. Collaborative autoethnography (CAE) was used to garner a richer understanding of student-staff experiences of the program. Through CAE, power emerged as central to our collective experiences, and a recognition that power asymmetry in students as partners programs is complex and multi-layered. We found that to address power imbalances in these programs requires considered strategies and intentional designs. Further, CAE, in and of itself, can be a powerful way to foster self-awareness, mutual trust, respect, and the acknowledgement of others in student-staff partnerships. We conclude by recommending the importance of deliberate design for equity and power towards consequential learning and transformational change.

Simpson, McTiernan et al. develop fully synthetic corneal implants. The resulting CLP-PEG-MPC implants show reduced corneal swelling, haze, and neovascularization compared to CLP-PEG only implants when grafted into a mini-pig cornea alkali burn model of inflammation over 12 months. This study suggests the superiority of CLP-PEG-MPC implants over the CLP-PEG only implants.

Sterilization of biodegradable, collagen-based implants is challenging as irradiation sterilization methods can alter their mechanical properties. Electron beam (EB) irradiation is a terminal sterilization method that has been used for biologically-derived implants. Here, recombinant human collagen type III-phosphorylcholine (RHCIII-MPC) hydrogels were irradiated with EB doses of 17, 19, or 21 kGy and their subsequent biocompatibility and ability to promote regeneration in rabbit corneas was evaluated. Unirradiated hydrogels stored in 1% chloroform in phosphate-buffered saline (C-PBS) were the controls. There were no significant differences between irradiated and non-irradiated samples in optical or physical properties (tensile strength, modulus, elasticity), or the ability to support cell growth. However, irradiated implants were more sensitive to high levels of collagenase than unirradiated controls and the C-PBS implants had increased cell growth compared to EB and controls at 72 h. Corneal implants e-beamed at 17 kGy or e-beamed and subsequently frozen (EB-F) to increase shelf-life showed no adverse biological effects of the irradiation. EB, EB-F, and C-PBS implanted corneas all rapidly re-epithelialized but showed mild neovascularization that resolved over 6 months. The regenerated neo-corneas were transparent at 6 months post-operation. In vivo confocal microscopy confirmed normal morphology for the epithelium, stroma, sub-basal nerves and unoperated endothelium. Histology showed that all the regenerated corneas were morphologically similar to the normal. Immunohistochemistry indicated the presence of a differentiated corneal epithelium and functional tear film. In conclusion, the e-beamed corneal implants performed as well as non-irradiated control implants, resulting in fully regenerated neo-corneas with new nerves and without blood vessels or inflammation that may impede vision or corneal function. Therefore, a complete validation study to establish EB irradiation as an effective means for corneal implant sterilization prior to clinical application is necessary as a next step.

Abstract Biofilms are complex microbial communities that present serious contamination risks to our environment and health. In this study, atmospheric air plasma and airborne acoustic ultrasound technology were applied to inactivate Escherichia coli and Listeria innocua biofilms. Both technologies were efficient in controlling, or completely inactivating, the target bacterial biofilms. Viability and metabolic assays, along with microscopy analysis, revealed that atmospheric air plasma and airborne acoustic ultrasound damaged both the bacterial biofilm cells and its structural integrity. Scanning electron microscopy images highlighted the disruption of the biofilms and pore formation in bacterial cells exposed to both the plasma and acoustic treatments. Elevated reactive oxygen and nitrogen species in bacterial cells treated with atmospheric air plasma, demonstrated their primary role in the observed bacterial inactivation process. Our findings provide potential antimicrobial strategies to combat bacterial biofilms in the food and healthcare sectors.

Abstract Background Campylobacter jejuni is the leading cause of bacterial gastroenteritis worldwide and the main source of infection is contaminated chicken meat. Although this important human pathogen is an obligate microaerophile, it must survive atmospheric oxygen conditions to allow transmission from contaminated chicken meat to humans. It is becoming increasingly evident that formation of biofilm plays a key role in the survival of this organism for extended periods on poultry products. We have recently demonstrated a novel inducible model for the study of adherent C. jejuni biofilm formation under aerobic conditions. By taking advantage of supercoiling mediated gene regulation, incubation of C. jejuni with subinhibitory concentrations of the Gyrase B inhibitor novobiocin was shown to promote the consistent formation of metabolically active adherent biofilm. Results In this study, we implement this model in conjunction with the fluorescent markers: TAMRA (live cells) and SytoX (dead cells, eDNA) to develop a novel systematic high-content imaging approach and describe how it can be implemented to gain quantifiable information about the integrity and extracellular polymeric substance (EPS) composition of adherent C. jejuni biofilm in aerobic conditions. We show that this produces a model with a consistent, homogenous biofilm that can be induced and used to screen a range of inhibitors of biofilm adherence and matrix formation. Conclusions This model allows for the first time a high throughput analysis of C. jejuni biofilms which will be invaluable in enabling researchers to develop mechanisms to disrupt these biofilms and reduce the viability of these bacteria under aerobic conditions.

Abstract Bacterial biofilms are difficult to inactivate due to their high antimicrobial resistance. Therefore, new approaches are required for more effective bacterial biofilm inactivation. Airborne acoustic ultrasound improves bactericidal or bacteriostatic activity which is safe and environmentally friendly. While, plasma activated water (PAW) is attracting increasing attention due to its strong antimicrobial properties. This study determined efficacy of combined airborne acoustic ultrasound and plasma activated water from both cold and thermal plasma systems in inactivating Escherichia coli K12 biofilms. The application of airborne acoustic ultrasound (15 min) alone was significantly more effective in reducing E. coli counts in 48 and 72 h biofilms compared to 30 min treatment with PAW. The effect of airborne acoustic ultrasound was more pronounced when used in combination with PAW. Airborne acoustic ultrasound treatment for 15 min of the E. coli biofilm followed by treatment with PAW significantly reduced the bacterial count by 2.2—2.62 Log10 CFU/mL when compared to control biofilm treated with distilled water. This study demonstrates that the synergistic effects of airborne acoustic ultrasound and PAW for enhanced antimicrobial effects. These technologies have the potential to prevent and control biofilm formation in food and bio-medical applications.

National Aeronautics and Space Administration’s (NASA) spacecraft assembly facilities are monitored for the presence of any bacteria or fungi that might conceivably survive a transfer to an extraterrestrial environment. Fungi present a broad and diverse range of phenotypic and functional traits to adapt to extreme conditions, hence the detection of fungi and subsequent eradication of them are needed to prevent forward contamination for future NASA missions. During the construction and assembly for the Mars 2020 mission, three fungal strains with unique morphological and phylogenetic properties were isolated from spacecraft assembly facilities. The reconstruction of phylogenetic trees based on several gene loci (ITS, LSU, SSU, RPB, TUB, TEF1) using multi-locus sequence typing (MLST) and whole genome sequencing (WGS) analyses supported the hypothesis that these were novel species. Here we report the genus or species-level classification of these three novel strains via a polyphasic approach using phylogenetic analysis, colony and cell morphology, and comparative analysis of WGS. The strain FJI-L9-BK-P1 isolated from the Jet Propulsion Laboratory Spacecraft Assembly Facility (JPL-SAF) exhibited a putative phylogenetic relationship with the strain Aaosphaeria arxii CBS175.79 but showed distinct morphology and microscopic features. Another JPL-SAF strain, FJII-L3-CM-DR1, was phylogenetically distinct from members of the family Trichomeriaceae and exhibited morphologically different features from the genera Lithohypha and Strelitziana. The strain FKI-L1-BK-DR1 isolated from the Kennedy Space Center facility was identified as a member of Dothideomycetes incertae sedis and is closely related to the family Kirschsteiniotheliaceae according to a phylogenetic analysis. The polyphasic taxonomic approach supported the recommendation for establishing two novel genera and one novel species. The names Aaosphaeria pasadenensis (FJI-L9-BK-P1 = NRRL 64424 = DSM 114621), Pasadenomyces melaninifex (FJII-L3-CM-DR1 = NRRL 64433 = DSM 114623), and Floridaphiala radiotolerans (FKI-L1-BK-DR1 = NRRL 64434 = DSM 114624) are proposed as type species. Furthermore, resistance to ultraviolet-C and presence of specific biosynthetic gene cluster(s) coding for metabolically active compounds are unique to these strains.

Objectives Human papillomavirus (HPV)-associated cancers account for about 9% of the cancer mortality burden in the United States; however, survival differs among sociodemographic factors. We determine sociodemographic and clinical variables associated with HPV-associated cancer survival. Methods Data derived from the Surveillance, Epidemiology, and End Results 18 cancer registry were analyzed for a cohort of adult patients diagnosed with a first primary HPV-associated cancer (anal, cervical, oropharyngeal, penile, vaginal, and vulvar cancers), between 2007 and 2015. Multivariable Fine and Gray proportional hazards regression models stratified by anatomic site estimated the association of sociodemographic and clinical variables and cancer-specific survival. Results A total of 77 774 adults were included (11 216 anal, 27 098 cervical, 30 451 oropharyngeal, 2221 penile, 1176 vaginal, 5612 vulvar; average age = 57.2 years). The most common HPV-associated cancer was cervical carcinoma (58%) for females and oropharyngeal (81%) for male. Among patients diagnosed with anal/rectal squamous cell carcinoma (SCC), males had a higher risk of death than females. NonHispanic (NH) blacks had a higher risk of death from anal/rectal SCC, oropharyngeal SCC, and cervical carcinoma; and Hispanics had a higher risk of death from oropharyngeal SCC than NH whites. Marital status was associated with risk of death for all anatomic sites except vulvar. Compared to nonMedicaid insurance, patients with Medicaid and uninsured had higher risk of death from anal/rectal SCC, oropharyngeal SCC, and cervical carcinoma. Conclusions There exists gender (anal) and racial and insurance (anal, cervical, and oropharyngeal) disparities in relative survival. Concerted efforts are needed to increase and sustain progress made in HPV vaccine uptake among these specific patient subgroups, to reduce cancer incidence.

Abstract Background Lichen sclerosus (LS) is a chronic, inflammatory dermatosis. Initial treatment with superpotent topical corticosteroids is the accepted and evidence‐based first‐line therapy. For those who do not respond after exclusion of other potentiating factors, the best second‐line therapy is unclear. Laser therapy is an emerging treatment for genital LS and despite uncertain efficacy its use is gaining popularity in the private sector. Objectives We aimed to review the effectiveness of laser therapy for genital LS in men, women and children. Methods We conducted a systematic review of all primary studies reporting the use of laser in genital LS. Ovid MEDLINE, PubMed, Ovid Embase, Cochrane CENTRAL, Web of Science, CINAHL and PsycINFO were searched from inception to February 2021. The quality of the studies was assessed using the revised Cochrane risk‐of‐bias tool for randomized trials, ROBINS‐I tool for non‐randomized trials and Joanna Briggs Institute checklist for case studies. Results A total of 24 studies, involving 616 adults, met inclusion criteria. These were six randomized controlled trials (RCTs), one non‐randomized trial, nine single arm trials and eight case series. Where assessed, most studies suggest that laser therapy in patients with LS may improve symptoms, clinical signs, quality of life and sexual function. However, results were highly heterogeneous and methodological quality was very low, therefore meta‐analysis was not possible. Conclusions There is poor evidence to support the use of laser therapy for genital LS at present. Effectiveness of laser needs to be robustly investigated in well‐conducted RCTs.

Abstract Draft genomes of the fungal species Fusarium xylarioides, Teratosphaeria gauchensis and T. zuluensis are presented. In addition an annotation of the genome of Ceratocystis fimbriata is presented. Overall these genomes provide a valuable resource for understanding the molecular processes underlying pathogenicity and potential management strategies of these economically important fungi.

Partial hospitalization programming (PHP) is a treatment option available for individuals with eating disorders (ED) who have made insufficient progress in outpatient settings or are behaviorally or medically unstable. Research demonstrates that this level of care yields efficacy for the majority of patients. However, not all patients achieve recovery in PHP and later admit to a higher level of care (HLOC) including residential treatment or inpatient hospitalization. Although PHP is an increasingly common treatment choice for ED, research concerning outcome predictors in outpatient, stepped levels of care remains limited. Thus, the current study sought to identify the predictors of patients first admitted to PHP that later enter residential or inpatient treatment. Participants were 788 patients (after exclusions) enrolled in adolescent or adult partial hospitalization programs in a specialized ED clinic. When compared to patients who maintained treatment in PHP, a significantly greater proportion of patients who discharged to a HLOC had previously received ED residential treatment. Moreover, patients who discharged to a HLOC were diagnosed with a comorbid anxiety disorder and reported greater anxious and depressive symptomatology. A logistic regression model predicting discharge from PHP to a HLOC was significant, and lower body mass index (BMI) was a significant predictor of necessitating a HLOC. Supplemental programming in partial hospitalization settings might benefit individuals with previous ED residential treatment experience, higher levels of anxiety and depression, and lower BMIs. Specialized intervention for these cases is both practically and economically advantageous, as it might reduce the risk of rehospitalization and at-risk patients needing to step up to a HLOC.

The use of film media involves considerably less preparation, waste, and incubator space than conventional agar-media-based assays and has proven in past studies to provide counts of cultivable microbes similar to those of traditional agar media. Film media also have the advantage of allowing sample volumes similar to those used in pour plates and, therefore, are well-suited for cultivable microbial counts in extremely low-biomass environments such as clean rooms or space habitats, particularly where the subsequent isolation of colonies is necessary. As the preparation of film media plates relies on water cohesion/adhesion rather than manual spreading, they may have future applications in low- or microgravity settings. In this study, cultivable microbial count performance was compared between agar media and film media in three kinds of samples: food items, surfaces in built environments on Earth (homes), and on the environmental surfaces of the International Space Station (ISS). Easy Plates (Kikkoman Corporation) and Petrifilm (3M) were compared with traditional agar plating for food and home surfaces, while only Easy Plates were compared with agar for ISS samples. For both food items and built environments on Earth, both types of film media performed comparably to agar media for bacterial counts, with R2 values of 0.94–0.96. Fungal counts for built-environment samples had a lower correlation between film and agar counts, with R2 values of 0.72–0.73. Samples from the ISS, which ranged from below detection to 103 CFU per 100 cm2, had R2 values of 0.80 for bacterial counts and 0.73 for fungal counts, partially due to multiple samples recording below the detection limit for agar or too numerous to count, and the growth of fungal species on R2A medium. The species compositions of isolates picked from agar vs. film media plates were similar; however, further phylogenetic analysis is needed to confirm the differential microbial diversity composition. Overall, film media such as Easy Plates and Petrifilm are viable alternatives to agar plates for low-biomass built environments as well as for food samples, and the two brands tested in this study performed equally well.

Case summary A 10-year-old spayed female Russian Blue cat was presented with a 3-month history of excessive otic discharge and scratching, only involving the right ear. Other than a moderate amount of ceruminous exudate present within the right ear on video-otoscopic examination, there were no other cutaneous abnormalities. The cat was deemed to be otherwise in good health based on physical examination and several laboratory profiles. A diagnosis of otodemodicosis was determined due to the presence of a large number of Demodex cati mites retrieved from cerumen. Treatment consisted only of monthly topical application of sarolaner/selamectin to the nape of the neck with a marked reduction in mite counts and otic pruritus after a single dose. Complete resolution was achieved after a total of four doses. Relevance and novel information This is the first report to describe the resolution of mite infestation owing to D cati after treatment with a sarolaner-containing spot-on product. In addition, to the best of the author’s knowledge, this is the first report of any isoxazoline product used in the successful treatment of demodicosis affecting the ear canal. In general, there is a lack of reports describing safe and effective treatments for feline otodemodicosis. Topically applied sarolaner/selamectin resulted in resolution of mites while avoiding any potential ototoxic events from medications applied directly into the ear, and provided a treatment that was easier to apply than oral or injectable macrocyclic lactones.

Insulin-stimulated glucose transport is a characteristic property of adipocytes and muscle cells and involves the regulated delivery of glucose transporter (GLUT4)-containing vesicles from intracellular stores to the cell surface. Fusion of these vesicles results in increased numbers of GLUT4 molecules at the cell surface. In an attempt to overcome some of the limitations associated with both primary and cultured adipocytes, we expressed an epitope- and GFP-tagged version of GLUT4 (HA–GLUT4–GFP) in HeLa cells. Here we report the characterisation of this system compared to 3T3-L1 adipocytes. We show that insulin promotes translocation of HA–GLUT4–GFP to the surface of both cell types with similar kinetics using orthologous trafficking machinery. While the magnitude of the insulin-stimulated translocation of GLUT4 is smaller than mouse 3T3-L1 adipocytes, HeLa cells offer a useful, experimentally tractable, human model system. Here, we exemplify their utility through a small-scale siRNA screen to identify GOSR1 and YKT6 as potential novel regulators of GLUT4 trafficking in human cells.