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Antibiotic resistance is a major worldwide concern, and new drugs with mechanistically novel modes of action are urgently needed. Here, we report the structure-based drug design, synthesis, and evaluation in vitro and in cellular systems of sialic acid derivatives able to inhibit the bacterial sialic acid symporter SiaT. We designed and synthesized 21 sialic acid derivatives and screened their affinity for SiaT by a thermal shift assay and elucidated the inhibitory mechanism through binding thermodynamics, computational methods, and inhibitory kinetic studies. The most potent compounds, which have a 180-fold higher affinity compared to the natural substrate, were tested in bacterial growth assays and indicate bacterial growth delay in methicillin-resistant

Supplementary Figure S1 presents the viability of SW48 and SW48PK cells after treatment with BEZ235 and TAK-228 at the 100-400nM concentrations.

PDF file - 30K, Relative gene expression is shown for three adenomas and three intramucosal carcinomas taken from the colons of DSS-treated F1 Min mice.

PDF file - 21K, Characteristics of F1 ApcMin/+ mice treated with 4% dextran sodium sulfate.

S1. AP spheroids were plated and allowed to mature for 48 hours. S2. To address the issue of potentially slower growth of the spheres with the largest initial sizes, a change-point analysis was performed. S3. AP mice were treated with control (A) or BEZ235 (B) for 14 days. S4. AP mice were treated with BEZ235 or control for 14 days. S5. AP spheroids were treated with NVP-BYL719, BEZ235, LY3023414, or control for 24 hours. S5. AP spheroids were treated with NVP-BYL719, BEZ235, LY3023414, or control for 24 hours. Supplementary Table S1. AP spheroids were treated with NVP-BYL719, GDC0941, BEZ235, LY3023414, or control for 48 hours. Supplemental Table S2. AP mice were treated with BEZ235, LY3023414 or control for 14 days.

PDF file - 54K, Panel A illustrates the colonoscopic surveillance protocol. Panel B shows the serial biopsy protocol.

PIK3CA mutations are common in clinical molecular profiling, yet an effective means to target these cancers has yet to be developed. MTORC1 inhibitors are often used off-label for patients with PIK3CA mutant cancers with only limited data to support this approach. Here we describe a cohort of patients treated with cancers possessing mutations activating the PI3K signaling cascade with minimal benefit to treatment with the MTORC1 inhibitor everolimus. Previously, we demonstrated that dual PI3K/mTOR inhibition could decrease proliferation, induce differentiation, and result in a treatment response in APC and PIK3CA mutant colorectal cancer. However, reactivation of AKT was identified, indicating that the majority of the benefit may be secondary to MTORC1/2 inhibition. TAK-228, an MTORC1/2 inhibitor, was compared with dual PI3K/mTOR inhibition using BEZ235 in murine colorectal cancer spheroids. A reduction in spheroid size was observed with TAK-228 and BEZ235 (−13% and −14%, respectively) compared with an increase of >200% in control (P < 0.001). These spheroids were resistant to MTORC1 inhibition. In transgenic mice possessing Pik3ca and Apc mutations, BEZ235 and TAK-228 resulted in a median reduction in colon tumor size of 19% and 20%, respectively, with control tumors having a median increase of 18% (P = 0.02 and 0.004, respectively). This response correlated with a decrease in the phosphorylation of 4EBP1 and RPS6. MTORC1/2 inhibition is sufficient to overcome resistance to everolimus and induce a treatment response in PIK3CA mutant colorectal cancers and deserves investigation in clinical trials and in future combination regimens.

PDF file - 42K, Complete list of differentially expressed genes between intramucosal carcinomas and adenomas.

Colorectal cancer often arises from adenomatous colonic polyps. Polyps can grow and progress to cancer, but may also remain static in size, regress, or resolve. Predicting which polyps progress and which remain benign is difficult. We developed a novel long-lived murine model of colorectal cancer with tumors that can be followed by colonoscopy. Our aim was to assess whether these tumors have similar growth patterns and histologic fates to human colorectal polyps to identify features to aid in risk stratification of colonic tumors. Long-lived ApcMin/+ mice were treated with dextran sodium sulfate to promote colonic tumorigenesis. Tumor growth patterns were characterized by serial colonoscopy with biopsies obtained for immunohistochemistry and gene expression profiling. Tumors grew, remained static, regressed, or resolved over time with different relative frequencies. Newly developed tumors demonstrated higher rates of growth and resolution than more established tumors that tended to remain static in size. Colonic tumors were hyperplastic lesions (3%), adenomas (73%), intramucosal carcinomas (20%), or adenocarcinomas (3%). Interestingly, the level of β-catenin was higher in adenomas that became intratumoral carcinomas than those that failed to progress. In addition, differentially expressed genes between adenomas and intramucosal carcinomas were identified. This novel murine model of intestinal tumorigenesis develops colonic tumors that can be monitored by serial colonoscopy, mirror growth patterns seen in human colorectal polyps, and progress to colorectal cancer. Further characterization of cellular and molecular features is needed to determine which features can be used to risk-stratify polyps for progression to colorectal cancer and potentially guide prevention strategies. Cancer Prev Res; 7(1); 105–13. ©2013 AACR.

PDF file - 28K, Differentially Expressed Gene Functional Categories between Adenomas and Intramucosal Carcinomas

Dot plots corresponding to population distribution curves and graphs of individual replicates for bar graphs in Figure 4.

Purpose:Cancer treatment is limited by inaccurate predictors of patient-specific therapeutic response. Therefore, some patients are exposed to unnecessary side effects and delays in starting effective therapy. A clinical tool that predicts treatment sensitivity for individual patients is needed.Experimental Design:Patient-derived cancer organoids were derived across multiple histologies. The histologic characteristics, mutation profile, clonal structure, and response to chemotherapy and radiation were assessed using bright-field and optical metabolic imaging on spheroid and single-cell levels, respectively.Results:We demonstrate that patient-derived cancer organoids represent the cancers from which they were derived, including key histologic and molecular features. These cultures were generated from numerous cancers, various biopsy sample types, and in different clinical settings. Next-generation sequencing reveals the presence of subclonal populations within the organoid cultures. These cultures allow for the detection of clonal heterogeneity with a greater sensitivity than bulk tumor sequencing. Optical metabolic imaging of these organoids provides cell-level quantification of treatment response and tumor heterogeneity allowing for resolution of therapeutic differences between patient samples. Using this technology, we prospectively predict treatment response for a patient with metastatic colorectal cancer.Conclusions:These studies add to the literature demonstrating feasibility to grow clinical patient-derived organotypic cultures for treatment effectiveness testing. Together, these culture methods and response assessment techniques hold great promise to predict treatment sensitivity for patients with cancer undergoing chemotherapy and/or radiation.

Supplementary Methods

Success rates for growing human cancers in spheroid culture

Supplementary Tables 1-9 from Genome-Wide Expression Profiling Reveals EBV-Associated Inhibition of MHC Class I Expression in Nasopharyngeal Carcinoma

Supplementary Methods, Figures 1-6 from Fundamental Differences in Cell Cycle Deregulation in Human Papillomavirus–Positive and Human Papillomavirus–Negative Head/Neck and Cervical Cancers

To identify the molecular mechanisms by which EBV-associated epithelial cancers are maintained, we measured the expression of essentially all human genes and all latent EBV genes in a collection of 31 laser-captured, microdissected nasopharyngeal carcinoma (NPC) tissue samples and 10 normal nasopharyngeal tissues. Global gene expression profiles clearly distinguished tumors from normal healthy epithelium. Expression levels of six viral genes (EBNA1, EBNA2, EBNA3A, EBNA3B, LMP1, and LMP2A) were correlated among themselves and strongly inversely correlated with the expression of a large subset of host genes. Among the human genes whose inhibition was most strongly correlated with increased EBV gene expression were multiple MHC class I HLA genes involved in regulating immune response via antigen presentation. The association between EBV gene expression and inhibition of MHC class I HLA expression implies that antigen display is either directly inhibited by EBV, facilitating immune evasion by tumor cells, and/or that tumor cells with inhibited presentation are selected for their ability to sustain higher levels of EBV to take maximum advantage of EBV oncogene-mediated tumor-promoting actions. Our data clearly reflect such tumor promotion, showing that deregulation of key proteins involved in apoptosis (BCL2-related protein A1 and Fas apoptotic inhibitory molecule), cell cycle checkpoints (AKIP, SCYL1, and NIN), and metastasis (matrix metalloproteinase 1) is closely correlated with the levels of EBV gene expression in NPC. (Cancer Res 2006; 66(16): 7999-8006)

Supplementary Methods from Genome-Wide Expression Profiling Reveals EBV-Associated Inhibition of MHC Class I Expression in Nasopharyngeal Carcinoma

Human papillomaviruses (HPV) are associated with nearly all cervical cancers, 20% to 30% of head and neck cancers (HNC), and other cancers. Because HNCs also arise in HPV-negative patients, this type of cancer provides unique opportunities to define similarities and differences of HPV-positive versus HPV-negative cancers arising in the same tissue. Here, we describe genome-wide expression profiling of 84 HNCs, cervical cancers, and site-matched normal epithelial samples in which we used laser capture microdissection to enrich samples for tumor-derived versus normal epithelial cells. This analysis revealed that HPV+ HNCs and cervical cancers differed in their patterns of gene expression yet shared many changes compared with HPV− HNCs. Some of these shared changes were predicted, but many others were not. Notably, HPV+ HNCs and cervical cancers were found to be up-regulated in their expression of a distinct and larger subset of cell cycle genes than that observed in HPV− HNC. Moreover, HPV+ cancers overexpressed testis-specific genes that are normally expressed only in meiotic cells. Many, although not all, of the hallmark differences between HPV+ HNC and HPV− HNC were a direct consequence of HPV and in particular the viral E6 and E7 oncogenes. This included a novel association of HPV oncogenes with testis-specific gene expression. These findings in primary human tumors provide novel biomarkers for early detection of HPV+ and HPV− cancers, and emphasize the potential value of targeting E6 and E7 function, alone or combined with radiation and/or traditional chemotherapy, in the treatment of HPV+ cancers. [Cancer Res 2007;67(10):4605–19]

Rheumatoid arthritis (RA) patients often develop rheumatoid factors (RFs), antibodies that bind IgG Fc, and anti-modified protein antibodies (AMPAs), multireactive autoantibodies that commonly bind citrullinated, homocitrullinated, and acetylated antigens. Recently, antibodies that bind citrulline-containing IgG epitopes were discovered in RA, suggesting that additional undiscovered IgG epitopes could exist and that IgG could be a shared antigen for RFs and AMPAs. This study was undertaken to reveal new IgG epitopes in rheumatic disease and to determine if multireactive AMPAs bind IgG.Using sera from patients with RA, systemic lupus erythematosus, Sjögren's disease (SjD), or spondyloarthropathy, IgG binding to native, citrulline-containing, and homocitrulline-containing linear epitopes of the IgG constant region was evaluated by peptide array, with highly bound epitopes further evaluated by enzyme-linked immunosorbent assay (ELISA). Binding of monoclonal AMPAs to IgG-derived peptides and IgG Fc was also evaluated by ELISA.Seropositive RA sera showed high IgG binding to multiple citrulline- and homocitrulline-containing IgG-derived peptides, whereas anti-SSA+ sera from SjD patients showed consistent binding to a single linear native epitope of IgG in the hinge region. Monoclonal AMPAs bound citrulline- and homocitrulline-containing IgG peptides and modified IgG Fc.The repertoire of epitopes bound by AMPAs includes modified IgG epitopes, positioning IgG as a common antigen that connects the otherwise divergent reactivities of RFs and AMPAs.

Antibody responses to off-target cancer-associated proteins have been detected following immunotherapies for cancer, suggesting these may be the result of antigen spread. We have previously reported that serum antibodies to prostate cancer-associated proteins were detectable using a high-throughput peptide array. We hypothesized that the breadth of antibody responses elicited by a vaccine could serve as a measure of the magnitude of its induced antigen spread. Consequently, sera from patients with prostate cancer, treated prior to or after vaccination in one of four separate clinical trials, were evaluated for antibody responses to an array of 177,604 peptides derived from over 1600 prostate cancer-associated gene products. Antibody responses to the same group of 5680 peptides previously reported were identified following vaccinations in which patients were administered GM-CSF as an adjuvant, but not with vaccine in the absence of GM-CSF. Hence, antibody responses to off-target proteins following vaccination may not necessarily serve as evidence of antigen spread and must be interpreted with particular caution following vaccine strategies that use GM-CSF, as GM-CSF appears to have direct effects on the production of antibodies. The evaluation of T cell responses to non-target antigens is likely a preferred approach for detection of immune-mediated antigen spread.

Colorectal cancer (CRC) is a leading cause of cancer death in the United States. Standard treatment for advanced stage CRC for decades has included 5-fluorouracil based chemotherapy. More recently, targeted therapies for metastatic CRC are being used based on the individual cancer's molecular profile. In the past few years, several different molecular subtype schemes for human CRC have been developed. The molecular subtypes can be distinguished by gene expression signatures and have the potential to be used to guide treatment decisions. However, many subtyping classification methods were developed using mRNA expression levels of hundreds to thousands of genes, making them impractical for clinical use. In this study, we assessed whether an immunohistochemical approach could be used for molecular subtyping of colorectal cancers. We validated two previously published, independent sets of immunohistochemistry classifiers and modified the published methods to improve the accuracy of the scoring methods. In addition, we evaluated whether protein and genetic signatures identified originally in the mouse were linked to clinical outcomes of patients with CRC. We found that low DDAH1 or low GAL3ST2 protein levels in human CRCs correlate with poor patient outcome. The results of this study have the potential to impact methods for determining the prognosis and therapy selection for CRC patients.

In bacteria and archaea, tripartite ATP-independent periplasmic (TRAP) transporters uptake essential nutrients. TRAP transporters receive their substrates via a secreted soluble substrate-binding protein. How a sodium ion-driven secondary active transporter is strictly coupled to a substrate-binding protein is poorly understood. Here we report the cryo-EM structure of the sialic acid TRAP transporter SiaQM from Photobacterium profundum at 2.97 Å resolution. SiaM comprises a “transport” domain and a “scaffold” domain, with the transport domain consisting of helical hairpins as seen in the sodium ion-coupled elevator transporter VcINDY. The SiaQ protein forms intimate contacts with SiaM to extend the size of the scaffold domain, suggesting that TRAP transporters may operate as monomers, rather than the typically observed oligomers for elevator-type transporters. We identify the Na+ and sialic acid binding sites in SiaM and demonstrate a strict dependence on the substrate-binding protein SiaP for uptake. We report the SiaP crystal structure that, together with docking studies, suggest the molecular basis for how sialic acid is delivered to the SiaQM transporter complex. We thus propose a model for substrate transport by TRAP proteins, which we describe herein as an ‘elevator-with-an-operator’ mechanism.

Anterior cruciate ligament rupture (ACLR) is a risk factor for the development of post-traumatic osteoarthritis (PTOA). While PTOA in the tibiofemoral joint compartment is well-characterized, very little is known about pathology in the patellofemoral compartment after ACL injury. Here, we evaluated the extent to which ACLR induces early patellofemoral joint damage in a rat model.Adult female Lewis rats were randomized to noninvasive ACLR or Sham. Two weeks post-injury, contrast-enhanced micro-computed tomography (µCT) of femoral and patellar cartilage was performed using 20% v/v ioxaglate. Morphometric parameters of femoral trochlear and patellar cartilage, subchondral bone, and trabecular bone were derived from µCT. Sagittal Safranin-O/Fast-Green-stained histologic sections were graded using the OARSI score in a blinded fashion.Cartilage and bone remodelling consistent with an early PTOA phenotype were observed in both femoral trochleas and patellae. ACLR caused osteophyte formation of the patella and pathology in the superficial zone of articular cartilage, including surface fibrillation, fissures, increased cellularity, and abnormal chondrocyte clustering. There were significant increases in thickness of patellar and trochlear cartilage. Loss of subchondral bone thickness, bone volume fraction, and tissue mineral density, as well as changes to patellar and trochlear trabecular microarchitecture, were indicative of catabolic bone remodelling. Several injury-induced changes, including increased cartilage thickness and trabecular spacing and decreased trabecular number were more severe in the patella compared to the trochlea.The patellofemoral joint develops mild but evident pathology in the early period following ACL rupture, extending the utility of this model to the study of patellofemoral PTOA.

We report the realization of an acoustic capacitive microphone formed by graphene/poly(methyl methacrylate) (PMMA). It is the first time that the ultra-large graphene/PMMA membrane suspended fully over the cavity has been fabricated by releasing the silicon dioxide sacrificial layer underneath the membrane. The novelty in the fabrication method is that the silicon dioxide layer has been etched by hydrogen fluoride vapor from the back of the partly etched silicon substrate. Using the new process, the ultra-large graphene/PMMA membrane, with a diameter to thickness ratio of 7800, has been suspended over the cavity with a 2 μm air gap. The spacing of 2 μm is the minimum gap over the graphene-based acoustic capacitive microphones which have been reported so far. The static deformation of the suspended graphene/PMMA membrane after silicon dioxide has been etched is estimated to be 270 nm. The aspect ratio of the membrane's diameter over its static deformation is around 13,000, which shows that the graphene/PMMA membrane with a diameter of a few millimeters can be transferred and suspended over the substrate with relatively small deformation by releasing the sacrificial silicon dioxide layer. The dynamic behavior of the device under electrostatic actuation has been characterized. The acoustic response of the graphene/PMMA capacitive microphone has been measured, and the sensitivity has been observed to be -47.5 dB V (4.22 mV/Pa) ± 10%. The strain in the graphene/PMMA membrane is estimated to be 0.034%.

Current jurisprudential trends empower the International Criminal Court (ICC) Prosecutor to override domestic investigative authorities in a manner that violates the letter and spirit of the Rome Statute. Sovereign states have primary responsibility to document, investigate and prevent atrocity crimes. Yet, current ICC practice subverts domestic enforcement efforts. No provision of the Rome Statute permits the Office of the Prosecutor (OTP) to substitute its unfettered judgment over the good-faith discretion of domestic prosecutors. ICC judges have created de facto institutional jurisdictional primacy by relying upon mere assertions regarding the insufficiency of domestic efforts. This trend is particularly problematic at the liminal phase from the preliminary examination (PE) to an authorised investigation because OTP policy preferences supersede good-faith domestic investigations and prosecutorial assessments. Juridical templates for assessing admissibility have been extrapolated from later phases of particularised cases into the PE phase. Current practice effectively eliminates sovereign prosecutorial discretion. Good-faith exercises of domestic prosecutorial discretion should not be constrained by post hoc Court-created straitjackets. This article dissects this problematic arc and proffers a model for harmonising domestic investigative efforts within the structure and intent of the Rome Statute. Its conclusions recommend reforms to ameliorate a foreseeable crisis of cooperation that could cripple an unreformed Court.

Summary Peptide microarrays have emerged as a powerful technology in immunoproteomics as they provide a tool to measure the abundance of different antibodies in patient serum samples. The high dimensionality and small sample size of many experiments challenge conventional statistical approaches, including those aiming to control the false discovery rate (FDR). Motivated by limitations in reproducibility and power of current methods, we advance an empirical Bayesian tool that computes local FDR statistics and local false sign rate statistics when provided with data on estimated effects and estimated standard errors from all the measured peptides. As the name suggests, the MixTwice tool involves the estimation of two mixing distributions, one on underlying effects and one on underlying variance parameters. Constrained optimization techniques provide for model fitting of mixing distributions under weak shape constraints (unimodality of the effect distribution). Numerical experiments show that MixTwice can accurately estimate generative parameters and powerfully identify non-null peptides. In a peptide array study of rheumatoid arthritis, MixTwice recovers meaningful peptide markers in one case where the signal is weak, and has strong reproducibility properties in one case where the signal is strong. Availabilityand implementation MixTwice is available as an R software package https://cran.r-project.org/web/packages/MixTwice/. Supplementary information Supplementary data are available at Bioinformatics online.

Non-dispersive infra-red (NDIR) detectors have become the dominant method for measuring atmospheric CO2, which is thought to be an important gas for honeybee colony health. In this work we describe a microcontroller-based system used to collect data from Senserion SCD41 NDIR sensors placed in the crown boards and queen excluders of honeybee colonies. The same sensors also provide relative humidity and temperature data. Several months of data have been recorded from four different hives. The mass change measurements, from hive scales, when foragers leave the hive were compared with the data from the gas sensors. Our data suggest that it is possible to estimate the colony size from the change in measured CO2, however no such link with the humidity is observed. Data are presented showing the CO2 decreasing over many weeks as a colony dies.

Spatial transcriptomics is a powerful and widely used approach for profiling the gene expression landscape across a tissue with emerging applications in molecular medicine and tumor diagnostics. Recent spatial transcriptomics experiments utilize slides containing thousands of spots with spot-specific barcodes that bind RNA. Ideally, unique molecular identifiers (UMIs) at a spot measure spot-specific expression, but this is often not the case in practice due to bleed from nearby spots, an artifact we refer to as spot swapping. To improve the power and precision of downstream analyses in spatial transcriptomics experiments, we propose SpotClean, a probabilistic model that adjusts for spot swapping to provide more accurate estimates of gene-specific UMI counts. SpotClean provides substantial improvements in marker gene analyses and in clustering, especially when tissue regions are not easily separated. As demonstrated in multiple studies of cancer, SpotClean improves tumor versus normal tissue delineation and improves tumor burden estimation thus increasing the potential for clinical and diagnostic applications of spatial transcriptomics technologies.

Recent advances in spatially resolved transcriptomics technologies enable both the measurement of genome-wide gene expression profiles and their mapping to spatial locations within a tissue. A first step in spatial transcriptomics data analysis is identifying genes with expression that varies spatially, and robust statistical methods exist to address this challenge. While useful, these methods do not detect spatial changes in the coordinated expression within a group of genes. To this end, we present SpatialCorr, a method for identifying sets of genes with spatially varying correlation structure. Given a collection of gene sets pre-defined by a user, SpatialCorr tests for spatially induced differences in the correlation of each gene set within tissue regions, as well as between and among regions. An application to cutaneous squamous cell carcinoma demonstrates the power of the approach for revealing biological insights not identified using existing methods.

Melanoma cells exhibit phenotypic plasticity that allows transition from a proliferative and differentiated phenotype to a more invasive and undifferentiated or transdifferentiated phenotype often also associated with drug resistance. The mechanisms involved in melanoma phenotype plasticity and its role in drug resistance are not fully understood. We previously demonstrated that the MAPK inhibitors (MAPKi)-resistance phenotype is associated with decreased expression of stem cell proliferation genes and increased expression of MAPK inactivation genes, including dual specificity phosphatases (DUSPs). Several members of the DUSP family genes, specifically DUSP1, -3, -8 and -9, are expressed in primary and metastatic melanoma cell lines and pre-and post BRAFi treated patient tumor cells. Here, we show that was knockdown of DUSP1 or DUSP8 or treatment with BCI, a pharmacological inhibitor of DUSP1/6 decreases the survival of MAPKi-resistant cells and sensitizes them to MAPKi. We also show that nestin, a neural crest stem cell marker, and MAP2, a neuronal differentiation marker, are downregulated in MAPKi-resistant cells whereas GFAP, a glial cell marker, present in both MAPKi-sensitive and -resistant cells. Inhibition of DUSP1/6 upregulated nestin expression in MAPKi-sensitive cells and cells with acquired resistance. In contrast, treatment with BCI caused upregulation of MAP2 in MAPKi-sensitive cells and downregulation of both MAP2 and GFAP in all MAPKi-resistant cell lines. These data suggest that DUSP1/6 are involved in melanoma neuronal transdifferentiation serve as mechanistic link between melanoma drug resistance and melanoma differentiation and targets for treatment of MAPKi-resistant melanoma.

Background: Excessive Daytime Sleepiness (EDS) is positively associated with the risk of cardiovascular disease (CVD) events and CVD mortality. However, the association between EDS and subclinical markers of CVD, such as arterial stiffness, is not fully understood and could have implications for CVD screening. EDS is multifactorial in origin, thus we incorporated measures of exhaustion and fatigue in order to comprehensively assess the association of lethargy and central artery stiffness. We examined the separate relationships between EDS, exhaustion, fatigue, and aortic stiffness via carotid-femoral pulse wave velocity (cfPWV) in a large population-based study. Methods: A cross-sectional analysis of ARIC Study participants (n=2500, mean age: 79.7, 57.2% female) who underwent arterial stiffness measurements (cfPWV) and completed the Epworth Sleep Scale (ESS) questionnaire in 2016-2019. EDS was defined as ESS 11. Exhaustion was derived from the Center for Epidemiological Study’s Depression (CES-D) scale, with positive exhaustion corresponding to increased effort in daily activities and noticeable difficulty in getting “going’. Fatigue was assessed at annual follow up calls in 2012-2013 and calculated as a summary score with participants responses about feeling tired in various scenarios in the past 7 days noted as a numeric value (1-5: never, rarely, sometimes, often or always). We used multivariable linear regression to estimate the association between ESS, exhaustion, and fatigue with cfPWV, adjusting for age, race-study center, body mass index, diabetes, heart rate, mean arterial pressure, and blood-pressure medication use. We evaluated potential effect modification by race, sex, diabetes, and hypertension. Results are presented as unstandardized beta coefficients (β) and 95% confidence intervals (CI). Results: A total of 14.2% participants reported EDS, 17.2% reported exhaustion, and the average fatigue score was 8.0. ESS was not associated with cfPWV when adjusting for covariates (β; 95% CI: -1.4; -4.7, 1.9). Similarly, EDS was not associated with cfPWV (β; 95% CI: -4.9; -40.4, 30.6), nor was there an association between ESS quartiles and cfPWV. However, both exhaustion (β; 95% CI: 40.4; 7.8, 73.0) and fatigue (β; 95% CI: 5.4; 0.6, 10.3) were positively associated with cfPWV after adjusting for covariates. There was no evidence of effect modification by race, sex, diabetes, or hypertension. Conclusion: EDS was not associated with arterial stiffness. A (positive) association was observed for both exhaustion and fatigue with cfPWV, suggesting a need for further investigation of fatigue related conditions and arterial stiffness in older adults.

Rat models of lumbar intervertebral disc (IVD) degeneration are widely employed to characterize biologic-based therapeutics, but their anatomy and small size preclude consistent delivery of injectable therapeutics to the lumbar spine via the traditional posterolateral approach. Here, we describe our experience with a repeat ventral transperitoneal approach in female Lewis rats, enabling induction of IVD degeneration and later intervention via an injectable therapeutic. In the initial surgery, the ventral aspect of the L5/L6 IVD was accessed, and an annular defect was created using a #11 scalpel blade. Eight weeks after the initial surgery, follow-up surgery was performed via the same approach, and an injectable gelatin hydrogel was delivered using a 31G needle. A custom injection guard was developed to control injection depth, ensuring consistent delivery to the nucleus pulposus. Notable challenges associated with repeat surgery were increased tissue adhesion, intraoperative bleeding, and difficulty placing the injection guard due to mobile gastrointestinal tissues. Complication rates were 9.4% and 15.6% for the initial and repeat surgeries, respectively. The most frequent complications associated with repeat surgery were transient neuropraxia and significant intraoperative bleeding (6.3% each). The repeat transperitoneal approach is a reproducible method to facilitate both injury and later intervention in a female rat model of lumbar IVD degeneration.

In environments where glucose is limited, some pathogenic bacteria metabolize host-derived sialic acid as a nutrient source. N-Acetylmannosamine kinase (NanK) is the second enzyme of the bacterial sialic acid import and degradation pathway and adds phosphate to N-acetylmannosamine using ATP to prime the molecule for future pathway reactions. Sequence alignments reveal that Gram-positive NanK enzymes belong to the Repressor, ORF, Kinase (ROK) family, but many lack the canonical Zn-binding motif expected for this function, and the sugar-binding EXGH motif is altered to EXGY. As a result, it is unclear how they perform this important reaction. Here, we study the Staphylococcus aureus NanK (SaNanK), which is the first characterization of a Gram-positive NanK. We report the kinetic activity of SaNanK along with the ligand-free, N-acetylmannosamine-bound and substrate analog GlcNAc-bound crystal structures (2.33, 2.20, and 2.20 Å resolution, respectively). These demonstrate, in combination with small-angle X-ray scattering, that SaNanK is a dimer that adopts a closed conformation upon substrate binding. Analysis of the EXGY motif reveals that the tyrosine binds to the N-acetyl group to select for the "boat" conformation of N-acetylmannosamine. Moreover, SaNanK has a stacked arginine pair coordinated by negative residues critical for thermal stability and catalysis. These combined elements serve to constrain the active site and orient the substrate in lieu of Zn binding, representing a significant departure from canonical NanK binding. This characterization provides insight into differences in the ROK family and highlights a novel area for antimicrobial discovery to fight Gram-positive and S. aureus infections.