Active Site Characterization of a Campylobacter jejuni Nitrate Reductase Variant Provides Insight into the Enzyme Mechanism.

Mo K-edge X-ray absorption spectroscopy (XAS) is used to probe the structure of wild-type Campylobacter jejuni nitrate reductase NapA and the C176A variant. The results of extended X-ray absorption fine structure (EXAFS) experiments on wt NapA support an oxidized Mo(VI) hexacoordinate active site coordinated by a single terminal oxo donor, four sulfur atoms from two separate pyranopterin dithiolene ligands, and an additional S atom from a conserved cysteine amino acid residue. We found no evidence of a terminal sulfido ligand in wt NapA. EXAFS analysis shows the C176A active site to be a 6-coordinate structure, and this is supported by EPR studies on C176A and small molecule analogs of Mo(V) enzyme forms. The SCys is replaced by a hydroxide or water ligand in C176A, and we find no evidence of a coordinated sulfhydryl (SH) ligand. Kinetic studies show that this variant has completely lost its catalytic activity toward nitrate. Taken together, the results support a critical role for the conserved C176 in catalysis and an oxygen atom transfer mechanism for the catalytic reduction of nitrate to nitrite that does not employ a terminal sulfido ligand in the catalytic cycle.

Synthetic MRI and MR Fingerprinting-Derived Relaxometry of Antenatal Human Brainstem Myelination: A Postmortem-Based Quantitative Imaging Study.

BACKGROUND AND PURPOSE: The radiologic evaluation of ongoing myelination is currently limited prenatally. Novel quantitative MR imaging modalities provide relaxometric properties that are linked to myelinogenesis. In this retrospective postmortem imaging study, the capability of Synthetic MR imaging and MR fingerprinting-derived relaxometry for tracking fetal myelin development was investigated. Moreover, the consistency of results for both MR approaches was analyzed. MATERIALS AND METHODS: In 26 cases, quantitative postmortem fetal brain MR data were available (gestational age range, 15 + 1 to 32 + 1; female/male ratio, 14/12). Relaxometric measurements (T1-/T2-relexation times) were determined in the medulla oblongata and the midbrain using Synthetic MR imaging/MR fingerprinting-specific postprocessing procedures (Synthetic MR imaging and MR Robust Quantitative Tool for MR fingerprinting). The Pearson correlations were applied to detect relationships between T1-relaxation times/T2-relaxation times metrics and gestational age at MR imaging. Intraclass correlation coefficients were calculated to assess the consistency of the results provided by both modalities. RESULTS: Both modalities provided quantitative data that revealed negative correlations with gestational age at MR imaging: Synthetic MR imaging-derived relaxation times (medulla oblongata [r = -0.459; P = .021]; midbrain [r = -0.413; P = .040]), T2-relaxation times (medulla oblongata [r = -0.625; P < .001]; midbrain [r = -0.571; P = .003]), and MR fingerprinting-derived T1-relaxation times (medulla oblongata [r = -0.433; P = .035]; midbrain [r = -0.386; P = .062]), and T2-relaxation times (medulla oblongata [r =-0.883; P < .001]; midbrain [r = -0.890; P < .001]).The intraclass correlation coefficient analysis for result consistency between both MR approaches ranged between 0.661 (95% CI, 0.351-0.841) (T2-relaxation times: medulla oblongata) and 0.920 (95% CI, 0.82-0.965) (T1-relaxation times: midbrain). CONCLUSIONS: There is a good-to-excellent consistency between postmortem Synthetic MR imaging and MR fingerprinting myelin quantifications in fetal brains older than 15 + 1 gestational age. The strong correlations between quantitative myelin metrics and gestational age indicate the potential of quantitative MR imaging to identify delayed or abnormal states of myelination at prenatal stages of cerebral development.

Patient-derived mini-colons enable long-term modeling of tumor-microenvironment complexity.

Existing organoid models fall short of fully capturing the complexity of cancer because they lack sufficient multicellular diversity, tissue-level organization, biological durability and experimental flexibility. Thus, many multifactorial cancer processes, especially those involving the tumor microenvironment, are difficult to study ex vivo. To overcome these limitations, we herein implemented tissue-engineering and microfabrication technologies to develop topobiologically complex, patient-specific cancer avatars. Focusing on colorectal cancer, we generated miniature tissues consisting of long-lived gut-shaped human colon epithelia ('mini-colons') that stably integrate cancer cells and their native tumor microenvironment in a format optimized for real-time, high-resolution evaluation of cellular dynamics. We demonstrate the potential of this system through several applications: a comprehensive evaluation of drug effectivity, toxicity and resistance in anticancer therapies; the discovery of a mechanism triggered by cancer-associated fibroblasts that drives cancer invasion; and the identification of immunomodulatory interactions among different components of the tumor microenvironment. Similar approaches should be feasible for diverse tumor types.

SMIM1 absence is associated with reduced energy expenditure and excess weight.

BACKGROUND: Obesity rates have nearly tripled in the past 50 years, and by 2030 more than 1 billion individuals worldwide are projected to be obese. This creates a significant economic strain due to the associated non-communicable diseases. The root cause is an energy expenditure imbalance, owing to an interplay of lifestyle, environmental, and genetic factors. Obesity has a polygenic genetic architecture; however, single genetic variants with large effect size are etiological in a minority of cases. These variants allowed the discovery of novel genes and biology relevant to weight regulation and ultimately led to the development of novel specific treatments. METHODS: We used a case-control approach to determine metabolic differences between individuals homozygous for a loss-of-function genetic variant in the small integral membrane protein 1 (SMIM1) and the general population, leveraging data from five cohorts. Metabolic characterization of SMIM1-/- individuals was performed using plasma biochemistry, calorimetric chamber, and DXA scan. FINDINGS: We found that individuals homozygous for a loss-of-function genetic variant in SMIM1 gene, underlying the blood group Vel, display excess body weight, dyslipidemia, altered leptin to adiponectin ratio, increased liver enzymes, and lower thyroid hormone levels. This was accompanied by a reduction in resting energy expenditure. CONCLUSION: This research identified a novel genetic predisposition to being overweight or obese. It highlights the need to investigate the genetic causes of obesity to select the most appropriate treatment given the large cost disparity between them. FUNDING: This work was funded by the National Institute of Health Research, British Heart Foundation, and NHS Blood and Transplant.

Proceedings of the dengue endgame summit: Imagining a world with dengue control.

The first dengue "endgame" summit was held in Syracuse, NY over August 9 and 10, 2023. Organized and hosted by the Institute for Global Health and Translational Sciences at SUNY Upstate Medical University, the gathering brought together researchers, clinicians, drug and vaccine developers, government officials, and other key stakeholders in the dengue field for a highly collaborative and discussion-oriented event. The objective of the gathering was to discuss the current state of dengue around the world, what dengue "control" might look like, and what a potential roadmap might look like to achieve functional dengue control. Over the course of 7 sessions, speakers with a diverse array of expertise highlighted both current and historic challenges associated with dengue control, the state of dengue countermeasure development and deployment, as well as fundamental virologic, immunologic, and medical barriers to achieving dengue control. While sustained eradication of dengue was considered challenging, attendees were optimistic that significant reduction in the burden of dengue can be achieved by integration of vector control with effective application of therapeutics and vaccines.

Neuronal Ceroid Lipofuscinosis in a Mixed-Breed Dog with a Splice Site Variant in CLN6.

A 23-month-old neutered male dog of unknown ancestry presented with a history of progressive neurological signs that included anxiety, cognitive impairment, tremors, seizure activity, ataxia, and pronounced visual impairment. The clinical signs were accompanied by global brain atrophy. Due to progression in the severity of disease signs, the dog was euthanized at 26 months of age. An examination of the tissues collected at necropsy revealed dramatic intracellular accumulations of autofluorescent inclusions in the brain, retina, and cardiac muscle. The inclusions were immunopositive for subunit c of mitochondrial ATP synthase, and their ultrastructural appearances were similar to those of lysosomal storage bodies that accumulate in some neuronal ceroid lipofuscinosis (NCL) diseases. The dog also exhibited widespread neuroinflammation. Based on these findings, the dog was deemed likely to have suffered from a form of NCL. A whole genome sequence analysis of the proband's DNA revealed a homozygous C to T substitution that altered the intron 3-exon 4 splice site of CLN6. Other mutations in CLN6 cause NCL diseases in humans and animals, including dogs. The CLN6 protein was undetectable with immunolabeling in the tissues of the proband. Based on the clinical history, fluorescence and electron-microscopy, immunohistochemistry, and molecular genetic findings, the disorder in this dog was classified as an NCL resulting from the absence of the CLN6 protein. Screening the dog's genome for a panel of breed-specific polymorphisms indicated that its ancestry included numerous breeds, with no single breed predominating. This suggests that the CLN6 disease variant is likely to be present in other mixed-breed dogs and at least some ancestral breeds, although it is likely to be rare since other cases have not been reported to date.

Embedded 3D printing of UV-curable thermosetting composites with continuous fiber.

Extrusion-based 3D printing methods with in-nozzle impregnation mechanisms have been extensively employed in the fabrication of continuous fiber composites. This study presents an innovative embedded 3D printing technique that addresses significant challenges associated with existing methods. The technique utilizes a deposition nozzle to precisely write continuous fibers below the resin. A laser beam is directed onto the resin surface, which simultaneously cures the resin around the fiber bundle. The printing method demonstrates its advantages in producing high-quality composite samples with well-aligned fibers, minimized void density, and outstanding mechanical properties. More importantly, it introduces several capabilities that are highly desirable in the fabrication of contemporary composites, but unattainable with existing methods, including the dynamic control of fiber volume fractions and the ability to change matrix materials during printing. Furthermore, it enables the printing of filaments along curved pathways and printing of overhanging filaments for hollow features without support materials. The developed printing method exhibits versatility in working with different commercially available feedstock resins and reinforcement fibers. It is anticipated to be an impactful approach for the future development of thermosetting composites with diverse structural or multifunctional applications.

Extended supercooled storage of red blood cells.

Red blood cell (RBC) transfusions facilitate many life-saving acute and chronic interventions. Transfusions are enabled through the gold-standard hypothermic storage of RBCs. Today, the demand for RBC units is unfulfilled, partially due to the limited storage time, 6 weeks, in hypothermic storage. This time limit stems from high metabolism-driven storage lesions at +1-6 °C. A recent and promising alternative to hypothermic storage is the supercooled storage of RBCs at subzero temperatures, pioneered by our group. Here, we report on long-term supercooled storage of human RBCs at physiological hematocrit levels for up to 23 weeks. Specifically, we assess hypothermic RBC additive solutions for their ability to sustain supercooled storage. We find that a commercially formulated next-generation solution (Erythro-Sol 5) enables the best storage performance and can form the basis for further improvements to supercooled storage. Our analyses indicate that oxidative stress is a prominent time- and temperature-dependent injury during supercooled storage. Thus, we report on improved supercooled storage of RBCs at -5 °C by supplementing Erythro-Sol 5 with the exogenous antioxidants, resveratrol, serotonin, melatonin, and Trolox. Overall, this study shows the long-term preservation potential of supercooled storage of RBCs and establishes a foundation for further improvement toward clinical translation.

Does the Paraphilia Scale Work for Everyone? Confirmatory Factor Analysis and Measurement Invariance Across Gender and Sexual Orientation Groups.

We conducted three studies to examine the factor structure and measurement invariance of the Paraphilia Scale, a measure of paraphilic interests used in multiple studies. In the first study, we conducted a confirmatory factor analysis (CFA) testing different a priori models with a community sample of 1,040 adults previously reported by Seto et al. (2021), and found support for a hierarchical four-factor model: An agonistic continuum involving coercion or physical pain (biastophilia, sexual sadism, masochism), chronophilias (pedophilia, hebephilia), courtship disorders (voyeurism, exhibitionism, and frotteurism), and fetishism (object fetishism, transvestic fetishism, urophilia-coprophilia). This factor structure was replicated in a second study comprising a combined sample of 400 mTurk participants and 870 university students. The third study analyzed the community sample and found evidence of configural invariance but not scalar or metric invariance across gender (man or woman) and sexual orientation for gender (heterosexual or other sexual orientation). This indicates that the factor structure of the Paraphilia Scale is robust for gender and sexual orientation for gender, but factor loadings differ across these groups, as do the loadings of individual items on the four factors. Implications for research on gender and sexual orientation differences in paraphilic interests are discussed.

Essential Fatty Acid Deficiency is Common after Total Pancreatectomy and Islet AutoTransplantation.

OBJECTIVES: Total pancreatectomy and islet autotransplantation (TPIAT) for pancreatitis may induce risk for essential fatty acid deficiency (EFAD) due to exocrine pancreatic insufficiency and intestinal alterations. The prevalence of EFAD post-TPIAT is currently unknown. METHODS: We abstracted essential fatty acid (EFA) profiles (n = 332 samples) for 197 TPIAT recipients (72% adult, 33% male). Statistical analyses determined the prevalence of, and associations with, EFAD post-operatively. EFAD was defined as a Triene-to-Tetraene ratio ≥ 0.05 if <18 years old, or ≥ 0.038 if ≥18 years old. RESULTS: Prevalence of EFAD was 33%, 49%, and 53.5% at 1, 2, and ≥ 3 years. At 1 year post-TPIAT, older age at transplant (p = 0.03), being an adult versus a child (p = 0.0024), and obstructive etiology (p = 0.0004) were significant predictors of EFAD. Only 6% of children had EFAD 1 year post-TPIAT vs. 46% of adults. ALA levels were lower with lower BMI at transplant (p = 0.011). EFAD was associated with the presence of other intestinal diseases (p < 0.0001). CONCLUSIONS: One-third of individuals had EFAD 1 year post-TPIAT, highlighting the need for systematic monitoring. Older age at transplant increased risk and adults were more affected than children. Other diagnoses affecting intestinal health may further increase risk for EFAD.

Thyroid Dysfunction Risk After Iodinated Contrast Media Administration: A Prospective Longitudinal Cohort Analysis.

CONTEXT: Iodinated contrast media (ICM) is a common source of excess iodine in medical settings, given the common use of iodinated radiologic procedures. OBJECTIVE: To determine the long-term risks of thyroid dysfunction following iodinated contrast administration in a prospective study. DESIGN, SETTING, PARTICIPANTS: A longitudinal cohort study was conducted of patients in the U.S. Veterans Affairs medical system who received ICM. MAIN OUTCOME MEASURES: Serum thyroid function, thyroid antibody, and inflammatory markers were measured at baseline. Thyroid function tests were repeated at 1 month, 3 months, and every 6 months thereafter until 36 months. Risk of thyroid dysfunction and longitudinal changes in thyroid hormone levels were assessed using mixed effect models. RESULTS: There were 122 participants (median age, 70.0 [IQR 62.2-74.0] years; 98.4% male). At baseline, six subjects had subclinical thyroid dysfunction prior to ICM receipt. During median follow-up of 18 months, iodine-induced thyroid dysfunction was observed in 11.5% (14/122); six (4.9%) developed hyperthyroidism (including one with overt hyperthyroidism) and eight (6.6%) subclinical hypothyroidism. At last follow-up, ten of 20 subjects with thyroid dysfunction (14 new-onset cases and six with preexisting thyroid dysfunction) had persistent subclinical hyperthyroidism or hypothyroidism. There were also subtle changes in thyroid hormones observed longitudinally within the reference ranges in the overall cohort. CONCLUSIONS: There is a rare long-term risk of an excess iodine load on thyroid dysfunction even among individuals from an overall iodine-sufficient region, supporting the need for targeted monitoring following iodinated contrast administration.

Improving Accuracy of Administrative Data for Perforated Appendicitis Classification.

INTRODUCTION: Reliance on International Classification of Diseases, Tenth Revision, Clinical Modification (ICD-10-CM) diagnosis codes may misclassify perforated appendicitis with resultant research, fiscal, and public health implications. We aimed to improve the accuracy of administrative data for perforated appendicitis classification relying on ICD-10-CM codes from 2015 to 2018. METHODS: We conducted a retrospective study of randomly sampled patients aged ≤18 years diagnosed with acute appendicitis from eight children's hospitals. Patients were identified using the Pediatric Health Information System, and true perforation status was determined by medical record review. We developed two algorithms by leveraging Pediatric Health Information System data elements and data mining (DM) approaches. The two developed algorithm performance was compared against algorithms that exclusively relied on ICD-10-CM codes using area under the curve and other measures. RESULTS: Of 1051 clinically validated encounters that were included, 383 (36.4%) patients were identified to have perforated appendicitis. The two algorithms developed using DM approaches primarily leveraged ICD-10-CM codes and length of stay. DM-developed algorithms had a significantly higher accuracy than algorithms relying exclusively on ICD-10-CM (P value < 0.01): sensitivity and specificity for DM-developed algorithms were 0.86-0.88 and 0.95-0.97, respectively, which were overall higher than algorithms that relied on only ICD-10-CM. CONCLUSIONS: This study provides an algorithm that can improve the accuracy of perforated appendicitis classification using commonly available elements in administrative data. We recommend that this algorithm is used in future appendicitis classification to ensure valid reporting, hospital-level benchmarking, and fiscal or public health assessments.

Hepatic steatosis induced by nicotine plus Coca-Cola™ is prevented by nicotinamide riboside (NR).

Introduction: Cigarettes containing nicotine (Nic) are a risk factor for the development of cardiovascular and metabolic diseases. We reported that Nic delivered via injections or e-cigarette vapor led to hepatic steatosis in mice fed with a high-fat diet. High-fructose corn syrup (HFCS) is the main sweetener in sugar-sweetened beverages (SSBs) in the US. Increased consumption of SSBs with HFCS is associated with increased risks of non-alcoholic fatty liver disease (NAFLD). Nicotinamide riboside (NR) increases mitochondrial nicotinamide adenine dinucleotide (NAD+) and protects mice against hepatic steatosis. This study evaluated if Nic plus Coca-Cola™ (Coke) with HFCS can cause hepatic steatosis and that can be protected by NR. Methods: C57BL/6J mice received twice daily intraperitoneal (IP) injections of Nic or saline and were given Coke (HFCS), or Coke with sugar, and NR supplementation for 10 weeks. Results: Our results show that Nic+Coke caused increased caloric intake and induced hepatic steatosis, and the addition of NR prevented these changes. Western blot analysis showed lipogenesis markers were activated (increased cleavage of the sterol regulatory element-binding protein 1 [SREBP1c] and reduction of phospho-Acetyl-CoA Carboxylase [p-ACC]) in the Nic+Coke compared to the Sal+Water group. The hepatic detrimental effects of Nic+Coke were mediated by decreased NAD+ signaling, increased oxidative stress, and mitochondrial damage. NR reduced oxidative stress and prevented mitochondrial damage by restoring protein levels of Sirtuin1 (Sirt1) and peroxisome proliferator-activated receptor coactivator 1-alpha (PGC1) signaling. Conclusion: We conclude that Nic+Coke has an additive effect on producing hepatic steatosis, and NR is protective. This study suggests concern for the development of NAFLD in subjects who consume nicotine and drink SSBs with HFCS.

Bile Acid Diarrhea Is Associated With an Increased Incidence of Gastrointestinal Cancers.

INTRODUCTION: Bile acid diarrhea (BAD) is an underrecognized and socially debilitating disease caused by high concentrations of bile acids in the colon. Bile acids directly and indirectly promote carcinogenesis. In this article, we investigated whether individuals with BAD have an increased risk of gastrointestinal (GI) cancers. METHODS: By using the Danish health registries, adult individuals with BAD were identified by International Classification of Diseases 10th revision code K90.8 or referral to the diagnostic 75selenium-homotaurocholic acid test followed by prescription of a bile acid sequestrant within 365 days (n = 5,245). Age- and sex-matched individuals without BAD were included for comparison (n = 52,450). We analyzed the cumulative incidence of GI cancers after BAD diagnosis and the odds ratios (ORs) of GI cancer 8 and 15 years before BAD diagnosis/matching. RESULTS: Cumulative incidence of GI cancer 6 years after BAD diagnosis/matching was 1.6% in the BAD group and 1.1% in controls ( P = 0.01). The ORs of total GI cancer 8 and 15 years before BAD diagnosis were 6.16 (5.08-7.48) and 5.19 (4.28-6.29), respectively. Furthermore, 47 individuals with BAD (0.9%) and 250 (0.5%) controls died of GI cancer. DISCUSSION: This nationwide cohort study indicates an association between BAD and GI cancers. We found both a higher incidence of GI cancer after BAD diagnosis compared with controls and increased OR of GI cancer before BAD diagnosis. Bearing in mind the underdiagnosis of BAD, the delay of BAD diagnosis, and the carcinogenic effect of bile acids, these findings warrant further investigations of the risk of GI cancer in individuals with BAD.

Manual scoring of periodic limb movements in children: is it still necessary?

OBJECTIVE: Although manual scoring has been classically considered the gold standard to identify periodic leg movements (PLM), it is a very time consuming and expensive process, also subject to variability in interpretation. In the last decades, different authors have observed reasonably good agreement between automated PSG scoring algorithms and manual scoring in adults, according to established criteria. We aim to compare the automatic software analysis of our polysomnogram with the manual staging in children with sleep-disordered breathing. METHODS: We performed a semiautomatic method, in which an experienced technician watched the video recording and removed from the automatic analysis those movements that did not correspond to true candidate leg movement (LM). RESULTS: A total of 131 PSGs were studied; applying the established criteria, 65 children were diagnosed of obstructive sleep apnea, and 66 presented snoring but with no sleep apnea. The mean age was 6.7 years (±1.7) and twenty-five children (19.08 %) had a PLMI >5/h. Statistical differences were found not only for PLMI (manual: 2.20 (0.7, 4.1) vs automatic (6.4 (3.85,9.5); p < 0.001), but for almost of all indexes assessed between the automatic and the manual scoring analysis. The level of concordance was only moderate for PLM index (0.63 [0.51-0.72]); showing that, unlike the articles published in the adult population, automatic analysis is not accurate in children and, manually or semi-automatically analysis as ours need to be done. CONCLUSION: It seems that PLM detection algorithm might work accurately but, the real need would be a true LM detection algorithm.

Victim Sexual Arousal During Nonconsensual Sex: A Scoping Review.

Sexual arousal in male and female victims during nonconsensual sex is an understudied phenomenon with many potential psychological, clinical, and legal implications for survivors. The aim of this scoping review was to assess the literature to determine whether we could estimate the frequency and circumstances of physiological sexual arousal (e.g., erection, lubrication, ejaculation, orgasm) among victims during nonconsensual sex. Six reference database and hand searches led to the screening of 13,894 articles and other reports. Eight articles and one book published between 1977 and 2019 included relevant data from 136 male survivors and 250 female survivors. Results confirmed that physiological sexual arousal (only genital responses were mentioned) can occur in both male and female victims during nonconsensual sex. The frequency of these responses could not be determined because of the widely different methodologies used. In addition, it was not possible to determine the circumstances in which victim sexual arousal was more likely to occur although some were inferred. The results of the scoping review highlight that physiological sexual arousal during nonconsensual sex does occur for victims but has not been studied systematically. There is a clear need to properly assess the type, circumstances, consequences, and frequency of sexual arousal during nonconsensual sex in large and diverse populations of male and female survivors.