NOX1 is essential for TNFα-induced intestinal epithelial ROS secretion and inhibits M cell signatures.

OBJECTIVE: Loss-of-function mutations in genes generating reactive oxygen species (ROS), such as NOX1, are associated with IBD. Mechanisms whereby loss of ROS drive IBD are incompletely defined. DESIGN: ROS measurements and single-cell transcriptomics were performed on colonoids stratified by NOX1 genotype and TNFα stimulation. Clustering of epithelial cells from human UC (inflamed and uninflamed) scRNASeq was performed. Validation of M cell induction was performed by immunohistochemistry using UEA1 (ulex europaeus agglutin-1 lectin) and in vivo with DSS injury. RESULTS: TNFα induces ROS production more in NOX1-WT versus NOX1-deficient murine colonoids under a range of Wnt-mediated and Notch-mediated conditions. scRNASeq from inflamed and uninflamed human colitis versus TNFα stimulated, in vitro colonoids defines substantially shared, induced transcription factors; NOX1-deficient colonoids express substantially lower levels of STAT3 (signal transducer and activator of transcription 3), CEBPD (CCAAT enhancer-binding protein delta), DNMT1 (DNA methyltransferase) and HIF1A (hypoxia-inducible factor) baseline. Subclustering unexpectedly showed marked TNFα-mediated induction of M cells (sentinel cells overlying lymphoid aggregates) in NOX1-deficient colonoids. M cell induction by UEA1 staining is rescued with H2O2 and paraquat, defining extra- and intracellular ROS roles in maintenance of LGR5+ stem cells. DSS injury demonstrated GP2 (glycoprotein-2), basal lymphoplasmacytosis and UEA1 induction in NOX1-deficiency. Principal components analyses of M cell genes and decreased DNMT1 RNA velocity correlate with UC inflammation. CONCLUSIONS: NOX1 deficiency plus TNFα stimulation contribute to colitis through dysregulation of the stem cell niche and altered cell differentiation, enhancing basal lymphoplasmacytosis. Our findings prioritise ROS modulation for future therapies.

Evidence-based guideline: Neuromuscular ultrasound for the diagnosis of ulnar neuropathy at the elbow.

INTRODUCTION/AIMS: The purpose of this literature review is to develop an evidence-based guideline for the use of neuromuscular ultrasound in the diagnosis of ulnar neuropathy at the elbow (UNE). The proposed research question was: "In patients with suspected UNE, does ulnar nerve enlargement as measured with ultrasound accurately identify those patients with UNE?" METHODS: A systematic review and meta-analysis was performed, and studies were classified according to American Academy of Neurology criteria for rating articles for diagnostic accuracy. RESULTS: Based on Class I evidence in four studies, it is probable that neuromuscular ultrasound measurement of the ulnar nerve at the elbow, either of diameter or cross-sectional area (CSA), is accurate for the diagnosis of UNE. RECOMMENDATION: For patients with symptoms and signs suggestive of ulnar neuropathy, clinicians should offer ultrasonographic measurement of ulnar nerve cross-sectional area or diameter to confirm the diagnosis and localize the site of compression (Level B).

Prognostic implications of immediate PSA response to early salvage radiotherapy.

INTRODUCTION: Up to 25% of men with prostate cancer who undergo radical prostatectomy will recur. In this setting, salvage radiotherapy may cure patients with local recurrence, but is unable to cure those with occult metastatic disease. The objective of this study is to examine how prostate-specific antigen (PSA) response to radiotherapy predicts subsequent disease progression and survival. MATERIALS AND METHODS: Using a prospectively populated database of 3089 men who underwent open radical prostatectomy, 212 patients (7%) were identified who received early salvage radiotherapy for biochemical recurrence. The main outcome was time to disease progression after salvage radiotherapy. Patients were stratified by PSA response after radiotherapy: 1) PSA < 0.1 ng/mL, 2) persistently detectable PSA, and 3) rising PSA. RESULTS: Patients received salvage radiotherapy at a median PSA of 0.20 ng/mL (IQR 0.10-0.30 ng/mL). At a median follow up of 47.3 months, a total of 52 (25%) patients experienced disease progression. On multivariable analysis, both persistent PSA (HR 5.12; 95% CI 1.98-13.23) and rising PSA (HR 16.55; 95% CI 6.61-41.48) were associated with increased risk of disease progression compared to those with PSA < 0.1 ng/mL after adjusting for pre-radiotherapy PSA, Gleason score, margin status, stage, and time to radiotherapy. Only rising PSA was associated with an increased risk of cancer-specific and all-cause mortality. CONCLUSIONS: PSA response is associated with the risk of disease progression following salvage radiotherapy. This information can be used to counsel patients on the potential need for additional therapy and identify those at greatest risk for progression and cancer-related mortality.

Mutational analysis of pulmonary tumours with neuroendocrine features using targeted massive parallel sequencing: a comparison of a neglected tumour group.

BACKGROUND: Lung cancer is the leading cause of cancer-related deaths worldwide. The typical and atypical carcinoid (TC and AC), the large-cell neuroendocrine carcinoma (LCNEC) and the small-cell lung cancers (SCLC) are subgroups of pulmonary tumours that show neuroendocrine differentiations. With the rising impact of molecular pathology in routine diagnostics the interest for reliable biomarkers, which can help to differentiate these subgroups and may enable a more personalised treatment of patients, grows. METHODS: A collective of 70 formalin-fixed, paraffin-embedded (FFPE) pulmonary neuroendocrine tumours (17 TCs, 17 ACs, 19 LCNECs and 17 SCLCs) was used to identify biomarkers by high-throughput sequencing. Using the Illumina TruSeq Amplicon-Cancer Panel on the MiSeq instrument, the samples were screened for alterations in 221 mutation hot spots of 48 tumour-relevant genes. RESULTS: After filtering >26 000 detected variants by applying strict algorithms, a total of 130 mutations were found in 29 genes and 49 patients. Mutations in JAK3, NRAS, RB1 and VHL1 were exclusively found in SCLCs, whereas the FGFR2 mutation was detected in LCNEC only. KIT, PTEN, HNF1A and SMO were altered in ACs. The SMAD4 mutation corresponded to the TC subtype. We prove that the frequency of mutations increased with the malignancy of tumour type. Interestingly, four out of five ATM-mutated patients showed an additional alteration in TP53, which was by far the most frequently altered gene (28 out of 130; 22%). We found correlations between tumour type and IASLC grade for ATM- (P=0.022; P=0.008) and TP53-mutated patients (P<0.001). Both mutated genes were also associated with lymph node invasion and distant metastasis (P⩽0.005). Furthermore, PIK3CA-mutated patients with high-grade tumours showed a reduced overall survival (P=0.040) and the mutation frequency of APC and ATM in high-grade neuroendocrine lung cancer patients was associated with progression-free survival (PFS) (P=0.020). CONCLUSIONS: The implementation of high-throughput sequencing for the analysis of the neuroendocrine lung tumours has revealed that, even if these tumours encompass several subtypes with varying clinical aggressiveness, they share a number of molecular features. An improved understanding of the biology of neuroendocrine tumours will offer the opportunity for novel approaches in clinical management, resulting in a better prognosis and prediction of therapeutic response.

SOX4, SOX11 and PAX6 mRNA expression was identified as a (prognostic) marker for the aggressiveness of neuroendocrine tumors of the lung by using next-generation expression analysis (NanoString).

BACKGROUND: Neuroendocrine tumors of the lung (NELC) account for 25% of all lung cancer cases and transcription factors may drive dedifferentiation of these tumors. This study was conducted to identify supportive diagnostic and prognostic biomarkers. MATERIALS & METHODS: A total of 16 TC, 13 AC, 16 large cell neuroendocrine carcinomas and 15 small cell lung cancer were investigated for the mRNA expression of 11 transcription factors and related genes (MYB, MYBBP1A, OCT4, PAX6, PCDHB, RBP1, SDCBP, SOX2, SOX4, SOX11, TEAD2). RESULTS: SOX4 (p = 0.0002), SOX11 (p < 0.0001) and PAX6 (p = 0.0002) were significant for tumor type. Elevated PAX6 and SOX11 expression correlated with poor outcome in large cell neuroendocrine carcinomas and small cell lung cancer (p < 0.0001 and p = 0.0232, respectively) based on survival data of 34 patients (57%). CONCLUSION: Aggressiveness of NELC correlated with increasing expression of transcription factors. SOX11 seems to be a highly valuable diagnostic and prognostic marker for aggressive NELC.

Different micro-RNA expression profiles distinguish subtypes of neuroendocrine tumors of the lung: results of a profiling study.

MicroRNAs (miRNAs) are a class of small (∼22 nucleotides), non-coding, highly conserved single-stranded RNAs with posttranscriptional regulatory features, including the regulation of cell proliferation, differentiation, survival, and apoptosis. They are deregulated in a broad variety of tumors showing characteristic expression patterns and can, thus, be used as a diagnostic tool. In contrast to non-small cell carcinoma of the lung neuroendocrine lung tumors, encompassing typical and atypical carcinoids, small cell lung cancer and large cell neuroendocrine lung cancer, no data about deregulation of tumor entity-specific miRNAs are available to date. miRNA expression differences might give useful information about the biological characteristics of these tumors, as well as serve as helpful markers.In 12 pulmonary neuroendocrine tumors classified as either typical carcinoid, atypical, large cell neuroendocrine or small cell lung cancer, screening for 763 miRNAs known to be involved in pulmonary cancerogenesis was conducted by performing 384-well TaqMan low-density array real-time qPCR. In the entire cohort, 44 miRNAs were identified, which showed a significantly different miRNA expression. For 12 miRNAs, the difference was highly significant (P<0.01). Eight miRNAs showed a negative (miR-22, miR-29a, miR-29b, miR-29c, miR-367*; miR-504, miR-513C, miR-1200) and four miRNAs a positive (miR-18a, miR-15b*, miR-335*, miR-1201) correlation to the grade of tumor biology. The miRNAs let-7d; miR-19; miR-576-5p; miR-340*; miR-1286 are significantly associated with survival. Members of the miR-29 family seem to be extremely important in this group of tumors. We found a number of miRNAs, which showed a highly significant deregulation in pulmonary neuroendocrine tumors. Moreover, some of these deregulated miRNAs seem to allow discrimination of the various subtypes of pulmonary neuroendocrine tumors. Thus, the analysis of specific sets of miRNAs can be proposed as diagnostic and/or predictive markers in this group of neoplasias.

Multimodal single-cell analyses reveal mechanisms of perianal fistula in diverse patients with Crohn's disease.

BACKGROUND: Crohn's disease complicated by perianal fistulae is more prevalent and severe in patients of African ancestry. METHODS: We profiled single cells from diverse patients with Crohn's disease with perianal fistula from colorectal mucosa and fistulous tracts. Immunofluorescence was performed to validate predicted cell-cell interactions. Unstimulated monocytes were chronically cultured in diverse cohorts. A subset was analyzed by single-nucleus RNA + ATAC sequencing. FINDINGS: Fistulous tract cells from complete proctectomies demonstrated enrichment of myeloid cells compared to paired rectal tissues. Ligand-receptor analysis highlights myeloid-stromal cross-talk and cellular senescence, with cellular co-localization validated by immunofluorescence. Chitinase-3 like-protein-1 (CHI3L1) is a top upregulated gene in stromal cells from fistulae expressing both destructive and fibrotic gene signatures. Monocyte cultures from patients of African ancestry and controls demonstrated differences in CHI3L1 and oncostatin M (OSM) expression upon differentiation compared to individuals of European ancestry. Activating protein-1 footprints are present in ATAC-seq peaks in stress response genes, including CHI3L1 and OSM; genome-wide chromatin accessibility including JUN footprints was observed, consistent with reported mechanisms of inflammatory memory. Regulon analyses confirm known cell-specific transcription factor regulation and implicate novel ones in fibroblast subsets. All pseudo-bulked clusters demonstrate enrichment of genetic loci, establishing multicellular contributions. In the most significant African American Crohn's genetic locus, upstream of prostaglandin E receptor 4, lymphoid-predominant ATAC-seq peaks were observed, with predicted RORC footprints. CONCLUSIONS: Population differences in myeloid-stromal cross-talk implicate fibrotic and destructive fibroblasts, senescence, epigenetic memory, and cell-specific enhancers in perianal fistula pathogenesis. The transcriptomic and epigenetic data provided here may guide optimization of promising mesenchymal stem cell therapies for perianal fistula. FUNDING: This work was supported by grants U01DK062422, U24DK062429, and R01DK123758.

Does the presence of the palmaris longus tendon influence median nerve function?

INTRODUCTION: The objective of this study was to determine if the presence or absence of a palmaris longis tendon influenced the function of the median nerve across the wrist. The primary hypothesis was that the presence of a palmaris longus tendon would be associated with more median nerve entrapment at the wrist. METHODS: This was a cross-sectional study. Subjects were recruited at a screening of dental professionals. The median and ulnar sensory nerve latencies across the wrist (relative prolongation of the median compared with the ulnar) and the presence or absence of the palmaris longus tendon were the primary outcome measures. RESULTS: A total of 462 subjects were recruited into the study of which 16.2% lacked a palmaris longus tendon. There was no difference in the median nerve function or the percentage with a 0.5 ms prolongation of the median sensory latency when comparing subjects with and without a palmaris longus tendon. CONCLUSIONS: The presence of a palmaris longus tendon does not influence the median nerve function across the wrist.

Use of hand diagrams in screening for ulnar neuropathy: comparison with electrodiagnostic studies.

INTRODUCTION: The objective of this study was to determine whether a hand diagram could be used to predict ulnar mononeuropathy. METHODS: This was a prospective study of 117 consecutive patients referred for hand symptoms. Each subject filled out a hand diagram of symptoms and had median and ulnar sensory and motor nerve conduction studies, including ulnar conduction across the elbow. RESULTS: The best model for predicting an ulnar mononeuropathy included hand diagram scores of definite or possible. The model had a sensitivity of 50% and specificity of 93% with an ROC area of 0.90. CONCLUSIONS: The ulnar hand diagram scoring system can be useful as a screening tool in the electrodiagnostic laboratory or for epidemiologic studies.

Agreement between clinical screening procedures for neuropathy in the feet.

INTRODUCTION: The correlation between monofilament testing, symptom surveys, and electrodiagnostic studies for the diagnosis of axonal polyneuropathy has not been well studied. This investigation was done to assess the agreement between these procedures in a non-random sample of volunteers. METHODS: The procedures evaluated included electrodiagnostic tests of the sural nerve, monofilament testing of the great toe, a symptom survey, and a body diagram. Kappa coefficients and sensitivity and specificity, using nerve conduction as a "gold standard," were used to determine the agreement between various combinations of procedures. RESULTS: Poor agreement (kappa values -0.12-0.44) and sensitivity (sensitivity <30%) were found for all combinations of symptoms and monofilament results in comparison with sural peak latency and amplitude. CONCLUSIONS: Overall, the results demonstrated a low discriminatory power for the screening procedures for identifying persons with impaired sural nerve function. The results highlight the need for further development and evaluation of screening methods for distal neuropathy in population-based studies.

Differential aging of median and ulnar sensory nerve parameters.

INTRODUCTION: Nerve conduction velocity slows and amplitude declines with aging. METHODS: Median and ulnar sensory nerves were tested at the annual meetings of the American Dental Association. Seven hundred four subjects had at least two observations. The rate of change in the nerve parameters was estimated while controlling for gender, age, change in hand temperature, baseline body mass index (BMI), and change in BMI. RESULTS: Amplitudes of the median sensory nerve action potentials decreased by 0.58 µV per year, whereas conduction velocity decreased at a rate of 0.41 m/s per year. Corresponding values for the ulnar nerve were 0.89 µV and 0.29 m/s per year. The rates of change in amplitudes did not differ, but the median nerve demonstrated a more rapid loss of conduction velocity. CONCLUSIONS: The rate of change for the median conduction velocity was higher than previously reported. The rate of change of median conduction velocity was significantly greater than for the ulnar nerve.

Evidence-based guideline: neuromuscular ultrasound for the diagnosis of carpal tunnel syndrome.

INTRODUCTION: The purpose of this study was to develop an evidence-based guideline for the use of neuromuscular ultrasound in the diagnosis of carpal tunnel syndrome (CTS). METHODS: Two questions were asked: (1) What is the accuracy of median nerve cross-sectional area enlargement as measured with ultrasound for the diagnosis of CTS? (2) What added value, if any, does neuromuscular ultrasound provide over electrodiagnostic studies alone for the diagnosis of CTS? A systematic review was performed, and studies were classified according to American Academy of Neurology criteria for rating articles of diagnostic accuracy (question 1) and for screening articles (question 2). RESULTS: Neuromuscular ultrasound measurement of median nerve cross-sectional area at the wrist is accurate and may be offered as a diagnostic test for CTS (Level A). Neuromuscular ultrasound probably adds value to electrodiagnostic studies when diagnosing CTS and should be considered in screening for structural abnormalities at the wrist in those with CTS (Level B).

Compliance with referral of sick children: a survey in five districts of Afghanistan.

BACKGROUND: Recognition and referral of sick children to a facility where they can obtain appropriate treatment is critical for helping reduce child mortality. A well-functioning referral system and compliance by caretakers with referrals are essential. This paper examines referral patterns for sick children, and factors that influence caretakers' compliance with referral of sick children to higher-level health facilities in Afghanistan. METHODS: The study was conducted in 5 rural districts of 5 Afghan provinces using interviews with parents or caretakers in 492 randomly selected households with a child from 0 to 2 years old who had been sick within the previous 2 weeks with diarrhea, acute respiratory infection (ARI), or fever. Data collectors from local nongovernmental organizations used a questionnaire to assess compliance with a referral recommendation and identify barriers to compliance. RESULTS: The number of referrals, 99 out of 492 cases, was reasonable. We found a high number of referrals by community health workers (CHWs), especially for ARI. Caretakers were more likely to comply with referral recommendations from community members (relative, friend, CHW, traditional healer) than with recommendations from health workers (at public clinics and hospitals or private clinics and pharmacies). Distance and transportation costs did not create barriers for most families of referred sick children. Although the average cost of transportation in a subsample of 75 cases was relatively high (US$11.28), most families (63%) who went to the referral site walked and hence paid nothing. Most caretakers (75%) complied with referral advice. Use of referral slips by health care providers was higher for urgent referrals, and receiving a referral slip significantly increased caretakers' compliance with referral. CONCLUSIONS: Use of referral slips is important to increase compliance with referral recommendations in rural Afghanistan.

Glutathione enzyme and selenoprotein polymorphisms associate with mercury biomarker levels in Michigan dental professionals.

Mercury is a potent toxicant of concern to both the general public and occupationally exposed workers (e.g., dentists). Recent studies suggest that several genes mediating the toxicokinetics of mercury are polymorphic in humans and may influence inter-individual variability in mercury accumulation. This work hypothesizes that polymorphisms in key glutathione synthesizing enzyme, glutathione S-transferase, and selenoprotein genes underlie inter-individual differences in mercury body burden as assessed by analytical mercury measurement in urine and hair, biomarkers of elemental mercury and methylmercury, respectively. Urine and hair samples were collected from a population of dental professionals (n=515), and total mercury content was measured. Average urine (1.06±1.24 microg/L) and hair mercury levels (0.49±0.63 microg/g) were similar to national U.S. population averages. Taqman assays were used to genotype DNA from buccal swab samples at 15 polymorphic sites in genes implicated in mercury metabolism. Linear regression modeling assessed the ability of polymorphisms to modify the relationship between mercury biomarker levels and exposure sources (e.g., amalgams, fish consumption). Five polymorphisms were significantly associated with urine mercury levels (GSTT1 deletion), hair mercury levels (GSTP1-105, GSTP1-114, GSS 5'), or both (SEPP1 3'UTR). Overall, this study suggests that polymorphisms in selenoproteins and glutathione-related genes may influence elimination of mercury in the urine and hair or mercury retention following exposures to elemental mercury (via dental amalgams) and methylmercury (via fish consumption).

Electrodiagnostic evaluation of carpal tunnel syndrome.

Carpal tunnel syndrome (CTS) is the most common nerve entrapment. Electrodiagnostic (EDX) studies are a valid and reliable means of confirming the diagnosis. This monograph addresses the various EDX techniques used to evaluate the median nerve at the wrist. It also demonstrates the limitations of EDX studies with a focus on the sensitivity and specificity of EDX testing for CTS. The need to use reference values for populations such as diabetics and active workers, where normative values differ from conventional cutoffs used to confirm suspected CTS, is presented. The value of needle electromyography (EMG) is examined.

Risk factors for lower extremity fatigue among assembly plant workers.

BACKGROUND: Work-related fatigue of the lower extremities is a known cause of lost productivity and significant employer costs. Common workplace solutions to reduce fatigue levels include anti-fatigue matting, shoe orthoses, or sit/stand work stations. However, assessment of these anti-fatigue measures within the workplace has been limited. METHODS: This was a cross sectional study in an automotive assembly plant on employees with at least 6 months tenure. Subject data were collected via questionnaires including Likert-scale questions to define fatigue severity. Jobs were evaluated for lower extremity ergonomic exposures via videotaping, pedometers, interviews, and industrial engineering records. RESULTS: Lower extremity fatigue at the end of the work day was associated with a higher prevalence of smoking, rheumatoid arthritis, job dissatisfaction, use of shoes with firmer outsoles, and increased time on the job spent standing or walking. Supervisor support and increased time spent on carpet were protective. Lower extremity fatigue that interfered with activities outside of work had additional risk factors including higher BMI, prior diagnosis of osteoarthritis, and increased hours per week spent working. CONCLUSIONS: While these results identify carpet as being protective against lower extremity fatigue, no similar relationship was identified for anti-fatigue mats. No adverse relationship was found between hard surfaces such as concrete and lower extremity fatigue. Given the high costs associated with work-related fatigue, future areas for potential intervention include smoking cessation, specific shoe recommendations, and enhancing psychosocial aspects of work such as supervisor support.

Risk factors for hip problems among assembly plant workers.

OBJECTIVE: The objective of this study was to determine the relative contributions of work activity (time spent standing, walking or sitting), floor surface characteristics, weight, BMI, age, foot biomechanics, and other demographic and medical history factors to the prevalence of hip disorders. METHODS: A cross-sectional observational study design was used to engine assembly plant workers. The main outcome measure was the finding of a hip disorder. The independent variables included baseline demographics, medical history, ergonomic exposures, psychosocial factors, shoe characteristics and foot biomechanics. RESULTS: Logistic regression revealed that increasing age, female gender, pes planus, smoking, history of a knee or hip injury, and a history of rheumatoid arthritis were significant risk factors while time on carpeted surfaces was protective. CONCLUSIONS: Hip disorders are associated with a history of biomechanical trauma to the hip but also from gait abnormalities such as pes planus.

HiSpike Method for High-Throughput Cost Effective Sequencing of the SARS-CoV-2 Spike Gene.

The changing nature of the SARS-CoV-2 pandemic poses unprecedented challenges to the world's health systems. Emerging spike gene variants jeopardize global efforts to produce immunity and reduce morbidity and mortality. These challenges require effective real-time genomic surveillance solutions that the medical community can quickly adopt. The SARS-CoV-2 spike protein mediates host receptor recognition and entry into the cell and is susceptible to generation of variants with increased transmissibility and pathogenicity. The spike protein is the primary target of neutralizing antibodies in COVID-19 patients and the most common antigen for induction of effective vaccine immunity. Tight monitoring of spike protein gene variants is key to mitigating COVID-19 spread and generation of vaccine escape mutants. Currently, SARS-CoV-2 sequencing methods are labor intensive and expensive. When sequence demands are high sequencing resources are quickly exhausted. Consequently, most SARS-CoV-2 strains are sequenced in only a few developed countries and rarely in developing regions. This poses the risk that undetected, dangerous variants will emerge. In this work, we present HiSpike, a method for high-throughput cost effective targeted next generation sequencing of the spike gene. This simple three-step method can be completed in < 30 h, can sequence 10-fold more samples compared to conventional methods and at a fraction of their cost. HiSpike has been validated in Israel, and has identified multiple spike variants from real-time field samples including Alpha, Beta, Delta and the emerging Omicron variants. HiSpike provides affordable sequencing options to help laboratories conserve resources for widespread high-throughput, near real-time monitoring of spike gene variants.