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Abstract Several hydroxysteroid dehydrogenase 17-beta 13 variants have previously been identified as protective against metabolic dysfunction-associated steatohepatitis (MASH) fibrosis, ballooning and inflammation, and as such this target holds significant therapeutic potential. However, over 5 years later, the function of 17B-HSD13 remains unknown. Structure-aided design enables the development of potent and selective sulfonamide-based 17B-HSD13 inhibitors. In order to probe their inhibitory potency in endogenous expression systems like primary human hepatocytes, inhibitors are transformed into synthetic surrogate substrates with distinct selectivity advantages over substrates previously published. Their application to cells endogenously expressing 17B-HSD13 enables quantitative measures of enzymatic inhibition in primary human hepatocytes which has never been reported to date. Application to multiple cellular systems expressing the protective human variants reveals that the most prevalent IsoD variant maintains NAD-dependent catalytic activity towards some but not all substrates, contradicting reports that the truncation results in loss-of-function.

Abstract Myelin loss induces neural dysfunction and contributes to the pathophysiology of neurodegenerative diseases, injury conditions, and aging. Because remyelination is often incomplete, better understanding endogenous remyelination and developing remyelination therapies that restore neural function are clinical imperatives. Here, we use in vivo two-photon microscopy and electrophysiology to study the dynamics of endogenous and therapeutic-induced cortical remyelination and functional recovery after cuprizone-mediated demyelination in mice. We focus on the visual pathway, which is uniquely positioned to provide insights into structure-function relationships during de/remyelination. We show endogenous remyelination is driven by recent oligodendrocyte loss and is highly efficacious following mild demyelination, but fails to restore the oligodendrocyte population when high rates of oligodendrocyte loss occur quickly. Testing a thyromimetic (LL-341070) compared to clemastine, we find it better enhances oligodendrocyte gain and hastens recovery of neuronal function. The therapeutic benefit of the thyromimetic is temporally restricted, and it acts exclusively following moderate to severe demyelination, eliminating the endogenous remyelination deficit. However, we find regeneration of oligodendrocytes and myelin to healthy levels is not necessary for recovery of visual neuronal function. These findings advance our understanding of remyelination and its impact on functional recovery to inform future therapeutic strategies.

Abstract We present a study on nanoscale skyrmionic spin textures in $$\hbox {NdMn}_{{2}}\hbox {Ge}_{{2}}$$ , a rare-earth complex noncollinear ferromagnet. We confirm, using X-ray microscopy, that $$\hbox {NdMn}_{{2}}\hbox {Ge}_{{2}}$$ can host lattices of metastable skyrmion bubbles at room temperature in the absence of a magnetic field, after applying a suitable field cooling protocol. The skyrmion bubbles are robust against temperature changes from room temperature to 330 K. Furthermore, the skyrmion bubbles can be distorted, deformed, and recovered by varying strength and orientation of the applied magnetic field. We have used nitrogen-vacancy nanoscale magnetic imaging to estimate and map the magnetic stray fields originating from our $$\hbox {NdMn}_{{2}}\hbox {Ge}_{{2}}$$ lamella samples and find stray field magnitudes on the order of a few mT near the sample surface. Micromagnetic simulations show an overall agreement with the observed behaviour of the sample under different magnetic field protocols. We also find that the presence of the Dzyaloshinskii-Moriya interaction is not required to reproduce our experimental results. Its inclusion in the simulation leads to a reversal of the skyrmionic object core polarity, which is not experimentally observed. Our results further corroborate the stability and robustness of the skyrmion bubbles formed in $${\hbox {NdMn}_2\hbox {Ge}_2}$$ and their potential for future spintronic applications.

Abstract Raman spectroscopy is a powerful method for probing electronic and vibrational properties of materials, particularly nanomaterials such as single-wall carbon nanotubes. Typically, Raman spectroscopy is conducted at a single, or few, excitation wavelengths, but that provides limited information about excitation resonance structure, and their dynamical evolution. Here, we extend a sensitive full-spectrum technique to rapidly obtain two-dimensional Raman excitation maps both statically and dynamically for chirality-pure single-wall carbon nanotube films. We demonstrate sensitive evaluation of structured resonance profiles even from weak vibrational modes, and sub-second time resolution of the dynamics of photo-driven defect production. Findings include the direct observation of bands and their profiles – including bands which could be missed in conventional Raman spectroscopy - and demonstration of differences for odd vs. even defect band combinations. This opens up possibilities to investigate the coupling of electronic states with vibrational modes in nanomaterials and track their dynamical evolution subject to intentional modulation.

Abstract Triple-negative breast cancer (TNBC) presents therapeutic challenges due to limited targeted treatment options and resistance to chemotherapy drugs, such as doxorubicin. This study investigated doxorubicin resistance mechanisms and a strategy to overcome it. A doxorubicin-resistant cell subline (231-DR) was developed from MDA-MB-231 TNBC cells, and enhanced expression of cellular FLICE-inhibitory protein (cFLIP) in 231-DR cells was identified as a potential driver of the resistance. Overexpression of cFLIP conferred resistance to doxorubicin-induced apoptosis, whereas siRNA-mediated cFLIP depletion induced apoptosis, particularly in 231-DR cells. Furthermore, the RNA-binding protein Hu antigen R (HuR) was found to regulate cFLIP expression. HuR Inhibition with KH-3 or RNA interference reduced cFLIP levels. Importantly, KH-3 sensitized TNBC cells to doxorubicin-induced apoptosis. In summary, this study delineates cFLIP’s role in mediating doxorubicin resistance and identifies HuR as a positive regulator of cFLIP, offering a novel therapeutic avenue against chemoresistance in TNBC by combining HuR inhibition with doxorubicin.

Abstract Hypertrophic cardiomyopathy (HCM) afflicts humans, cats, pigs, and rhesus macaques. Disease sequelae include congestive heart failure, thromboembolism, and sudden cardiac death (SCD). Sarcomeric mutations explain some human and cat cases, however, the molecular basis in rhesus macaques remains unknown. RNA-Seq of the LV tissues of five HCM-affected and seven healthy control rhesus macaques was employed for differential transcriptomic analyses. DNA from 15 severely HCM-affected and 21 healthy geriatric rhesus macaques were selected for whole-genome sequencing. A genome-wide association study (GWAS) of disease status and SCD outcome was performed. 614 down- and 1,065 upregulated differentially expressed genes (DEGs) were identified between groups. The top DEG (MAFF) was overexpressed in affected animals (log2FoldChange = 4.71; P Adjusted-value = 1.14E-133). Channelopathy-associated enriched terms were identified in ~ 57% of downregulated DEGs providing transcriptomic evidence of hypertrophic and arrhythmic disease processes. For GWAS, no putative variant withstood segregation. Polygenic modeling analysis resulted in poor prediction power and burden testing could not explain HCM by an association of multiple variants in any gene. Neither single nor compound genetic variant(s), or identified polygenic profile, suggest complex genotype–phenotype interactions in rhesus macaques. Brought forth is an established dataset of robustly phenotyped rhesus macaques as an open-access resource for future cardiovascular disease genetic studies.

Abstract Background Vancomycin, an antibiotic with activity against methicillin-resistant Staphylococcus aureus (MRSA), is frequently included in empiric treatment for community-acquired pneumonia (CAP) despite the fact that MRSA is rarely implicated in CAP. Conducting polymerase chain reaction (PCR) testing on nasal swabs to identify the presence of MRSA colonization has been proposed as an antimicrobial stewardship intervention to reduce the use of vancomycin. Observational studies have shown reductions in vancomycin use after implementation of MRSA colonization testing, and this approach has been adopted by CAP guidelines. However, the ability of this intervention to safely reduce vancomycin use has yet to be tested in a randomized controlled trial. Methods STOP-Vanc is a pragmatic, prospective, single center, non-blinded randomized trial. The objective of this study is to test whether the use of MRSA PCR testing can safely reduce inappropriate vancomycin use in an intensive care setting. Adult patients with suspicion for CAP who are receiving vancomycin and admitted to the Medical Intensive Care Unit at Vanderbilt University Medical Center will be screened for eligibility. Eligible patients will be enrolled and randomized in a 1:1 ratio to either receive MRSA nasal swab PCR testing in addition to usual care (intervention group), or usual care alone (control group). PCR testing results will be transmitted through the electronic health record to the treating clinicians. Primary providers of intervention group patients with negative swab results will also receive a page providing clinical guidance recommending discontinuation of vancomycin. The primary outcome will be vancomycin-free hours alive, defined as the expected number of hours alive and free of the use of vancomycin within the first 7 days following trial enrollment estimated using a proportional odds ratio model. Secondary outcomes include 30-day all-cause mortality and time alive off vancomycin. Discussion STOP-Vanc will provide the first randomized controlled trial data regarding the use of MRSA nasal swab PCR testing to guide antibiotic de-escalation. This study will provide important information regarding the effect of MRSA PCR testing and antimicrobial stewardship guidance on clinical outcomes in an intensive care unit setting. Trial registration ClinicalTrials.gov NCT06272994. Registered on February 22, 2024.

Abstract Many studies have explored different loading and rehabilitation strategies, yet rehabilitation intensity and its impact on the local strain environment and bone healing have largely not been investigated. This study combined implantable strain sensors and subject-specific finite element models in a 2 mm rodent segmental bone defect model. After injury animals were underwent high or low intensity rehabilitation. High intensity rehabilitation increased local strains within the regenerative niche by an average of 44% compared to the low intensity rehabilitation. Finite element modeling demonstrated that resistance rehabilitation significantly increased compressive strain by a factor of 2.0 at week 2 and 4.45 after 4 weeks of rehabilitation. Animals that underwent resistance running had the greatest bone volume and improved functional recovery with regenerated femurs that matched intact failure torque and torsional stiffness values. These results demonstrate the potential for early resistance rehabilitation to improve bone healing.

Abstract Background The primary objective of this study was to assess the frequency of body composition increases and their relationships to changes in body weight in two cohorts of real world, treatment‐naïve, advanced non‐small cell lung cancer (NSCLC) patients. One cohort received the current standard of care (CSOC), which consisted of immunotherapy and newer chemotherapy regimens, and the other cohort was treated with the former standard of care (FSOC), consisting only of older platinum‐containing regimens. Methods CSOC (n = 106) and FSOC (n = 88) cohorts of advanced NSCLC patients were included in this study. Weights were collected at each clinical visit, and body composition analysis from routine chest computed tomography via automated segmentation software assessed at baseline and at 6 and 12 weeks. Standard statistical methods were used to calculate relationships between changes in weight and in body composition. Results The CSOC cohort contained 106 stage IV NSCLC patients treated between 16/12/2014 and 22/10/2020 while the FSOC cohort contained 88 stage III/IV NSCLC patients treated between 16/6/2006 and 18/11/2014. While each cohort exhibited decreases in median weight, body mass index (BMI), mean skeletal muscle index (SMI) and subcutaneous adipose tissue index (SATI) at the 6 and 12 week time points, a subset of patients experienced increases in these parameters. Using a threshold of ≥2.5% increase for weight, BMI, SMI, and SATI at the 12 week time point, both cohorts showed similar (20.5% and 27.3%) increases in these parameters. With a cut point of ≥5% increase at 12 weeks follow‐up, 8.0% to 25.0% of the patients gained ≥5% in weight, BMI, SMI and SATI. Comparing these results in each cohort showed no significant differences. Pearson coefficients for weight change related to changes in SMI and SATI at 6 and 12 weeks ranged from 0.31 to 0.58 with all P values

Abstract Additional and refurbished pumping stations are required to mitigate the intensifying occurrence of droughts and floodings. These installations negatively impact threatened freshwater fish populations due to the increased risk of injury and mortality when fish pass through them. Fish-friendly pumping installations have been proposed as a potential solution to reduce these risks. However, published assessments of these new types of pumps remains lacking, and the few available studies do not enable a cross-comparison with conventional pump types. The promising, yet understudied, Fairbank Nijhuis ‘fish-friendly’ axial flow pump has been assessed in previous works, however the results remain ambiguous due to low recapture rates, unconsidered parameters, fixed operating conditions, and the inability to identify the likely sources of injury and mortality. In this study, we address the limitations of previous works by implementing a standardized protocol for live fish in conjunction with passive barotrauma detection sensors. The major finding of this work is that safe passage of eel (100% survival) is confirmed, but that bream and roach had a much lower survival probability (24% and 70% survival respectively) than expected, albeit higher than for a conventional axial flow pump (roach survival: 13%). Furthermore, roach and bream passing at higher rpm suffered significantly higher mortalities. The impact of the impeller was found to be the most common source of severe injury for both pumps. These results are significant because they conclusively show that fish-friendly pumps may be considered safe for eel, but not for other endemic European fish species such as roach and bream.

Abstract The RNA-binding protein Hu antigen R (HuR) plays a pivotal role in cancer progression, and previous studies have demonstrated its involvement in suppressing cell death in cancer. However, the precise mechanisms underlying HuR inhibition-induced cell death remain elusive. Here, we investigated the impacts of HuR functional inhibition via the small molecule inhibitor KH-3 on cell proliferation, colony formation, and cell death across multiple cancer cell lines, with an emphasis on breast and prostate cancers. KH-3 treatment induced apoptotic cell death of various cancer cell lines, as well as autophagy-associated cell death and ferroptosis. Remarkably, KH-3-induced cell death was partially rescued by an autophagy inhibitor and a ferroptosis inhibitor. The anti-tumor effects of KH-3 were further validated in two mouse xenograft models of human prostate cancer. Mechanistically, KH-3 reduced the expression of HuR targets involved in apoptosis and ferroptosis suppression, including cFLIP and SLC7A11, respectively. Moreover, cFLIP silencing enhanced Caspase-8 activation as well as PARP cleavage in both breast cancer and prostate cancer cells. Both KH-3-induced pharmacological HuR inhibition and RNA interference-mediated HuR knockdown reduced the expression of SLC7A11. Additionally, KH-3 also reduced XIAP and Survivin, enhancing the activation of multiple caspases and leading to apoptosis. This study highlights the critical roles of HuR in programmed cell death regulation, advocating HuR inhibition as a promising anti-tumor strategy for cell-death-inducing cancer therapy.

Abstract Background Mobile health (mHealth) applications have been utilized in chronic diseases to improve medication adherence, though factors driving engagement remain understudied. We previously demonstrated the benefits of mHealth app utilization in increasing adherence to hydroxyurea. In the “Integration of mHealth into Sickle Cell Disease (SCD) Care to Increase Hydroxyurea Utilization” (meSH) study, greater engagement with the InCharge Health app increased the percentage of days covered (PDC) for hydroxyurea. However, factors influencing app engagement were not investigated. This study aimed to identify patient-level factors associated with mHealth engagement during and post-study among adolescents and adults with SCD. Methods All meSH participants were included. The InCharge Health app use was measured for 24 weeks (study period) and 12 weeks (post-study period). Baseline demographics and validated measures of self-efficacy, financial hardship, and pain severity, impact, and frequency were evaluated as predictors of app engagement during the study period and post-study using linear models. Results Complete app use data were obtained from 284 of 293 participants, including 150 (52.8%) females and 240 (84.8%) who were HbSS/HbSβ0-thalassemia with a median age of 27 years. During the study period, 232 (81.7%) used InCharge Health at least once (median use excluding zeros: 26 days; IQR: 7–72 days). During the post-study period, 132 (46.5%) participants used the app at least once (median use excluding zeros: 16 days; IQR: 3–44 days). During the study period, the HbSS genotype, age > 25 years, higher education, higher self-efficacy score, and lower pain severity were associated with greater app engagement. Only age (p = 0.0032) and financial hardship (p = 0.0125) were significant in a multivariable model after backward elimination. During the post-study period, older age (p = 0.035), lower pain severity (p = 0.021) and lower frequency of pain (p = 0.033) predicted mHealth engagement. All three remained significant in a multivariable model after backward elimination. Conclusions Older age, lower financial hardship, lower pain severity, and greater self-efficacy were associated with increased engagement with a mHealth intervention for improving medication adherence among people living with SCD. This research can shift future app development from focusing on factors that drive patient interaction with mHealth targeting modifiable factors influencing app engagement. Trial registration The trial was registered with ClinicalTrials.gov. Registration Identifier: NCT04080167. Registration date: November 11, 2019.

Generation of cardiomyocytes from human pluripotent stem cells (hPSCs) is of high interest for disease modelling and regenerative medicine. hPSCs can provide an unlimited source of patient-specific cardiomyocytes that are otherwise difficult to obtain from individuals. Moreover, the low proliferation rate of adult cardiomyocytes and low viability ex vivo limits the quantity of study material. Most protocols for the differentiation of cardiomyocytes from hPSCs are based on the temporal modulation of the Wnt pathway. However, during the initial stage of GSK-3 inhibition, a substantial number of cells are lost due to detachment. In this study, we aimed to increase the efficiency of generating cardiomyocytes from hPSCs. We identified cell death as a detrimental factor during this initial stage of in vitro cardiomyocyte differentiation. Through pharmacological targeting of different types of cell death, we discovered that ferroptosis was the main cell death type during the first 48 h of the in vitro differentiation procedure. Inhibiting ferroptosis using ferrostatin-1 during cardiomyocyte differentiation resulted in increased robustness and cell yield.

Abstract Plasmodium vivax transmission occurs throughout the tropics and is an emerging threat in areas of Plasmodium falciparum decline, causing relapse infections that complicate treatment and control. Targeted sequencing for P. falciparum has been widely deployed to detect population structure and the geographic spread of antimalarial and diagnostic resistance. However, there are fewer such tools for P. vivax. Leveraging global variation data, we designed four molecular inversion probe (MIP) genotyping panels targeting geographically differentiating SNPs, neutral SNPs, putative antimalarial resistance genes, and vaccine candidate genes. We deployed these MIP panels on 866 infections from the Peruvian Amazon and identified transmission networks with clonality (IBD[identity by descent]>0.99), copy number variation in Pvdbp and multiple Pvrbps, mutations in antimalarial resistance orthologs, and balancing selection in 13 vaccine candidate genes. Our MIP panels are the broadest genotyping panel currently available and are poised for successful deployment in other regions of P. vivax transmission.

Abstract Plasmodium ovale curtisi (Poc) and Plasmodium ovale wallikeri (Pow) are relapsing malaria parasites endemic to Africa and Asia that were previously thought to represent a single species. Amid increasing detection of ovale malaria in sub-Saharan Africa, we present a population genomic study of both species across the continent. We conducted whole-genome sequencing of 25 isolates from Central and East Africa and analyzed them alongside 20 previously published African genomes. Isolates are predominantly monoclonal (43/45), with their genetic similarity aligning with geography. Pow shows lower average nucleotide diversity (1.8×10−4) across the genome compared to Poc (3.0×10−4) (p

Abstract Background Immune checkpoint inhibitors (ICIs) have revolutionized the treatment landscape for non-small cell lung cancer (NSCLC). The variability in patient responses necessitates a blood-based, multi-cohort gene signature to predict ICI response in NSCLC. Methods We performed transcriptomic profiling of peripheral blood mononuclear cell (PBMC) and buffy coat (BC) samples from three independent cohorts of NSCLC patients treated with ICIs: a retrospective cohort (PMBCR, n = 59), a retrospective validation cohort (BC, n = 44), and a prospective validation cohort (PBMCP, n = 42). We identified a 5-gene signature (UQCRB, NDUFA3, CDKN2D, FMNL1-DT, and APOL3) predictive of ICI response and validated its clinical utility in the prospective PBMCP cohort. Response was evaluated using RECIST criteria, and patients were followed up for progression-free survival (PFS) and overall survival (OS). Results In the prospective PBMCP cohort, the 5-gene signature demonstrated high accuracy in stratifying patients into responders and non-responders (AUC = 0.89, 95% CI: 0.80–0.99). Predicted responders exhibited significantly longer PFS compared to predicted non-responders (median: 13.8 months vs. 4.2 months, HR = 0.21, 95% CI: 0.07–0.58, p = 0.005). Conclusion Our study confirms a 5-gene signature as a key biomarker for ICI response in NSCLC, enhancing treatment precision.

Abstract The emergence of antimalarial drug resistance is an impediment to malaria control and elimination in Africa. Analysis of temporal trends in molecular markers of resistance is critical to inform policy makers and guide malaria treatment guidelines. In a low and seasonal transmission region of southern Zambia, we successfully genotyped 85.5% (389/455) of Plasmodium falciparum samples collected between 2013 and 2018 from 8 spatially clustered health centres using molecular inversion probes (MIPs) targeting key drug resistance genes. Aside from one sample from 2016 carrying K13 622I, no other World Health Organization-validated or candidate artemisinin partial resistance (ART-R) mutations were observed. However, in the more recent years (2016–2017) five novel K13-propeller-domain mutations, C532S, A578S, Q613E, D680N and G718S were identified at low prevalence. Moreover, 13% (CI, 9.6–17.2) of isolates had the AP2MU 160N mutation, which has been associated with delayed clearance following artemisinin combination therapy in Africa. This mutation increased in prevalence between 2015 and 2018 and bears a genomic signature of selection. During this time period, there was an increase in the MDR1 NFD haplotype that is associated with reduced susceptibility to lumefantrine. Sulfadoxine-pyrimethamine polymorphisms were near fixation. While validated ART-R mutations are rare, a mutation associated with slow parasite clearance in Africa appears to be under selection in southern Zambia.

Introduction: Immune checkpoint inhibitors (ICIs) have substantially advanced the treatment of patients with malignant melanoma. However, improving therapeutic efficacy requires identifying drug combinations that elicit durable responses without inducing intolerable toxicity. Within that context, selinexor emerges as a possible combination option that has been shown in preclinical studies to enhance the efficacy of ICI therapy. Methods: In this phase 1b study, we investigated selinexor in combination with pembrolizumab in 25 patients with advanced non-uveal melanoma. Patients received selinexor at a dosage of 60 mg taken orally twice weekly, and pembrolizumab intravenously at a dosage of 200 mg every 3 weeks. Results: Despite the high incidence of adverse events (96%), most treatment-related toxicities were manageable with supportive care and dose reductions. The most common adverse events of any grade were nausea (n = 20; 80%), decreased white blood cell count (n = 15; 60%), vomiting (n = 14; 56%), anemia (n = 12; 48%), fatigue (n = 12; 48%), and decreased platelet count (n = 12; 48%). The 10 patients with treatment-naïve evaluable disease had an objective response rate (ORR) of 70% (n = 7, including three patients with complete response), which was significantly higher than that of the 14 patients with prior anti–programmed cell death protein 1 (anti-PD-1) therapy, whose ORR was 7% (n = 1; p = 0.002). Stable disease was observed in two patients (20%) with treatment-naïve disease and seven patients (50%) with prior anti-PD-1 therapy. Conclusion: Selinexor combined with pembrolizumab showed promising antitumor activity in patients with treatment-naïve metastatic melanoma. The toxicity profile of the combination was consistent with that reported for individual agents, with no additional safety concerns.

The North Pacific Transition Zone (NPTZ) is known as a global marine hotspot for many endangered and commercially significant highly mobile marine species. In the last few decades, the region has undergone unprecedented physical and biological transformations in response to climate variability and change. Although it is anticipated that many highly mobile species will need to adapt and shift their distributions, current predictions have relied on short-term data sets or modeled simulations. This has left a critical gap in our understanding of long-term (decadal or longer) change and species’ responses within the NPTZ. Here, we integrate nearly 3 decades of satellite tracking data from a climate sentinel, the juvenile North Pacific loggerhead sea turtle (Caretta caretta), with concurrent observations of sea surface temperature (SST) and chlorophyll-a concentrations to examine higher trophic level response to climate-induced changes within the eastern bounds of the NPTZ. Between 1997–2024, the NPTZ has warmed by 1.6°C and experienced an approximately 19% decline in mean surface chlorophyll-a concentration, a proxy for reduced productivity, resulting in a 28% (1.65 million km2) increase in total oligotrophic habitat in the eastern NPTZ. Over the same period, the average latitude of loggerhead sea turtle foraging habitat in the NPTZ has shifted northwards by 450–600 km. This represents a distributional shift rate of 116–200km/decade. In most years both the southern and northern range limits for the loggerhead turtle have shifted northward in tandem, indicating a habitat range shift rather than a range expansion. Our findings reveal significant physical and biological change to the NPTZ over the last quarter century and the first empirical evidence illustrating the substantial spatial response of a highly mobile megafaunal species. As the NPTZ continues to become more oligotrophic, these insights can provide vital information for dynamic conservation and management strategies within this critically important ecosystem.

Over the past two decades, therapeutic antibodies have emerged as a rapidly expanding domain within the field of biologics. In silico tools that can streamline the process of antibody discovery and optimization are critical to support a pipeline that is growing more numerous and complex every year. High-quality structural information remains critical for the antibody optimization process, but antibody-antigen complex structures are often unavailable and in silico antibody docking methods are still unreliable. In this study, DeepAb, a deep learning model for predicting antibody Fv structure directly from sequence, was used in conjunction with single-point experimental deep mutational scanning (DMS) enrichment data to design 200 potentially optimized variants of an anti-hen egg lysozyme (HEL) antibody. We sought to determine whether DeepAb-designed variants containing combinations of beneficial mutations from the DMS exhibit enhanced thermostability and whether this optimization affected their developability profile. The 200 variants were produced through a robust high-throughput method and tested for thermal and colloidal stability (Tonset, Tm, Tagg), affinity (KD) relative to the parental antibody, and for developability parameters (nonspecific binding, aggregation propensity, self-association). Of the designed clones, 91% and 94% exhibited increased thermal and colloidal stability and affinity, respectively. Of these, 10% showed a significantly increased affinity for HEL (5- to 21-fold increase) and thermostability (>2.5C increase in Tm1), with most clones retaining the favorable developability profile of the parental antibody. Additional in silico tests suggest that these methods would enrich for binding affinity even without first collecting experimental DMS measurements. These data open the possibility of in silico antibody optimization without the need to predict the antibody–antigen interface, which is notoriously difficult in the absence of crystal structures.

Position statement The International Society of Sports Nutrition (ISSN) provides an objective and critical review of the use of a ketogenic diet in healthy exercising adults, with a focus on exercise performance and body composition. However, this review does not address the use of exogenous ketone supplements. The following points summarize the position of the ISSN:1. A ketogenic diet induces a state of nutritional ketosis, which is generally defined as serum ketone levels above 0.5 mM. While many factors can impact what amount of daily carbohydrate intake will result in these levels, a broad guideline is a daily dietary carbohydrate intake of less than 50 grams per day.2. Nutritional ketosis achieved through carbohydrate restriction and a high dietary fat intake is not intrinsically harmful and should not be confused with ketoacidosis, a life-threatening condition most commonly seen in clinical populations and metabolic dysregulation.3. A ketogenic diet has largely neutral or detrimental effects on athletic performance compared to a diet higher in carbohydrates and lower in fat, despite achieving significantly elevated levels of fat oxidation during exercise (~1.5 g/min).4. The endurance effects of a ketogenic diet may be influenced by both training status and duration of the dietary intervention, but further research is necessary to elucidate these possibilities. All studies involving elite athletes showed a performance decrement from a ketogenic diet, all lasting six weeks or less. Of the two studies lasting more than six weeks, only one reported a statistically significant benefit of a ketogenic diet.5. A ketogenic diet tends to have similar effects on maximal strength or strength gains from a resistance training program compared to a diet higher in carbohydrates. However, a minority of studies show superior effects of non-ketogenic comparators.6. When compared to a diet higher in carbohydrates and lower in fat, a ketogenic diet may cause greater losses in body weight, fat mass, and fat-free mass, but may also heighten losses of lean tissue. However, this is likely due to differences in calorie and protein intake, as well as shifts in fluid balance.7. There is insufficient evidence to determine if a ketogenic diet affects males and females differently. However, there is a strong mechanistic basis for sex differences to exist in response to a ketogenic diet.

Abstract Primary cultures of proximal tubule cells are widely used to model the behavior of kidney epithelial cells in vitro. However, de-differentiation of primary cells upon culture has been observed and appreciated for decades, yet the mechanisms driving this phenomenon remain poorly understood. This confounds the interpretation of experiments using primary kidney epithelial cells and prevents their use to engineer functional kidney tissue ex vivo. In this report, we measure the dynamics of cell-state transformations in early primary culture of mouse proximal tubules to identify key pathways and processes that correlate with and may drive de-differentiation. Our data show that the loss of proximal-tubule-specific genes is rapid, uniform, and sustained even after confluent, polarized epithelial monolayers develop. This de-differentiation occurs uniformly across many common culture condition variations. Changes in early culture were strongly associated with the loss of HNF4A. Exogenous re-expression of HNF4A can promote expression of a subset of proximal tubule genes in a de-differentiated proximal tubule cell line. Using genetically labeled proximal tubule cells, we show that selective pressures very early in culture influence which cells grow to confluence. Together, these data indicate that the loss of in vivo function in proximal tubule cultures occurs very early and suggest that the sustained loss of HNF4A is a key regulatory event mediating this change.

Abstract In this review, we discuss current research on forest carbon risk from natural disturbance under climate change for the United States, with emphasis on advancements in analytical mapping and modeling tools that have potential to drive research for managing future long-term stability of forest carbon. As a natural mechanism for carbon storage, forests are a critical component of meeting climate mitigation strategies designed to combat anthropogenic emissions. Forests consist of long-lived organisms (trees) that can store carbon for centuries or more. However, trees have finite lifespans, and disturbances such as wildfire, insect and disease outbreaks, and drought can hasten tree mortality or reduce tree growth, thereby slowing carbon sequestration, driving carbon emissions, and reducing forest carbon storage in stable pools, particularly the live and standing dead portions that are counted in many carbon offset programs. Many forests have natural disturbance regimes, but climate change and human activities disrupt the frequency and severity of disturbances in ways that are likely to have consequences for the long-term stability of forest carbon. To minimize negative effects and maximize resilience of forest carbon, disturbance risks must be accounted for in carbon offset protocols, carbon management practices, and carbon mapping and modeling techniques. This requires detailed mapping and modeling of the quantities and distribution of forest carbon across the United States and hopefully one day globally; the frequency, severity, and timing of disturbances; the mechanisms by which disturbances affect carbon storage; and how climate change may alter each of these elements. Several tools (e.g. fire spread models, imputed forest inventory models, and forest growth simulators) exist to address one or more of the aforementioned items and can help inform management strategies that reduce forest carbon risk, maintain long-term stability of forest carbon, and further explore challenges, uncertainties, and opportunities for evaluating the continued potential of, and threats to, forests as viable mechanisms for forest carbon storage, including carbon offsets. A growing collective body of research and technological improvements have advanced the science, but we highlight and discuss key limitations, uncertainties, and gaps that remain.

Abstract During the 2022 mpox outbreak, our study conducted 30 interviews with a recruited sample of Chicago gay men (age 18+) during June-September to investigate their experiences of mpox, HIV/AIDS, and COVID-19. Participants were interviewed with a semi-structured guide about gay sexual identity and social experiences; HIV/AIDS, ART, and PrEP; and COVID-19 behaviors and vaccination. All 30 interview respondents had been fully vaccinated for COVID-19 and expressed minimal COVID-19 vaccine hesitancy. All the men living with HIV in our study were on ART with HIV well controlled. A majority of HIV- participants (70%) were on PrEP, with participants universally aware of PrEP benefits. Additionally, most participants had already received at least one shot of the Jynneos mpox vaccine, with many interviewees enduring long lines, sometimes at multiple locations, before vaccination in primarily gay social spaces. These Chicago gay men demonstrated widespread enthusiasm for mpox vaccination as a disease prevention strategy and most of them had already been vaccinated despite significant barriers. The enthusiasm of the participants in our study emerged within a medical landscape shaped by both COVID-19 vaccination and HIV/AIDS-related health interventions including ART and PrEP, which may have helped instill increased medical trust among this population. Our study suggests that out urban gay men may comprise a distinctive minority population with increased medical trust due to specific social, sexual, and historical experiences.

Abstract Background INCbase is an international, multicenter prospective observational study using a customizable web-based modular registry to study the clinical, biological and electrophysiological variation and boundaries of chronic inflammatory demyelinating polyneuropathy (CIDP). The primary objective of INCbase is to develop and validate a clinical prediction model for treatment response. Methods All patients meeting clinical criteria for CIDP can be included in INCbase. Collected data include demographics, clinical history, diagnostics and various domains of clinical outcomes. Data is collected at a minimum of every 6 months for two years, and more frequently at the discretion of the investigational site to allow for assessment of unexpected changes in treatment response or clinical status. Participants can be enrolled in various sub-studies designed to capture data relevant to specific groups of interest. Data is entered directly into the web-based data entry system by local investigators and/or participants. Collection and local storage of biomaterial is optional. To develop a clinical prediction model for treatment response, newly diagnosed patients with active disease warranting start of first-line treatment will be included. The study population will be split into a development and validation cohort. Univariate and multivariate logistic regression analysis will be used to identify and combine predictors at start of treatment for treatment response at six months. Model performance will be assessed through discrimination and calibration in an external validation cohort. The externally validated prediction model will be made available to researchers and clinicians on the INCbase website. Discussion With this study, we aim to create a clinically relevant and implementable prediction model for treatment response to first line treatments in CIDP. INCbase enrollment started in April 2021, with 29 centers across 8 countries and 303 patients participating to date. This collaborative effort between academia, patient advocacy organizations and pharmaceutical industry will deepen our understanding of how to diagnose and treat CIDP.

Abstract Recent findings suggest that adding a visual depiction of a nose to virtual reality displays (virtual nose) can reduce motion sickness. If so, this would be a simple intervention that could improve the experience of a variety of VR applications. However, only one peer-reviewed study has reported a benefit from a virtual nose, and the effect was observed in a single low-powered experiment. To further test the effectiveness of a virtual nose for mitigating motion sickness in VR, we performed a preregistered experiment with higher power and better control. Subjects were presented with simulated movement in a virtual environment using a head-mounted display, and the resulting motion sickness was measured using the Fast Motion Sickness Scale (FMS) and the Simulator Sickness Questionnaire (SSQ). Conditions with and without a virtual nose were tested in separate sessions on different days, and the mean habituation effects were removed analytically. Awareness of the manipulation was assessed with a funnel debriefing procedure. The sample size (n = 32) was chosen to have over 90% power to detect the estimated effect size based on previous data (d z = 0.6). We found no significant difference between motion sickness in conditions with and without the virtual nose. The estimated effect size was close to zero, d z = − 0.02, with a 95% credible interval [− 0.37, 0.33]. Results from a Bayesian analysis imply that any benefit from a virtual nose is unlikely to be more than a 26% reduction in FMS scores, and any cost is unlikely to be more than a 23% increase. Our results do not support the hypothesis that a virtual nose is a general and effective way to relieve motion sickness in virtual reality.

ABSTRACT Environmental DNA (eDNA) sampling is a powerful method for detecting aquatic species at low densities. However, eDNA may remain close to the source in lentic systems, decreasing the effectiveness of eDNA surveys. We conducted cage experiments with salamanders and simultaneous detailed hydrologic and wind measurements to investigate the influence of the physical environment on detection patterns of eDNA in ponds. We found much higher detection rates in the surface layer than at depth, and that aquatic vegetation reduced detection of eDNA produced in open water by 80%–94%. Within the surface mixed layer, detection rates were highest close to the source in the direction of water flow in the bottom half of the layer, and detections farthest from the source occurred when velocities in this sublayer were high. Detections were near zero even close to the source when this sublayer was flowing fast and away from the sampling point. The direction of water flow in this lower half of the surface mixed layer was negatively correlated with wind direction for most of the study. These spatial and temporal dynamics indicate that eDNA transport processes in ponds are highly complex. Sampling away from aquatic vegetation, in the surface mixed layer, and upwind of potential sources, in addition to sampling at many locations within a pond and considering temporal patterns, may improve detection of rare pond species. This work contributes to a growing body of literature characterizing the variability of eDNA detection in lentic systems.

Objective This study describes recency of suicide attempt (SA) and suicidal ideation (SI) at index emergency department (ED) visit and post‐ED‐discharge reattempt among pediatric patients. Methods Secondary analysis from the ED Screen for Teens at Risk for Suicide study included patients ages 12–17 who reported a past SA during screening and received a phone call 3‐month post‐ED‐discharge assessing reattempt. Logistic regressions tested past SA/SI recency as predictors of reattempt. Descriptive statistics on SA/SI recency and reattempt are provided. Results Sample included 351 patients reporting a past SA during screening (70% cisgender female, 59% White, 53% psychiatric chief complaint, mean [SD] age = 15.3 [1.5]). 17% (60/351) reattempted suicide post‐ED‐discharge. Compared to patients who attempted over a year ago, patients with recent SA within 3 months, especially 1 week before screening, had greater odds of reattempting (3 months: p

Abstract The continued advancement of electrochemical technologies requires an increasingly detailed understanding of the microscopic processes that control their performance, inspiring the development of new multi-modal diagnostic techniques. Here, we introduce a neutron imaging approach to enable the quantification of spatial and temporal variations in species concentrations within an operating redox flow cell. Specifically, we leverage the high attenuation of redox-active organic materials (high hydrogen content) and supporting electrolytes (boron-containing) in solution and perform subtractive neutron imaging of active species and supporting electrolyte. To resolve the concentration profiles across the electrodes, we employ an in-plane imaging configuration and correlate the concentration profiles to cell performance with polarization experiments under different operating conditions. Finally, we use time-of-flight neutron imaging to deconvolute concentrations of active species and supporting electrolyte during operation. Using this approach, we evaluate the influence of cell polarity, voltage bias and flow rate on the concentration distribution within the flow cell and correlate these with the macroscopic performance, thus obtaining an unprecedented level of insight into reactive mass transport. Ultimately, this diagnostic technique can be applied to a range of (electro)chemical technologies and may accelerate the development of new materials and reactor designs.

Abstract Western blot is a popular biomolecular analysis method for measuring the relative quantities of independent proteins in complex biological samples. However, variability in quantitative western blot data analysis poses a challenge in designing reproducible experiments. The lack of rigorous quantitative approaches in current western blot statistical methodology may result in irreproducible inferences. Here we describe best practices for the design and analysis of western blot experiments, with examples and demonstrations of how different analytical approaches can lead to widely varying outcomes. To facilitate best practices, we have developed the blotRig tool for designing and analyzing western blot experiments to improve their rigor and reproducibility. The blotRig application includes functions for counterbalancing experimental design by lane position, batch management across gels, and analytics with covariates and random effects.

Abstract Fibrosis is involved in 45% of deaths in the United States, and no treatment exists to reverse the progression of lung or kidney fibrosis. Myofibroblasts are key to the progression and maintenance of fibrosis. We investigated features of cell adhesion necessary for monocytes to differentiate into myofibroblasts, seeking to identify pathways key to myofibroblast differentiation. Blocking antibodies against integrins α3, αM, and αMβ2 de-differentiate myofibroblasts in vitro, lower the pro-fibrotic secretome of myofibroblasts, and treat lung fibrosis and inhibit kidney fibrosis in vivo. Decorin’s collagen-binding peptide can be used to direct functionalized blocking antibodies (against integrins-α3, -αM, -αMβ2) to both fibrotic lungs and fibrotic kidneys, reducing the dose of antibody necessary to treat fibrosis. This targeted immunotherapy blocking key integrins may be an effective therapeutic for the treatment of fibrosis.

Abstract While the critical role of NKX2-1 and its transcriptional targets in lung morphogenesis and pulmonary epithelial cell differentiation is increasingly known, mechanisms by which chromatin accessibility alters the epigenetic landscape and how NKX2-1 interacts with other co-activators required for alveolar epithelial cell differentiation and function are not well understood. Combined deletion of the histone methyl transferases Prdm3 and Prdm16 in early lung endoderm causes perinatal lethality due to respiratory failure from loss of AT2 cells and the accumulation of partially differentiated AT1 cells. Combination of single-cell RNA-seq, bulk ATAC-seq, and CUT&RUN data demonstrate that PRDM3 and PRDM16 regulate chromatin accessibility at NKX2-1 transcriptional targets critical for perinatal AT2 cell differentiation and surfactant homeostasis. Lineage specific deletion of PRDM3/16 in AT2 cells leads to lineage infidelity, with PRDM3/16 null cells acquiring partial AT1 fate. Together, these data demonstrate that NKX2-1-dependent regulation of alveolar epithelial cell differentiation is mediated by epigenomic modulation via PRDM3/16.

Abstract Background Physical activity and metformin pharmacotherapy are associated with improved clinical outcomes in breast and colorectal cancer survivors. Myokines are cytokines secreted from skeletal muscle that may mediate these associations. Methods This hypothesis‐generating analysis used biospecimens collected from a multi‐centre 2 × 2 factorial randomized design of 116 patients with stage I–III breast and colorectal cancer who were randomized to 12 weeks of (1) aerobic exercise (moderate intensity titrated to 220 min/week); (2) metformin (850 mg daily for 2 weeks and then titrated to 850 mg twice per day); (3) aerobic exercise and metformin; or (4) control. Fourteen myokines were quantified using a multiplex panel. Myokine concentrations were log‐transformed, and main effects analyses were conducted using linear mixed‐effects regression models. The type I error rate was controlled with the Holm sequential testing procedure. Results Randomization to exercise increased leukaemia inhibitory factor (1.26 pg/mL, 95% confidence interval [CI]: 0.69, 1.84; adjusted P = 0.001) and interleukin‐15 (2.23 pg/mL, 95% CI: 0.87, 3.60; adjusted P = 0.013) compared with randomization to no exercise. Randomization to metformin decreased apelin (−2.69 pg/mL, 95% CI: −4.31, −1.07; adjusted P = 0.014) and interleukin‐15 (−1.74 pg/mL, 95% CI: −2.79, −0.69; adjusted P = 0.013) compared with randomization to no metformin. Metformin decreased myostatin, irisin, oncostatin M, fibroblast growth factor 21 and osteocrin; however, these changes were not statistically significant after correction for multiple comparisons. Conclusions This pilot study demonstrates that randomization to exercise and metformin elicit unique effects on myokine concentrations in cancer patients. This hypothesis‐generating observation warrants further basic, translational and clinical investigation and replication.

Abstract Globally, catadromous freshwater eels of the genus Anguilla are of conservation concern, including critically endangered European eel (Anguilla anguilla). Pumping stations that move river water to a higher elevation severely impact eels during their seaward spawning migration. Fish-friendly pumps can mitigate fish injury and mortality but here we uniquely rethink a fish-friendly pump as a fish passage solution. In this pluriannual study, the seasonal timing of pump operation was misaligned with the typical silver eel migration period. Eels were almost exclusively nocturnal but night-time pumping represented as little as 5.6% a year. Night-time eel approaches were primarily influenced by pump duration and temperature, but did not align with lunar phase, unlike in unregulated rivers. After reaching the pumping station, eel passage was influenced by weedscreen aperture and increased when the aperture was increased. Passive sensor collision suggested non-pump infrastructure could cause injury and mortality to eels. It is therefore recommended pump operation should align with the timing of silver eel migration, weedscreen and pump entrance efficiencies should be maximised, and non-pump infrastructure must have low fish injury risk. Ultimately, considering the entire structure a fish passage solution will help ensure fish-friendly pumps have high conservation value for anguillid eels globally.

Abstract Background One major goal of total knee arthroplasty (TKA) is to achieve balanced medial and lateral gaps in flexion and extension. While bone resections are planned by the surgeon, soft tissue laxity is largely intrinsic and patient-specific in the absence of additional soft tissue releases. We sought to determine the variability in soft tissue laxity in patients undergoing TKA. Methods We retrospectively reviewed 113 patients undergoing TKA. Data on preoperative knee deformity were collected. Data from a dynamic intraoperative stress examination were collected by a robotic tracking system to quantify maximal medial and lateral opening in flexion (85–95 degrees) and extension (-5–20 degrees). T-tests were used to assess the differences between continuous variables. Results A valgus stress opened the medial compartment a mean of 4.3 ± 2.3 mm (0.0–12.4 mm) in extension and 4.6 ± 2.3 mm (0.0–12.9 mm) in flexion. A varus stress opened the lateral compartment a mean of 5.4 ± 2.4 mm (0.3–12.6 mm) in extension and 6.2 ± 2.5 mm (0.0–13.4 mm) in flexion. The medial compartment of varus knees opened significantly more in response to valgus stress than valgus knees in both extension (5.2 mm vs. 2.6 mm; P

Abstract Preclinical studies suggest that simultaneous HER2/VEGF blockade may have cooperative effects in gastroesophageal adenocarcinomas. In a single-arm investigator initiated clinical trial for patients with untreated advanced HER2+ gastroesophageal adenocarcinoma, bevacizumab was added to standard of care capecitabine, oxaliplatin, and trastuzumab in 36 patients (NCT01191697). Primary endpoint was objective response rate and secondary endpoints included safety, duration of response, progression free survival, and overall survival. The study met its primary endpoint with an objective response rate of 81% (95% CI 65–92%). Median progression free and overall survival were 14.0 (95% CI, 11.3–36.4) and 23.2 months (95% CI, 16.6–36.4), respectively. The median duration of response was 14.9 months. The regimen was well tolerated without unexpected or severe toxicities. In post-hoc ctDNA analysis, baseline ctDNA features were prognostic: Higher tumor fraction and alternative MAPK drivers portended worse outcomes. ctDNA at resistance identified oncogenic mutations and these were detectable 2–8 cycles prior to radiographic progression. Capecitabine, oxaliplatin, trastuzumab and bevacizumab shows robust clinical activity in HER2+ gastroesophageal adenocarcinoma. Combination of VEGF inhibitors with chemoimmunotherapy and anti-PD1 regimens is warranted.

Abstract Staphylococcus aureus is responsible for a substantial number of invasive infections globally each year. These infections are problematic because they are frequently recalcitrant to antibiotic treatment. Antibiotic tolerance, the ability of bacteria to persist despite normally lethal doses of antibiotics, contributes to antibiotic treatment failure in S. aureus infections. To understand how antibiotic tolerance is induced, S. aureus biofilms exposed to multiple anti-staphylococcal antibiotics are examined using both quantitative proteomics and transposon sequencing. These screens indicate that arginine metabolism is involved in antibiotic tolerance within a biofilm and support the hypothesis that depletion of arginine within S. aureus communities can induce antibiotic tolerance. Consistent with this hypothesis, inactivation of argH, the final gene in the arginine synthesis pathway, induces antibiotic tolerance. Arginine restriction induces antibiotic tolerance via inhibition of protein synthesis. In murine skin and bone infection models, an argH mutant has enhanced ability to survive antibiotic treatment with vancomycin, highlighting the relationship between arginine metabolism and antibiotic tolerance during S. aureus infection. Uncovering this link between arginine metabolism and antibiotic tolerance has the potential to open new therapeutic avenues targeting previously recalcitrant S. aureus infections.

Abstract Genome-wide association studies implicate multiple loci in risk for systemic lupus erythematosus (SLE), but few contain exonic variants, rendering systematic identification of non-coding variants essential to decoding SLE genetics. We utilized SNP-seq and bioinformatic enrichment to interrogate 2180 single-nucleotide polymorphisms (SNPs) from 87 SLE risk loci for potential binding of transcription factors and related proteins from B cells. 52 SNPs that passed initial screening were tested by electrophoretic mobility shift and luciferase reporter assays. To validate the approach, we studied rs2297550 in detail, finding that the risk allele enhanced binding to the transcription factor Ikaros (encoded by IKZF1), thereby modulating expression of IKBKE. Correspondingly, primary cells from genotyped healthy donors bearing the risk allele expressed higher levels of the interferon / NF-κB regulator IKKε. Together, these findings define a set of likely functional non-coding lupus risk variants and identify a regulatory pathway involving rs2297550, Ikaros, and IKKε implicated by human genetics in risk for SLE.

Abstract Background The COVID-19 pandemic brought greater focus to the rural mortality penalty in the U.S., which describes the greater mortality rate in rural compared to urban areas. Although it is widely thought that issues such as access to care, age structure of the population, and differences in behavior are likely drivers of the rural mortality penalty, few studies have attempted to tie delayed access to care in rural populations to healthcare outcomes quantitatively. Therefore, it is critical to try and understand these factors to enable more effective public health policy. Methods We performed a cross-sectional analysis of a population of patients with COVID-19 who were admitted to hospitals in the United States between 3/1/2020 and 2/26/2023 to better understand factors leading to outcome disparities amongst groups that all had some level of access to hospital care. Nevertheless, it is widely thought that rural populations often experience delayed access to care, due to transportation and other constraints. Therefore, we hypothesized that deteriorated patient condition at admission likely explained some of the observed difference in mortality between rural and urban populations. Results Our results supported our hypothesis, showing that the rural mortality penalty persists in this population and that by multiple measures, rural patients were likely to be admitted in worse condition, had worse overall health, and were older. Conclusions Although the pandemic threw the rural mortality penalty into sharp relief, it is important to remember that it existed prior to the pandemic and will continue to exist until effective interventions are implemented. This study demonstrates the critical need to address the underlying factors that resulted in rural-dwelling patients being admitted to the hospital in worse condition than their urban-dwelling counterparts during the COVID-19 pandemic, which likely affected other healthcare outcomes as well.

The future of humanity depends on successfully adapting key cropping systems for food security, such as corn (Zea mays L.), to global climatic changes, including changing air temperatures. We monitored the effects of climate change on harvested yields using long-term research plots that were established in 2001 near Fort Collins, Colorado, and long-term average yields in the region (county). We found that the average temperature for the growing period of the irrigated corn (May to September) has increased at a rate of 0.023 °C yr−1, going from 16.5 °C in 1900 to 19.2 °C in 2019 (p < 0.001), but precipitation did not change (p = 0.897). Average minimum (p < 0.001) temperatures were positive predictors of yields. This response to temperature depended on N fertilizer rates, with the greatest response at intermediate fertilizer rates. Maximum (p < 0.05) temperatures and growing degree days (GDD; p < 0.01) were also positive predictors of yields. We propose that the yield increases with higher temperatures observed here are likely only applicable to irrigated corn and that irrigation is a good climate change mitigation and adaptation practice. However, since pan evaporation significantly increased from 1949 to 2019 (p < 0.001), the region’s dryland corn yields are expected to decrease in the future from heat and water stress associated with increasing temperatures and no increases in precipitation. This study shows that increases in GDD and the minimum temperatures that are contributing to a changing climate in the area are important parameters that are contributing to higher yields in irrigated systems in this region.

The introduction of pluripotent stem cells into the field of disease modelling resulted in numerous opportunities to study and uncover disease mechanisms in a petri dish. This promising avenue has also been applied to model Marfan syndrome, a disease affecting multiple organ systems, including the skeletal and cardiovascular system. Marfan syndrome is caused by pathogenic variants in FBN1, the gene encoding for the extracellular matrix protein fibrillin-1 which ensembles into microfibrils. There is a poor genotype-phenotype correlation displayed by the diverse clinical manifestations of this disease in patients. Up to now, 52 different human pluripotent stem cells lines have been established and reported for Marfan syndrome. These stem cells have been employed to model aortopathy, skeletal abnormalities and cardiomyopathy in vitro. These models were able to recapitulate key features of the disease that are also observed in patients. The use of pluripotent stem cells will help to uncover disease mechanisms and to identify new therapeutic strategies in Marfan syndrome.

Introduction: Racial and ethnic disparities in the presentation and outcomes of lung cancer are widely known. To evaluate potential factors contributing to these observations, we measured systemic immune parameters in Black and White patients with lung cancer. Methods: Patients scheduled to receive cancer immunotherapy were enrolled in a multi-institutional prospective biospecimen collection registry. Clinical and demographic information were obtained from electronic medical records. Pretreatment peripheral blood samples were collected and analyzed for cytokines using a multiplex panel and for immune cell populations using mass cytometry. Differences between Black and White patients were determined and corrected for multiple comparisons. Results: A total of 187 patients with NSCLC (Black, 19; White, 168) were included in the analysis. Compared with White patients, Black patients had greater comorbidity (median Charlson Comorbidity Index 5 versus 3; p = 0.04) and were more likely to have received previous chemotherapy (79% versus 47%; p = 0.03). Black patients had significantly lower levels of CCL23 and CCL27 and significantly higher levels of CCL8, CXCL1, CCL26, CCL25, CCL1, IL-1b, CXCL16, and IFN-γ (all p < 0.05, false discovery rate < 0.1). Black patients also exhibited greater populations of nonclassical CD16+ monocytes, NKT-like cells, CD4+ cells, CD38+ monocytes, and CD57+ gamma delta T cells (all p < 0.05). Conclusions: Black and White patients with lung cancer exhibit several differences in immune parameters, with Black patients exhibiting greater levels of numerous proinflammatory cytokines and cell populations. The etiology and clinical significance of these differences warrant further evaluation.

Category: Trauma; Ankle Introduction/Purpose: To compare the quality of syndesmotic reduction with the ankle in maximal dorsiflexion versus neutral plantarflexion (normal resting position). Methods: Two fellowship-trained orthopaedic surgeons disrupted syndesmoses of 10 cadaveric ankle specimens from 5 donors. All ankles were initially placed in neutral plantarflexion and were subsequently reduced and stabilized with one 0.062-inch K-wire in a quadricortical fashion. Post-reduction computed tomography (CT) scans were then obtained. This process was repeated with the ankles placed in maximal dorsiflexion. Post-reduction CT scans were compared to baseline CT imaging obtained prior to syndesmotic disruption. Mixed-effects linear regression was used to assess differences between baseline scans and reduction in neutral plantarflexion and maximal dorsiflexion with significance set at P< 0.05. Results: Syndesmotic reduction and stabilization in maximal dorsiflexion led to increased external rotation of the fibula compared to baseline scans [13.0 degrees ± 5.4 degrees (mean ± SD) vs. 7.5 degrees ± 2.4 degrees, P=0.002]. There was a tendency towards lateral translation of the fibula with the ankle placed in maximal dorsiflexion (3.3 mm ± 1.0 mm vs. 2.7 mm ± 0.7 mm, P=0.096). No other statistically significant differences between measurements of reduction with the ankle placed in neutral plantarflexion or maximal dorsiflexion and baseline were present (P>0.05). Conclusion: Reducing the syndesmosis with the ankle in maximal dorsiflexion leads to malreduction with external rotation of the fibula. There was no statistically significant difference in reduction quality with the ankle placed in neutral plantarflexion. Future studies should assess the clinical implications of ankle positioning during syndesmotic fixation.

Background The use of immune checkpoint inhibitors (CPIs) has become a dominant regimen in modern cancer therapy, however immune resistance induced by tumor-associated macrophages (TAMs) with immune suppressive and evasion properties limits responses. Therefore, the rational design of immune modulators that can control the immune suppressive properties of TAMs and polarize them, as well as dendritic cells (DCs), toward a more proinflammatory phenotype is a principal objective in cancer immunotherapy.Methods Here, using a protein engineering approach to enhance cytokine residence in the tumor microenvironment, we examined combined stimulation of the myeloid compartment via tumor stroma-binding granulocyte–macrophage colony-stimulating factor (GM-CSF) to enhance responses in both DCs and T cells via stroma-binding interleukin-12 (IL-12). We evaluated tumor responses at the levels of outcome, cellular responses, and cytokine responses in both the tumors and the tumor-draining lymph nodes. We further investigated the potentiation of DC response to IL-12 by GM-CSF stimulation ex vivo.Results Engineered GM-CSF restored an antitumorigenic tumor myeloid microenvironment otherwise suppressed by TAMs, while engineered IL-12 provided effector signals to T cells, thereby boosting both tumor-resident antitumor macrophage and CD8+ T cell populations. Furthermore, engineered GM-CSF potentiated DC response to IL-12, upregulating DC expression of IL-12 receptor and enhancing their expression of proinflammatory cytokines and chemokines on IL-12 exposure. This resulted in remarkable synergistic efficacy in multiple solid tumor models treated with the dual cytokine combination. The combination therapy also improved the efficacy of CPI in a CPI-resistant genetically-engineered melanoma model and exhibited synergistic antitumor efficacy in a pulmonary metastasis model.Conclusion Our strategy provides a rational design for combination immunotherapy targeting both myeloid and lymphoid compartments through complementary mechanisms.

Category: Ankle Arthritis; Trauma Introduction/Purpose: Intraarticular pilon fractures have a high rate of posttraumatic osteoarthritis (PTOA) despite anatomic reduction of the articular surface. Elevated synovial inflammatory proteins triggered by injury contribute to PTOA development.MicroRNAs (miRNA), regulators of gene expression, are dysregulated in the cartilage of osteoarthritic joints, and their modulation may deter cartilage destruction. Specifically, miRNA-146a is associated with normal cartilage development and prevents overexpression of inflammatory proteins in degenerative osteoarthritis but is yet to be studied in clinical PTOA. This novel study aims to determine the effect of acute intraarticular pilon fracture on miRNA-146a expression and its association with inflammatory proteins. Methods: Bilateral ankles of patients with unilateral pilon fracture were aspirated for synovial fluid at the time of external fixation and/or open reduction internal fixation (ORIF). After processing, synovial fluid samples were sequenced for small RNA analysis. Measured levels of miRNA-146a in injured ankles were compared to the contralateral uninjured ankles, which served as baseline controls. Results: Five patients with unilateral pilon fractures consented to participate in the study. miRNA-146a was readily detectable in all patient samples. Using uninjured ankles (n=5) as a control, mean fold miRNA-146a expression was 1.0 ±0.93 [Mean ± Standard Deviation (SD)]. In comparison, mean fold miRNA-146a in injured ankles (n=5) was 0.4 ± 0.53(p=0.07). Additional results will be analyzed and available to present at the 2024 AOFAS Annual Meeting. Conclusion: Though not statistically significant, our preliminary data demonstrated that miRNA-146a was lower in injured ankles when compared to uninjured ankles. This study aims to better characterize the effect of acute pilon fracture on miRNA-146 expression and its association with inflammatory proteins. Anticipated results will help elucidate the role of miRNA-146a in the pathogenesis of PTOA following pilon fracture, impacting future PTOA research on the diagnostic and therapeutic capabilities of miRNAs.

ABSTRACT Phylosymbiosis, the association between the phylogenetic relatedness of hosts and the composition of their microbial communities, is a widespread phenomenon in diverse animal taxa. However, the generality of the existence of such a pattern has been questioned in many animals across the tree of life, including small‐sized aquatic invertebrates. This study aims to investigate the microbial communities associated with poorly known marine interstitial nemerteans to uncover their microbiota diversity and assess the occurrence of phylosymbiosis. Specimens from various Central American sites were analyzed using morphology‐based taxonomy and molecular techniques targeting the host 18S rRNA gene whereas their microbial association was analyzed by targeting the prokaryotic 16S rRNA gene. Phylogenetic and statistical analyses were conducted to examine the potential effects of host nemertean taxa and sampling locations on the host‐associated microbial communities. The results provide compelling evidence of phylosymbiosis in meiofaunal nemertean species, highlighting the significant impact of host genetic relatedness on microbiome diversity in small‐sized animals. This finding supports previous studies that demonstrate how certain nemertean species harbor distinct microbial communities with functional and ecological implications. Given the remarkable diversity of meiofaunal animals—spanning numerous phyla with varying lifestyles and co‐existing in the same habitat—combined with advancements in multi‐omics approaches, there is a promising opportunity to deepen our understanding of the evolutionary and ecological interactions between hosts and their microbiota throughout the animal tree of life.

Abstract ESK‐001 is a highly selective allosteric inhibitor of tyrosine kinase 2 (TYK2), which plays an essential role in mediating cytokine signaling in multiple immune‐mediated diseases. In 2 phase I studies, a first‐in‐human single ascending dose (SAD) and multiple ascending dose (MAD) study and a multiple‐dose (MD) study, we evaluated the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of orally administered ESK‐001 in healthy participants using a randomized, double‐blind, placebo‐controlled study design. ESK‐001 was rapidly absorbed with systemic exposures generally increasing dose‐proportionally across all cohorts. The mean terminal half‐life ranged from 8 to 13 h with no to minimal accumulation of ESK‐001 following q.d. doses and ~2‐fold accumulation following Q12 doses. Less than 1% of unchanged ESK‐001 was eliminated in urine. ESK‐001 inhibited the downstream TYK2 pathway as shown by inhibition of pSTAT1 expression. Transcriptomic analysis of unstimulated whole blood samples confirmed dose‐dependent inhibition of Type I IFN‐induced genes and SIGLEC1, a novel TYK2‐responsive biomarker. By correlating PK exposure data with PD readouts, a strong PK/PD relationship was demonstrated. There were no deaths, serious treatment‐emergent adverse events (TEAEs), nor severe TEAEs, and most TEAEs were mild in severity. In conclusion, ESK‐001 was generally safe and well‐tolerated in healthy participants, showed linear dose‐dependent PK characteristics, and maximally inhibited TYK2‐dependent pathways with a predictable concentration‐dependent PK/PD relationship. These findings were used to select the dose range of ESK‐001 for the STRIDE phase II trial in plaque psoriasis and to support further clinical development of ESK‐001 in other TYK2‐mediated diseases.