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Abstract Peptidoglycan (PG) is an essential structural component of the bacterial cell wall that is synthetized during cell division and elongation. PG forms an extracellular polymer crucial for cellular viability, the synthesis of which is the target of many antibiotics. PG assembly requires a glycosyltransferase (GT) to generate a glycan polymer using a Lipid II substrate, which is then crosslinked to the existing PG via a transpeptidase (TP) reaction. A Shape, Elongation, Division and Sporulation (SEDS) GT enzyme and a Class B Penicillin Binding Protein (PBP) form the core of the multi-protein complex required for PG assembly. Here we used single particle cryo-electron microscopy to determine the structure of a cell elongation-specific E. coli RodA-PBP2 complex. We combine this information with biochemical, genetic, spectroscopic, and computational analyses to identify the Lipid II binding sites and propose a mechanism for Lipid II polymerization. Our data suggest a hypothesis for the movement of the glycan strand from the Lipid II polymerization site of RodA towards the TP site of PBP2, functionally linking these two central enzymatic activities required for cell wall peptidoglycan biosynthesis.

Abstract This study examined the latent structure of the broad range of complex neuropsychiatric morbidities occurring 1 year after COVID-19 infection. As part of the CU-COVID19 study, 248 (response rate=39.3%) of 631 adults hospitalized for COVID-19 infection in Hong Kong completed an online survey between March-2021 and January-2022. Disorder prevalence was compared against a random non-infected household sample (n=1834). 248 surveys were received on average 321 days post-infection (Mean age: 48.9, 54% female, moderate/severe/critical infection: 58.2%). 32.4% were screened to have at least one mental disorder, 78.7% of whom had concurrent fatigue/subjective cognitive impairment (SCI). Only PTSD (19.1%) was significantly more common than control (14%, p=0.047). Latent profile analysis classified individuals into P1 (12·4%)-no current neuropsychiatric morbidities, P2 (23.1%)-SCI/fatigue, P3 (45.2%)-anxiety/PTSD, P4 (19.3%)-depression. SCI and fatigue pervaded in all profiles (P2-4) with neuropsychiatric morbidities one-year post-infection. PTSD, anxiety and depressive symptoms were most important in differentiating P2-4. Past mental health and P4 independently predicted functional impairment. Neuropsychiatric morbidity was associated with past mental health, reduced resilience, financial problems, but not COVID-19 severity. Their confluence with depressive and anxiety symptoms predicted impairment and are associated with psychological and environmental factors.

Abstract We aimed to determine a method for prescribing a standing prosthetic leg length (ProsL) that results in an equivalent running biological leg length (BioL) for athletes with unilateral (UTTA) and bilateral transtibial amputations (BTTA). We measured standing leg length of ten non-amputee (NA) athletes, ten athletes with UTTA, and five athletes with BTTA. All athletes performed treadmill running trials from 3 m/s to their maximum speed. We calculated standing and running BioL and ProsL lengths and assessed the running-to-standing leg length ratio (Lratio) at three instances during ground contact: touchdown, mid-stance, and take-off. Athletes with UTTA had 2.4 cm longer standing ProsL than BioL length (p = 0.030), but their ProsL length were up to 3.3 cm shorter at touchdown and 4.1 cm shorter at mid-stance than BioL, at 3–11.5 m/s. At touchdown, mid-stance, and take-off, athletes with BTTA had 0.01–0.05 lower Lratio at 3 m/s (p

Summary: Mutated Ras and Raf kinases are well-known to promote cancer metastasis via flux through the Ras/Raf/MEK/ERK (mitogen-activated protein kinase [MAPK]) pathway. A role for non-mutated Raf in metastasis is also emerging, but the key mechanisms remain unclear. Elevated expression of any of the three wild-type Raf family members (C, A, or B) can drive metastasis. We utilized an in vivo model to show that wild-type C-Raf overexpression can promote metastasis of immortalized prostate cells in a gene dosage-dependent manner. Analysis of the transcriptomic and phosphoproteomic landscape indicated that C-Raf-driven metastasis is accompanied by upregulated MAPK signaling. Use of C-Raf mutants demonstrated that the dimerization domain, but not its kinase activity, is essential for metastasis. Endogenous Raf monomer knockouts revealed that C-Raf’s ability to form dimers with endogenous Raf molecules is important for promoting metastasis. These data identify wild-type C-Raf heterodimer signaling as a potential target for treating metastatic disease.

Abstract In 1977, a sample of diseased adult honeybees (Apis mellifera) from Egypt was found to contain large amounts of a previously unknown virus, Egypt bee virus, which was subsequently shown to be serologically related to deformed wing virus (DWV). By sequencing the original isolate, we demonstrate that Egypt bee virus is in fact a fourth unique, major variant of DWV (DWV-D): more closely related to DWV-C than to either DWV-A or DWV-B. DWV-A and DWV-B are the most common DWV variants worldwide due to their close relationship and transmission by Varroa destructor. However, we could not find any trace of DWV-D in several hundred RNA sequencing libraries from a worldwide selection of honeybee, varroa and bumblebee samples. This means that DWV-D has either become extinct, been replaced by other DWV variants better adapted to varroa-mediated transmission, or persists only in a narrow geographic or host range, isolated from common bee and beekeeping trade routes.

Abstract Background To examine the antidepressant efficacy and response predictors of R-DLPFC-LF rTMS for antidepressant-nonresponding BD. Methods We conducted a single-blind randomized sham-controlled trial for 54 (28 sham, 26 active) patients with antidepressant-nonresponding BD (baseline MADRS ≥ 20). Patients received 15 daily sessions of active or sham neuronavigated rTMS (Figure-of-8 coil, five 1 Hz 60 s 110% RMT trains). Outcome measures included depressive response (≥ 50% MADRS reduction, CGI ≤ 2) and remission (MADRS 0.05). There was no significant group * time interaction (ps > 0.05) in a multivariate ANOVA with MADRS, HAMA and YMRS as dependent variables. Exploratory analysis found MADRS improvement to be moderated by baseline anxiety (p = 0.02) and melancholia (p = 0.03) at week 3, and depressive onset at weeks 6 (p = 0.03) and 12 (p = 0.04). In subjects with below-mean anxiety (HAMA

Athletes with transtibial amputation (TTA) use running-specific prostheses (RSPs) to run. RSP configuration likely affects the biomechanics of such athletes across speeds. We determined how the use of three RSP models (Catapult, Sprinter and Xtend) with three stiffness categories (recommended, ±1), and three heights (recommended, ±2 cm) affected contact length (Lc), stance average vertical ground reaction force (Favg), step frequency (fstep) and asymmetry between legs for 10 athletes with unilateral TTA at 3–7 m s−1. The use of the Xtend versus Catapult RSP decreased Lc (p = 2.69 × 10−7) and Favg asymmetry (p = 0.032); the effect on Lc asymmetry diminished with faster speeds (p = 0.0020). The use of the Sprinter versus Catapult RSP decreased Favg asymmetry (p = 7.00 × 10−5); this effect was independent of speed (p = 0.90). The use of a stiffer RSP decreased Lc asymmetry (p ≤ 0.00033); this effect was independent of speed (p ≥ 0.071). The use of a shorter RSP decreased Lc (p = 5.86 × 10−6), Favg (p = 8.58 × 10−6) and fstep asymmetry (p = 0.0011); each effect was independent of speed (p ≥ 0.15). To minimize asymmetry, athletes with unilateral TTA should use an Xtend or Sprinter RSP with 2 cm shorter than recommended height and stiffness based on intended speed.

The pearl whipray Fontitrygon margaritella (Compagno & Roberts, 1984) is a common elasmobranch in coastal western African waters. However, knowledge on their life-history and trophic ecology remains limited. Therefore, we aimed to determine the growth, maturity and diet of F. margaritella from the Bijagós Archipelago in Guinea-Bissau. Growth was modelled with: von Bertalanffy, Gompertz and logistic functions. Model selection revealed no model significantly outperformed another. The sampled age ranged from less than 1 to 7 years (1.8 ± 1.9 cm, mean ± standard deviation) and size (disc width) ranged from 12.2 to 30.6 cm (18.7 ± 5.2 cm). Size-at-maturity was estimated at 20.3 cm (95% CI [18.8–21.8 cm]) for males and 24.3 cm for females (95% CI [21.9–26.5 cm]), corresponding ages of 2.2 and 3.9 years. The diet differed significantly among young-of-the-year (YOY), juveniles and adults (p = 0.001). Diet of all life stages consisted mainly of crustaceans (27.4%, 28.5%, 33.3%) and polychaetes (12.5%, 26.7%, 20.3%), for YOY, juveniles and adults respectively. This study shows that F. margaritella is relatively fast-growing, matures early and experiences ontogenetic diet shifts. These results contribute to status assessments and conservation efforts of F. margaritella and closely related species.

Exposure to teachers and teaching-related activities is vital for young children’s learning. When COVID-19 closed schools, teachers responded with a mix of live- and prerecorded lessons and one-on-one communication with students, which necessitated shifts in planning time. The current study identifies pre-COVID predictors of time teachers devoted to each of these teaching-related activities to illuminate actionable levers for supporting educators during widespread educational disruption. Teachers with higher prepandemic job commitment devoted more overall time to pandemic-induced remote teaching. Teachers’ prepandemic executive functioning and observed classroom instructional and organizational quality—features of successful teachers during normal times—predicted more during-pandemic time remote teaching, while teacher older age and having more high-needs students was associated with less remote teaching time. These results contribute to an emerging literature that spotlights potential promising avenues for supporting teachers via professional development during normal times as well as in future widespread educational disruptions.

Running-prostheses have enabled exceptional athletes with bilateral leg amputations to surpass Olympic 400 m athletics qualifying standards. Due to the world-class performances and relatively fast race finishes of these athletes, many people assume that running-prostheses provide users an unfair advantage over biologically legged competitors during long sprint races. These assumptions have led athletics governing bodies to prohibit the use of running-prostheses in sanctioned non-amputee (NA) competitions, such as at the Olympics. However, here we show that no athlete with bilateral leg amputations using running-prostheses, including the fastest such athlete, exhibits a single 400 m running performance metric that is better than those achieved by NA athletes. Specifically, the best experimentally measured maximum running velocity and sprint endurance profile of athletes with prosthetic legs are similar to, but not better than those of NA athletes. Further, the best experimentally measured initial race acceleration (from 0 to 20 m), maximum velocity around curves, and velocity at aerobic capacity of athletes with prosthetic legs were 40%, 1–3% and 19% slower compared to NA athletes, respectively. Therefore, based on these 400 m performance metrics, use of prosthetic legs during 400 m running races is not unequivocally advantageous compared to the use of biological legs.

Abstract Since the early 2000s, researchers have been trying to develop lower-limb exoskeletons that augment human mobility by reducing the metabolic cost of walking and running versus without a device. In 2013, researchers finally broke this ‘metabolic cost barrier’. We analyzed the literature through December 2019, and identified 23 studies that demonstrate exoskeleton designs that improved human walking and running economy beyond capable without a device. Here, we reviewed these studies and highlighted key innovations and techniques that enabled these devices to surpass the metabolic cost barrier and steadily improve user walking and running economy from 2013 to nearly 2020. These studies include, physiologically-informed targeting of lower-limb joints; use of off-board actuators to rapidly prototype exoskeleton controllers; mechatronic designs of both active and passive systems; and a renewed focus on human-exoskeleton interface design. Lastly, we highlight emerging trends that we anticipate will further augment wearable-device performance and pose the next grand challenges facing exoskeleton technology for augmenting human mobility.

Adenocarcinomas from multiple tissues can converge to treatment-resistant small cell neuroendocrine (SCN) cancers composed of ASCL1, POU2F3, NEUROD1, and YAP1 subtypes. We investigated how mitochondrial metabolism influences SCN cancer (SCNC) progression. Extensive bioinformatics analyses encompassing thousands of patient tumors and human cancer cell lines uncovered enhanced expression of proliferator-activatedreceptor gamma coactivator 1-alpha (PGC-1α), a potent regulator of mitochondrial oxidative phosphorylation (OXPHOS), across several SCNCs. PGC-1α correlated tightly with increased expression of the lineage marker Achaete-scute homolog 1, (ASCL1) through a positive feedback mechanism. Analyses using a human prostate tissue-based SCN transformation system showed that the ASCL1 subtype has heightened PGC-1α expression and OXPHOS activity. PGC-1α inhibition diminished OXPHOS, reduced SCNC cell proliferation, and blocked SCN prostate tumor formation. Conversely, PGC-1α overexpression enhanced OXPHOS, validated by small-animal Positron Emission Tomography mitochondrial imaging, tripled the SCN prostate tumor formation rate, and promoted commitment to the ASCL1 lineage. These results establish PGC-1α as a driver of SCNC progression and subtype determination, highlighting metabolic vulnerabilities in SCNCs across different tissues.

Combining a T cell-targeting mRNA vaccine encoding the conserved SARS-CoV-2 RNA-dependent RNA polymerase, RdRp, with a Spike-encoding mRNA vaccine may offer an additional pathway toward COVID-19 protection. Here, we show that a nucleoside-modified RdRp mRNA vaccine raises robust and durable CD8+ T cell responses in mice. Immunization drives a CD8+ T cell response enriched toward a specific RdRp epitope. Unexpectedly, coadministration of mRNA vaccines encoding RdRp or the Spike Receptor Binding Domain (RBD) dampens RBD-specific immune responses. Contralateral administration reduces the suppression of RBD-specific T cell responses while type I interferon signaling blockade restores RBD-specific antibodies. A staggered immunization strategy maintains both RBD vaccine-mediated antibody and T cell responses as well as protection against lethal SARS-CoV-2 challenge in human ACE2 transgenic mice. In HLA-A2.1 transgenic mice, the RdRp vaccine elicits CD8+ T cell responses against HLA-A*02:01-restricted epitopes recognized by human donor T cells. These results highlight RdRp as a candidate antigen for COVID-19 vaccines. The findings also offer insights into crafting effective multivalent mRNA vaccines to broaden CD8+ T cell responses against SARS-CoV-2 and potentially other viruses with pandemic potential.

Background:. Closed incisional negative pressure therapy (ciNPT) has been shown to improve surgical outcomes. Functional reduction mammaplasty has a wound dehiscence rate of 25% and higher in most series, requiring extra care and delayed secondary healing. We aimed to determine if shifting from standard care dressings to ciNPT reduced early dehiscence after breast reduction. Methods:. This multisurgeon retrospective study compared consecutive patients undergoing primary breast reduction dressed with ciNPT to similar patients with standard dressing materials. Perioperative management was otherwise unchanged. Early dehiscence was defined as incisional disruption requiring wound care within the first 30 postoperative days. Statistical analyses were performed using t-test and Fisher exact test. Results:. We analyzed 79 patients with 158 breasts (114 standard and 44 ciNPT). Both groups were similar. Mean ages were 35 and 34 years; body mass index, 28.5 and 27.4 kg/m2; and reduction volumes, 565 and 610 g, respectively. None were active smokers, and 9.5% were former smokers. Wise pattern skin incisions were used in all, and parenchymal resections mostly utilized superomedial pedicles. Median ciNPT treatment was 6 days. Early dehiscence was significantly lower with ciNPT, occurring in only 1 of 44 (2%) breasts, compared to 16 of 114 in the standard group (14%), P = 0.003, a relative risk reduction of 84%. Two control patients required debridement, whereas none of the ciNPT patients did. Conclusion:. Application of ciNPT markedly decreased early dehiscence requiring wound care, compared to using standard dressings, in otherwise similarly risk-stratified breast reduction patients.

BackgroundGABA-deficit characterizes depression (MDD), which is highly comorbid with Functional Dyspepsia (FD). We examined brain GABA concentrations and resting activities in post-prandial distress subtype FD (FD-PDS) patients with and without MDD.Methods24 female age/education-matched FD-PDS with comorbid MDD (FD-PDS-MDD), non-depressed FD-PDS, and healthy controls each were compared on GABA concentrations, resting fMRI (fALFF) in bilateral pregenual anterior cingulate (pgACC), left dorsolateral prefrontal cortex (DLPFC), insula, and somatosensory cortex (SSC).ResultsFD-PDS-MDD patients had mild though elevated depressive symptoms. FD-PDS patients had generally mild dyspeptic symptoms. No significant between-group differences in GABA or fALFF were found. No significant correlations were found between GABA and depressive/dyspeptic symptoms after Bonferroni correction. In patients, GABA correlated positively with left insula fALFF (r = 0.38, Bonferroni-corrected p = .03).ConclusionWe did not find altered GABA concentrations or brain resting activity in FD-PDS or its MDD comorbidity. The neurochemical link between MDD and FD remains elusive.

Abstract Widespread antibiotic resistance, especially of Gram-negative bacteria, has become a severe concern for human health. Tripartite efflux pumps are one of the major contributors to resistance in Gram-negative pathogens, by efficiently expelling a broad spectrum of antibiotics from the organism. In Neisseria gonorrhoeae, one of the first bacteria for which pan-resistance has been reported, the most expressed efflux complex is MtrCDE. Here we present the electrophysiological characterisation of the outer membrane component MtrE and the membrane fusion protein MtrC, obtained by a combination of planar lipid bilayer recordings and in silico techniques. Our in vitro results show that MtrE can be regulated by periplasmic binding events and that the interaction between MtrE and MtrC is sufficient to stabilize this complex in an open state. In contrast to other efflux conduits, the open complex only displays a slight preference for cations. The maximum conductance we obtain in the in vitro recordings is comparable to that seen in our computational electrophysiology simulations conducted on the MtrE crystal structure, indicating that this state may reflect a physiologically relevant open conformation of MtrE. Our results suggest that the MtrC/E binding interface is an important modulator of MtrE function, which could potentially be targeted by new efflux inhibitors.

Abstract Snake fungal disease (SFD) is an emerging disease of conservation concern in eastern North America. Ophidiomyces ophiodiicola, the causative agent of SFD, has been isolated from over 30 species of wild snakes from six families in North America. Whilst O. ophiodiicola has been isolated from captive snakes outside North America, the pathogen has not been reported from wild snakes elsewhere. We screened 33 carcasses and 303 moulted skins from wild snakes collected from 2010–2016 in Great Britain and the Czech Republic for the presence of macroscopic skin lesions and O. ophiodiicola. The fungus was detected using real-time PCR in 26 (8.6%) specimens across the period of collection. Follow up culture and histopathologic analyses confirmed that both O. ophiodiicola and SFD occur in wild European snakes. Although skin lesions were mild in most cases, in some snakes they were severe and were considered likely to have contributed to mortality. Culture characterisations demonstrated that European isolates grew more slowly than those from the United States, and phylogenetic analyses indicated that isolates from European wild snakes reside in a clade distinct from the North American isolates examined. These genetic and phenotypic differences indicate that the European isolates represent novel strains of O. ophiodiicola. Further work is required to understand the individual and population level impact of this pathogen in Europe.

Running-specific prostheses (RSPs) have facilitated an athlete with bilateral transtibial amputations to compete in the Olympic Games. However, the performance effects of using RSPs compared to biological legs remains controversial. Further, the use of different prosthetic configurations such as shape, stiffness, and height likely influence performance. We determined the effects of using 15 different RSP configurations on the maximum speed of five male athletes with bilateral transtibial amputations. These athletes performed sets of running trials up to maximum speed using three different RSP models (Freedom Innovations Catapult FX6, Össur Flex-Foot Cheetah Xtend and Ottobock 1E90 Sprinter) each with five combinations of stiffness category and height. We measured ground reaction forces during each maximum speed trial to determine the biomechanical parameters associated with different RSP configurations and maximum sprinting speeds. Use of the J-shaped Cheetah Xtend and 1E90 Sprinter RSPs resulted in 8.3% and 8.0% (p

ABSTRACT: Background: Millions of households in the world depend on wood and biomass for cooking and heating. This dependence leads to undesirable toxic effects, such as ocular and pulmonary toxicity. Objectives: The present study examined the potential oculoprotective and pulmonary protective activity of naringin (NRG), a naturally occurring flavonoid, against wood smoke (WS)-induced toxicity in a rat model. Methods: Forty-eight adult male albino rats were randomly distributed into six (n=8) groups. All rats were fed, given water, and observed for 21 days, Group I (control) received only distilled water and no WS exposure, Group II was exposed to WS, Group III was exposed to WS and given 50 mg/kg/d α-tocopherol (vitamin E), Group IV was exposed to WS and given 80 mg/kg/day NRG, Group V was administered only 80 mg/kg/d NRG only, and Group VI was administered only 50 mg/kg/d vitamin E. WS exposure was for 20 min/d. The effect of NRG treatment on acetylcholinesterase activity, nitric oxide radical production, malondialdehyde level, and antioxidant enzymes (ie, superoxide dismustase and catalase) in WS-exposed rats was examined. Results: Subchronic (21 day) exposure to WS induced ocular and pulmonary toxicity manifested by the infiltration of parenchyma, atrophy, and inflammation of the cells, which was correlated with alterations in antioxidant enzyme concentrations. Cell damage was associated with an increase in acetylcholinesterase activity and nitric oxide radical concentrations. The toxicity triggered by WS was modulated by the coadministration of NRG. Conclusion: These results suggest that NRG treatment may be useful to reduce WS-induced oxidative stress and related ocular and pulmonary damage in rats. (Curr Ther Res Clin Exp. 2012; 73:XXX–XXX)

Summary: Neoantigen-specific T cells are increasingly viewed as important immunotherapy effectors, but physically isolating these rare cell populations is challenging. Here, we describe a sensitive method for the enumeration and isolation of neoantigen-specific CD8+ T cells from small samples of patient tumor or blood. The method relies on magnetic nanoparticles that present neoantigen-loaded major histocompatibility complex (MHC) tetramers at high avidity by barcoded DNA linkers. The magnetic particles provide a convenient handle to isolate the desired cell populations, and the barcoded DNA enables multiplexed analysis. The method exhibits superior recovery of antigen-specific T cell populations relative to literature approaches. We applied the method to profile neoantigen-specific T cell populations in the tumor and blood of patients with metastatic melanoma over the course of anti-PD1 checkpoint inhibitor therapy. We show that the method has value for monitoring clinical responses to cancer immunotherapy and might help guide the development of personalized mutational neoantigen-specific T cell therapies and cancer vaccines. : Peng et al. report a sensitive method to detect tumor-associated neoantigen-specific T cells. Neoantigens and fluorescent DNA barcodes, presented on nanoparticle scaffolds, permit multiplex capture and analysis of specific T cell populations from blood or tumor. Neoantigen-specific T cell numbers track tumor volume in a melanoma patient responding to immunotherapy. Keywords: neoantigens, cancer immunotherapy, nanotechnology, microfluidics, T cell receptor

Summary: Cancer progression to an aggressive phenotype often co-opts aspects of stem cell biology. Here, we developed gene signatures for normal human stem cell populations to understand the relationship between epithelial cancers and stem cell transcriptional programs. Using a pan-cancer approach, we reveal that aggressive epithelial cancers are enriched for a transcriptional signature shared by epithelial adult stem cells. The adult stem cell signature selected for epithelial cancers with worse overall survival and alterations of oncogenic drivers. Lethal small cell neuroendocrine lung, prostate, and bladder cancers transcriptionally converged onto the adult stem cell signature and not other stem cell signatures tested. We found that DNA methyltransferase expression correlated with adult stem cell signature status and was enriched in small cell neuroendocrine cancers. DNA methylation analysis uncovered a shared epigenomic profile between small cell neuroendocrine cancers. These pan-cancer findings establish a molecular link between human adult stem cells and aggressive epithelial cancers. : Smith et al. use a systems-level, human pan-stem cell approach to find that aggressive epithelial cancers, independent of tissue of origin, activate an adult stem cell expression program. They report that small cell neuroendocrine cancers from different tissues are molecularly linked to adult stem cells through a shared transcriptional and epigenomic profile. Keywords: stem cell signature, pan-cancer, neuroendocrine prostate cancer, small cell lung cancer, adult stem cells

Trans-differentiation from an adenocarcinoma to a small cell neuroendocrine state is associated with therapy resistance in multiple cancer types. To gain insight into the underlying molecular events of the trans-differentiation, we perform a multi-omics time course analysis of a pan-small cell neuroendocrine cancer model (termed PARCB), a forward genetic transformation using human prostate basal cells and identify a shared developmental, arc-like, and entropy-high trajectory among all transformation model replicates. Further mapping with single cell resolution reveals two distinct lineages defined by mutually exclusive expression of ASCL1 or ASCL2. Temporal regulation by groups of transcription factors across developmental stages reveals that cellular reprogramming precedes the induction of neuronal programs. TFAP4 and ASCL1/2 feedback are identified as potential regulators of ASCL1 and ASCL2 expression. Our study provides temporal transcriptional patterns and uncovers pan-tissue parallels between prostate and lung cancers, as well as connections to normal neuroendocrine cell states.

Mutated Ras and Raf kinases are well-known to promote cancer metastasis via flux through the Ras/Raf/MEK/ERK (mitogen-activated protein kinase [MAPK]) pathway. A role for non-mutated Raf in metastasis is also emerging, but the key mechanisms remain unclear. Elevated expression of any of the three wild-type Raf family members (C, A, or B) can drive metastasis. We utilized an in vivo model to show that wild-type C-Raf overexpression can promote metastasis of immortalized prostate cells in a gene dosage-dependent manner. Analysis of the transcriptomic and phosphoproteomic landscape indicated that C-Raf-driven metastasis is accompanied by upregulated MAPK signaling. Use of C-Raf mutants demonstrated that the dimerization domain, but not its kinase activity, is essential for metastasis. Endogenous Raf monomer knockouts revealed that C-Raf's ability to form dimers with endogenous Raf molecules is important for promoting metastasis. These data identify wild-type C-Raf heterodimer signaling as a potential target for treating metastatic disease.

CAR (chimeric antigen receptor) T cell therapy has shown clinical success in treating hematological malignancies, but its treatment of solid tumors has been limited. One major challenge is on-target, off-tumor toxicity, where CAR T cells also damage normal tissues that express the targeted antigen. To reduce this detrimental side-effect, Boolean-logic gates like AND-NOT gates have utilized an inhibitory CAR (iCAR) to specifically curb CAR T cell activity at selected nonmalignant tissue sites. However, the strategy seems inefficient, requiring high levels of iCAR and its target antigen for inhibition. Using a TROP2-targeting iCAR with a single PD1 inhibitory domain to inhibit a CEACAM5-targeting CAR (CEACAR), we observed that the inefficiency was due to a kinetic delay in iCAR inhibition of cytotoxicity. To improve iCAR efficiency, we modified three features of the iCAR-the avidity, the affinity, and the intracellular signaling domains. Increasing the avidity but not the affinity of the iCAR led to significant reductions in the delay. iCARs containing twelve different inhibitory signaling domains were screened for improved inhibition, and three domains (BTLA, LAIR-1, and SIGLEC-9) each suppressed CAR T function but did not enhance inhibitory kinetics. When inhibitory domains of LAIR-1 or SIGLEC-9 were combined with PD-1 into a single dual-inhibitory domain iCAR (DiCARs) and tested with the CEACAR, inhibition efficiency improved as evidenced by a significant reduction in the inhibitory delay. These data indicate that a delicate balance between CAR and iCAR signaling strength and kinetics must be achieved to regulate AND-NOT gate CAR T cell selectivity.

Reports of severe disease outbreaks in amphibian communities in mainland Europe due to strains of the common midwife toad virus (CMTV)-like clade of Ranavirus are increasing and have created concern due to their considerable population impacts. In Great Britain, viruses in another clade of Ranavirus-frog virus 3 (FV3)-like-have caused marked declines of common frog (Rana temporaria) populations following likely recent virus introductions. The British public has been reporting mortality incidents to a citizen science project since 1992, with carcasses submitted for post-mortem examination, resulting in a long-term tissue archive spanning 25 years. We screened this archive for ranavirus (458 individuals from 228 incidents) using molecular methods and undertook preliminary genotyping of the ranaviruses detected. In total, ranavirus was detected in 90 individuals from 41 incidents focused in the north and south of England. The majority of detections involved common frogs (90%) but also another anuran, a caudate and a reptile. Most incidents were associated with FV3-like viruses but two, separated by 300 km and 16 years, involved CMTV-like viruses. These British CMTV-like viruses were more closely related to ranaviruses from mainland Europe than to each other and were estimated to have diverged at least 458 years ago. This evidence of a CMTV-like virus in Great Britain in 1995 represents the earliest confirmed case of a CMTV associated with amphibians and raises important questions about the history of ranavirus in Great Britain and the epidemiology of CMTV-like viruses. Despite biases present in the opportunistic sample used, this study also demonstrates the role of citizen science projects in generating resources for research and the value of maintaining long-term wildlife tissue archives.

[This corrects the article DOI: 10.1371/journal.pone.0168298.].

Exposure to teachers and teaching-related activities is vital for young children's learning. When COVID-19 closed schools, teachers responded with a mix of live- and prerecorded lessons and one-on-one communication with students, which necessitated shifts in planning time. The current study identifies pre-COVID predictors of time teachers devoted to each of these teaching-related activities to illuminate actionable levers for supporting educators during widespread educational disruption. Teachers with higher prepandemic job commitment devoted more overall time to pandemic-induced remote teaching. Teachers' prepandemic executive functioning and observed classroom instructional and organizational quality--features of successful teachers during normal times--predicted more during-pandemic time remote teaching, while teacher older age and having more high-needs students was associated with less remote teaching time. These results contribute to an emerging literature that spotlights potential promising avenues for supporting teachers via professional development during normal times as well as in future widespread educational disruptions.

Systemic targeted therapy in prostate cancer is primarily focused on ablating androgen signaling. Androgen deprivation therapy and second-generation androgen receptor (AR)-targeted therapy selectively favor the development of treatment-resistant subtypes of metastatic castration-resistant prostate cancer (mCRPC), defined by AR and neuroendocrine (NE) markers. Molecular drivers of double-negative (AR-/NE-) mCRPC are poorly defined. In this study, we comprehensively characterized treatment-emergent mCRPC by integrating matched RNA sequencing, whole-genome sequencing, and whole-genome bisulfite sequencing from 210 tumors. AR-/NE- tumors were clinically and molecularly distinct from other mCRPC subtypes, with the shortest survival, amplification of the chromatin remodeler CHD7, and PTEN loss. Methylation changes in CHD7 candidate enhancers were linked to elevated CHD7 expression in AR-/NE+ tumors. Genome-wide methylation analysis nominated Krüppel-like factor 5 (KLF5) as a driver of the AR-/NE- phenotype, and KLF5 activity was linked to RB1 loss. These observations reveal the aggressiveness of AR-/NE- mCRPC and could facilitate the identification of therapeutic targets in this highly aggressive disease.SignificanceComprehensive characterization of the five subtypes of metastatic castration-resistant prostate cancer identified transcription factors that drive each subtype and showed that the double-negative subtype has the worst prognosis.

The MYC proto-oncogene contributes to the pathogenesis of more than half of human cancers. Malignant transformation by MYC transcriptionally up-regulates the core pre-mRNA splicing machinery and causes misregulation of alternative splicing. However, our understanding of how splicing changes are directed by MYC is limited. We performed a signaling pathway-guided splicing analysis to identify MYC-dependent splicing events. These included an HRAS cassette exon repressed by MYC across multiple tumor types. To molecularly dissect the regulation of this HRAS exon, we used antisense oligonucleotide tiling to identify splicing enhancers and silencers in its flanking introns. RNA-binding motif prediction indicated multiple binding sites for hnRNP H and hnRNP F within these cis-regulatory elements. Using siRNA knockdown and cDNA expression, we found that both hnRNP H and F activate the HRAS cassette exon. Mutagenesis and targeted RNA immunoprecipitation implicate two downstream G-rich elements in this splicing activation. Analyses of ENCODE RNA-seq datasets confirmed hnRNP H regulation of HRAS splicing. Analyses of RNA-seq datasets across multiple cancers showed a negative correlation of HNRNPH gene expression with MYC hallmark enrichment, consistent with the effect of hnRNP H on HRAS splicing. Interestingly, HNRNPF expression showed a positive correlation with MYC hallmarks and thus was not consistent with the observed effects of hnRNP F. Loss of hnRNP H/F altered cell cycle progression and induced apoptosis in the PC3 prostate cancer cell line. Collectively, our results reveal mechanisms for MYC-dependent regulation of splicing and point to possible therapeutic targets in prostate cancers.

The mechanical stiffness of running-specific prostheses likely affects the functional abilities of athletes with leg amputations. However, each prosthetic manufacturer recommends prostheses based on subjective stiffness categories rather than performance based metrics. The actual mechanical stiffness values of running-specific prostheses (i.e. kN/m) are unknown. Consequently, we sought to characterize and disseminate the stiffness values of running-specific prostheses so that researchers, clinicians, and athletes can objectively evaluate prosthetic function. We characterized the stiffness values of 55 running-specific prostheses across various models, stiffness categories, and heights using forces and angles representative of those measured from athletes with transtibial amputations during running. Characterizing prosthetic force-displacement profiles with a 2nd degree polynomial explained 4.4% more of the variance than a linear function (p

Alternative splicing (AS) is prevalent in cancer, generating an extensive but largely unexplored repertoire of novel immunotherapy targets. We describe Isoform peptides from RNA splicing for Immunotherapy target Screening (IRIS), a computational platform capable of discovering AS-derived tumor antigens (TAs) for T cell receptor (TCR) and chimeric antigen receptor T cell (CAR-T) therapies. IRIS leverages large-scale tumor and normal transcriptome data and incorporates multiple screening approaches to discover AS-derived TAs with tumor-associated or tumor-specific expression. In a proof-of-concept analysis integrating transcriptomics and immunopeptidomics data, we showed that hundreds of IRIS-predicted TCR targets are presented by human leukocyte antigen (HLA) molecules. We applied IRIS to RNA-seq data of neuroendocrine prostate cancer (NEPC). From 2,939 NEPC-associated AS events, IRIS predicted 1,651 epitopes from 808 events as potential TCR targets for two common HLA types (A*02:01 and A*03:01). A more stringent screening test prioritized 48 epitopes from 20 events with "neoantigen-like" NEPC-specific expression. Predicted epitopes are often encoded by microexons of ≤30 nucleotides. To validate the immunogenicity and T cell recognition of IRIS-predicted TCR epitopes, we performed in vitro T cell priming in combination with single-cell TCR sequencing. Seven TCRs transduced into human peripheral blood mononuclear cells (PBMCs) showed high activity against individual IRIS-predicted epitopes, providing strong evidence of isolated TCRs reactive to AS-derived peptides. One selected TCR showed efficient cytotoxicity against target cells expressing the target peptide. Our study illustrates the contribution of AS to the TA repertoire of cancer cells and demonstrates the utility of IRIS for discovering AS-derived TAs and expanding cancer immunotherapies.

The ability to selectively bind to antigenic peptides and secrete cytokines can define populations of cells with therapeutic potential in emerging T cell receptor (TCR) immunotherapies. We leverage cavity-containing hydrogel microparticles, called nanovials, each coated with millions of peptide-major histocompatibility complex (pMHC) monomers to isolate antigen-reactive T cells. T cells are captured and activated by pMHCs and secrete cytokines on nanovials, allowing sorting based on both affinity and function. The TCRs of sorted cells on nanovials are sequenced, recovering paired αβ-chains using microfluidic emulsion-based single-cell sequencing. By labeling nanovials having different pMHCs with unique oligonucleotide-barcodes we could link TCR sequence to targets with 100% accuracy. We identified with high specificity an expanded repertoire of functional TCRs targeting viral antigens compared to standard techniques.

Multiple sclerosis (MS) is an autoimmune disease driven by lymphocyte activation against myelin autoantigens in the central nervous system leading to demyelination and neurodegeneration. The deoxyribonucleoside salvage pathway with the rate-limiting enzyme deoxycytidine kinase (dCK) captures extracellular deoxyribonucleosides for use in intracellular deoxyribonucleotide metabolism. Previous studies have shown that deoxyribonucleoside salvage activity is enriched in lymphocytes and required for early lymphocyte development. However, specific roles for the deoxyribonucleoside salvage pathway and dCK in autoimmune diseases such as MS are unknown. Here we demonstrate that dCK activity is necessary for the development of clinical symptoms in the MOG35-55 and MOG1-125 experimental autoimmune encephalomyelitis (EAE) mouse models of MS. During EAE disease, deoxyribonucleoside salvage activity is elevated in the spleen and lymph nodes. Targeting dCK with the small molecule dCK inhibitor TRE-515 limits disease severity when treatments are started at disease induction or when symptoms first appear. EAE mice treated with TRE-515 have significantly fewer infiltrating leukocytes in the spinal cord, and TRE-515 blocks activation-induced B and T cell proliferation and MOG35-55 -specific T cell expansion without affecting innate immune cells or naïve T and B cell populations. Our results demonstrate that targeting dCK limits symptoms in EAE mice and suggest that dCK activity is required for MOG35-55 -specific lymphocyte activation-induced proliferation.

Efficient and adequate generation of deoxyribonucleotides is critical to successful DNA repair. We show that ataxia telangiectasia mutated (ATM) integrates the DNA damage response with DNA metabolism by regulating the salvage of deoxyribonucleosides. Specifically, ATM phosphorylates and activates deoxycytidine kinase (dCK) at serine 74 in response to ionizing radiation (IR). Activation of dCK shifts its substrate specificity toward deoxycytidine, increases intracellular dCTP pools post IR, and enhances the rate of DNA repair. Mutation of a single serine 74 residue has profound effects on murine T and B lymphocyte development, suggesting that post-translational regulation of dCK may be important in maintaining genomic stability during hematopoiesis. Using [(18)F]-FAC, a dCK-specific positron emission tomography (PET) probe, we visualized and quantified dCK activation in tumor xenografts after IR, indicating that dCK activation could serve as a biomarker for ATM function and DNA damage response in vivo. In addition, dCK-deficient leukemia cell lines and murine embryonic fibroblasts exhibited increased sensitivity to IR, indicating that pharmacologic inhibition of dCK may be an effective radiosensitization strategy.

Impaired walking performance is a key predictor of morbidity among older adults. A distinctive characteristic of impaired walking performance among older adults is a greater metabolic cost (worse economy) compared to young adults. However, older adults who consistently run have been shown to retain a similar running economy as young runners. Unfortunately, those running studies did not measure the metabolic cost of walking. Thus, it is unclear if running exercise can prevent the deterioration of walking economy.To determine if and how regular walking vs. running exercise affects the economy of locomotion in older adults.15 older adults (69 ± 3 years) who walk ≥ 30 min, 3x/week for exercise, "walkers" and 15 older adults (69 ± 5 years) who run ≥ 30 min, 3x/week, "runners" walked on a force-instrumented treadmill at three speeds (0.75, 1.25, and 1.75 m/s). We determined walking economy using expired gas analysis and walking mechanics via ground reaction forces during the last 2 minutes of each 5 minute trial. We compared walking economy between the two groups and to non-aerobically trained young and older adults from a prior study.Older runners had a 7-10% better walking economy than older walkers over the range of speeds tested (p = .016) and had walking economy similar to young sedentary adults over a similar range of speeds (p = .237). We found no substantial biomechanical differences between older walkers and runners. In contrast to older runners, older walkers had similar walking economy as older sedentary adults (p = .461) and ∼ 26% worse walking economy than young adults (p

Existing approaches that quantify cytotoxic T cell responses rely on bulk or surrogate measurements which impede the direct identification of single activated T cells of interest. Single cell microscopy or flow cytometry methodologies typically rely on fluorescent labeling, which limits applicability to primary cells such as human derived T lymphocytes. Here, we introduce a quantitative method to track single T lymphocyte mediated cytotoxic events within a mixed population of cells using live cell interferometry (LCI), a label-free microscopy technique that maintains cell viability. LCI quantifies the mass distribution within individual cells by measuring the phase shift caused by the interaction of light with intracellular biomass. Using LCI, we imaged cytotoxic T cells killing cognate target cells. In addition to a characteristic target cell mass decrease of 20-60% over 1-4 h following attack by a T cell, there was a significant 4-fold increase in T cell mass accumulation rate at the start of the cytotoxic event and a 2-3 fold increase in T cell mass relative to the mass of unresponsive T cells. Direct, label-free measurement of CD8+ T and target cell mass changes provides a kinetic, quantitative assessment of T cell activation and a relatively rapid approach to identify specific, activated patient-derived T cells for applications in cancer immunotherapy.

New therapies for late stage and castration resistant prostate cancer (CRPC) depend on defining unique properties and pathways of cell sub-populations capable of sustaining the net growth of the cancer. One of the best enrichment schemes for isolating the putative stem/progenitor cell from the murine prostate gland is Lin(-);Sca1(+);CD49f(hi) (LSC(hi)), which results in a more than 10-fold enrichment for in vitro sphere-forming activity. We have shown previously that the LSC(hi) subpopulation is both necessary and sufficient for cancer initiation in the Pten-null prostate cancer model. To further improve this enrichment scheme, we searched for cell surface molecules upregulated upon castration of murine prostate and identified CD166 as a candidate gene. CD166 encodes a cell surface molecule that can further enrich sphere-forming activity of WT LSC(hi) and Pten null LSC(hi). Importantly, CD166 could enrich sphere-forming ability of benign primary human prostate cells in vitro and induce the formation of tubule-like structures in vivo. CD166 expression is upregulated in human prostate cancers, especially CRPC samples. Although genetic deletion of murine CD166 in the Pten null prostate cancer model does not interfere with sphere formation or block prostate cancer progression and CRPC development, the presence of CD166 on prostate stem/progenitors and castration resistant sub-populations suggest that it is a cell surface molecule with the potential for targeted delivery of human prostate cancer therapeutics.

Analysis of DNA methylation is a valuable tool to understand disease progression and is increasingly being used to create diagnostic and prognostic clinical biomarkers. While conversion of cytosine to 5-methylcytosine (5mC) commonly results in transcriptional repression, further conversion to 5-hydroxymethylcytosine (5hmC) is associated with transcriptional activation. Here we perform the first study integrating whole-genome 5hmC with DNA, 5mC, and transcriptome sequencing in clinical samples of benign, localized, and advanced prostate cancer. 5hmC is shown to mark activation of cancer drivers and downstream targets. Furthermore, 5hmC sequencing revealed profoundly altered cell states throughout the disease course, characterized by increased proliferation, oncogenic signaling, dedifferentiation, and lineage plasticity to neuroendocrine and gastrointestinal lineages. Finally, 5hmC sequencing of cell-free DNA from patients with metastatic disease proved useful as a prognostic biomarker able to identify an aggressive subtype of prostate cancer using the genes TOP2A and EZH2, previously only detectable by transcriptomic analysis of solid tumor biopsies. Overall, these findings reveal that 5hmC marks epigenomic activation in prostate cancer and identify hallmarks of prostate cancer progression with potential as biomarkers of aggressive disease.SignificanceIn prostate cancer, 5-hydroxymethylcytosine delineates oncogene activation and stage-specific cell states and can be analyzed in liquid biopsies to detect cancer phenotypes. See related article by Wu and Attard, p. 3880.

Endothelium-leukocyte interaction is critical for inflammatory responses. Whereas the tissue microenvironments are often acidic at inflammatory sites, the mechanisms by which cells respond to acidosis are not well understood. Using molecular, cellular and biochemical approaches, we demonstrate that activation of GPR4, a proton-sensing G protein-coupled receptor, by isocapnic acidosis increases the adhesiveness of human umbilical vein endothelial cells (HUVECs) that express GPR4 endogenously. Acidosis in combination with GPR4 overexpression further augments HUVEC adhesion with U937 monocytes. In contrast, overexpression of a G protein signaling-defective DRY motif mutant (R115A) of GPR4 does not elicit any increase of HUVEC adhesion, indicating the requirement of G protein signaling. Downregulation of GPR4 expression by RNA interference reduces the acidosis-induced HUVEC adhesion. To delineate downstream pathways, we show that inhibition of adenylate cyclase by inhibitors, 2',5'-dideoxyadenosine (DDA) or SQ 22536, attenuates acidosis/GPR4-induced HUVEC adhesion. Consistently, treatment with a cAMP analog or a G(i) signaling inhibitor increases HUVEC adhesiveness, suggesting a role of the G(s)/cAMP signaling in this process. We further show that the cAMP downstream effector Epac is important for acidosis/GPR4-induced cell adhesion. Moreover, activation of GPR4 by acidosis increases the expression of vascular adhesion molecules E-selectin, VCAM-1 and ICAM-1, which are functionally involved in acidosis/GPR4-mediated HUVEC adhesion. Similarly, hypercapnic acidosis can also activate GPR4 to stimulate HUVEC adhesion molecule expression and adhesiveness. These results suggest that acidosis/GPR4 signaling regulates endothelial cell adhesion mainly through the G(s)/cAMP/Epac pathway and may play a role in the inflammatory response of vascular endothelial cells.

Tissue-specific antigens can serve as targets for adoptive T cell transfer-based cancer immunotherapy. Recognition of tumor by T cells is mediated by interaction between peptide-major histocompatibility complexes (pMHCs) and T cell receptors (TCRs). Revealing the identity of peptides bound to MHC is critical in discovering cognate TCRs and predicting potential toxicity. We performed multimodal immunopeptidomic analyses for human prostatic acid phosphatase (PAP), a well-recognized tissue antigen. Three physical methods, including mild acid elution, coimmunoprecipitation, and secreted MHC precipitation, were used to capture a thorough signature of PAP on HLA-A*02:01. Eleven PAP peptides that are potentially A*02:01-restricted were identified, including five predicted strong binders by NetMHCpan 4.0. Peripheral blood mononuclear cells (PBMCs) from more than 20 healthy donors were screened with the PAP peptides. Seven cognate TCRs were isolated which can recognize three distinct epitopes when expressed in PBMCs. One TCR shows reactivity toward cell lines expressing both full-length PAP and HLA-A*02:01. Our results show that a combined multimodal immunopeptidomic approach is productive in revealing target peptides and defining the cloned TCR sequences reactive with prostatic acid phosphatase epitopes.

Cross-reactivity and direct killing of target cells remain underexplored for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-specific CD8+ T cells. Isolation of T cell receptors (TCRs) and overexpression in allogeneic cells allows for extensive T cell reactivity profiling. We identify SARS-CoV-2 RNA-dependent RNA polymerase (RdRp/NSP12) as highly conserved, likely due to its critical role in the virus life cycle. We perform single-cell TCRαβ sequencing in human leukocyte antigen (HLA)-A∗02:01-restricted, RdRp-specific T cells from SARS-CoV-2-unexposed individuals. Human T cells expressing these TCRαβ constructs kill target cell lines engineered to express full-length RdRp. Three TCR constructs recognize homologous epitopes from common cold coronaviruses, indicating CD8+ T cells can recognize evolutionarily diverse coronaviruses. Analysis of individual TCR clones may help define vaccine epitopes that can induce long-term immunity against SARS-CoV-2 and other coronaviruses.

Cancer transcriptomes frequently exhibit RNA dysregulation. As the resulting aberrant transcripts may be translated into cancer-specific proteins, there is growing interest in exploiting RNA dysregulation as a source of tumor antigens (TAs) and thus novel immunotherapy targets. Recent advances in high-throughput technologies and rapid accumulation of multiomic cancer profiling data in public repositories have provided opportunities to systematically characterize RNA dysregulation in cancer and identify antigen targets for immunotherapy. However, given the complexity of cancer transcriptomes and proteomes, important conceptual and technological challenges exist. Here, we highlight the expanding repertoire of TAs arising from RNA dysregulation and introduce multiomic and big data strategies for identifying optimal immunotherapy targets. We discuss extant barriers for translating these targets into effective therapies as well as the implications for future research.