Tonic TNF conditioning of macrophages safeguards stimulus-specific inflammatory responses.

Tumor necrosis factor (TNF) is a key inflammatory cytokine that warns recipient cells of a nearby infection or tissue damage. Acute exposure to TNF activates characteristic oscillatory dynamics of the transcription factor NFκB and induces a characteristic gene expression program; these are distinct from the responses of cells directly exposed to pathogen-associated molecular patterns (PAMPs). Here, we report that tonic TNF exposure is critical for safeguarding TNF's specific functions. In the absence of tonic TNF conditioning, acute exposure to TNF causes (i) NFκB signaling dynamics that are less oscillatory and more like PAMP-responsive NFκB dynamics, (ii) immune gene expression that is more similar to the Pam3CSK4 response program, and (iii) broader epigenomic reprogramming that is characteristic of PAMP-responsive changes. We show that the absence of tonic TNF signaling effects subtle changes to TNF receptor availability and dynamics such that enhanced pathway activity results in non-oscillatory NFκB. Our results reveal tonic TNF as a key tissue determinant of the specific cellular responses to acute paracrine TNF exposure, and their distinction from responses to direct exposure to PAMPs.

Pharmacy, physical therapy, occupational therapy, and physician assistant professional students' perspectives on interprofessional roles and responsibilities.

Understanding roles and responsibilities within the interprofessional practice is a key competency of interprofessional education (IPE). Students in health professions programs can have limited understanding and perceptions of health professions, including their own and other professions. The purpose of this study was to understand students' perceptions of the roles and responsibilities of other health-care professionals. Students enrolled in occupational therapy, pharmacy, physical therapy, and physician assistant programs at a university participated in a three-hour IPE workshop. Throughout this workshop, they worked in small interprofessional teams to identify unique and shared roles and responsibilities of health professions. Students used a "dream catcher" graphic organizer to compare and contrast these roles and responsibilities. Researchers used thematic analysis of completed graphic organizers to identify themes in students' perceptions. Students identified many shared and unique characteristics about their professions' values and expertise, patient care process, practice settings, patient populations, education, and regulations. While students correctly identified many aspects of their professions, there were some inaccuracies that were addressed by small group faculty facilitators.

Deletion of serine/arginine-rich splicing factor 3 in hepatocytes predisposes to hepatocellular carcinoma in mice.

UNLABELLED: Alterations in RNA splicing are associated with cancer, but it is not clear whether they result from malignant transformation or have a causative role. We show here that hepatocyte-specific deletion of serine/arginine-rich splicing factor 3 (SRSF3) impairs hepatocyte maturation and metabolism in early adult life, and mice develop spontaneous hepatocellular carcinoma (HCC) with aging. Tumor development is preceded by chronic liver disease with progressive steatosis and fibrosis. SRSF3 protects mice against CCl4 -induced fibrosis and carcinogenesis and suppresses inclusion of the profibrogenic EDA exon in fibronectin 1. Loss of SRSF3 increases expression of insulin-like growth factor 2 and the A-isoform of the insulin receptor, allowing aberrant activation of mitogenic signaling, promotes aberrant splicing and expression of epithelial to mesenchymal transition (EMT) genes, and activates Wnt/ß-catenin signaling leading to c-Myc induction. Finally, SRSF3 expression is either decreased or the protein mislocalized in human HCC. CONCLUSION: Our data suggest a potential role for SRSF3 in preventing hepatic carcinogenesis by regulating splicing to suppress fibrosis, mitogenic splicing, and EMT. Thus, these mice may provide an attractive model to discover the pathogenic mechanisms linking aberrant pre-messenger RNA splicing with liver damage, fibrosis, and HCC.

Stimulus-response signaling dynamics characterize macrophage polarization states.

The functional state of cells is dependent on their microenvironmental context. Prior studies described how polarizing cytokines alter macrophage transcriptomes and epigenomes. Here, we characterized the functional responses of 6 differentially polarized macrophage populations by measuring the dynamics of transcription factor nuclear factor κB (NF-κB) in response to 8 stimuli. The resulting dataset of single-cell NF-κB trajectories was analyzed by three approaches: (1) machine learning on time-series data revealed losses of stimulus distinguishability with polarization, reflecting canalized effector functions. (2) Informative trajectory features driving stimulus distinguishability ("signaling codons") were identified and used for mapping a cell state landscape that could then locate macrophages conditioned by an unrelated condition. (3) Kinetic parameters, inferred using a mechanistic NF-κB network model, provided an alternative mapping of cell states and correctly predicted biochemical findings. Together, this work demonstrates that a single analyte's dynamic trajectories may distinguish the functional states of single cells and molecular network states underlying them. A record of this paper's transparent peer review process is included in the supplemental information.

Thyroglossal Duct Cyst Carcinoma: Lessons from a 20-Case Series.

Aims and Objectives: Thyroglossal duct cyst (TDC) is a common congenital cyst with an incidence of about 7%. Thyroglossal duct cyst carcinoma (TDCC) is a rare sequel which arises from TDC and has an incidence of about 1%. As these are rare, they do not have well-defined management guidelines. The aim of this study was to analyse the clinical profile and pathological characteristics of patients with thyroglossal duct cyst carcinoma and to propose a protocol for their treatment and follow-up. Materials and Methods: A retrospective study was done from January 2000 to December 2019. All the clinical details, imaging characteristics, treatment and histopathology were analysed. Results: The mean age group in our study was 37.9 years with a female preponderance. The clinical features like rapid increase in size, fixity of the lump and lymph node metastasis were not very common. Seventy-five percent of our patients who underwent imaging had suspicious characteristics. Fifty-six percent of our patients had FNAC suggestive of TDCC. Fifty percent of our patients had concomitant thyroid carcinoma. None of our patients had distant metastasis at follow-up. Conclusions: TDCC is rare and a disease of young adulthood and usually has good prognosis. It may be a clinical surprise or a small lesion which can be detected with ultrasound and targeted FNAC. There is high rate of concomitant thyroid carcinoma and hence needs careful assessment. Sistrunk's procedure with total thyroidectomy either staged or simultaneously has good outcome and permits adjuvant treatment.

Kinetics of mRNA nuclear export regulate innate immune response gene expression.

The abundance and stimulus-responsiveness of mature mRNA is thought to be determined by nuclear synthesis, processing, and cytoplasmic decay. However, the rate and efficiency of moving mRNA to the cytoplasm almost certainly contributes, but has rarely been measured. Here, we investigated mRNA export rates for innate immune genes. We generated high spatio-temporal resolution RNA-seq data from endotoxin-stimulated macrophages and parameterized a mathematical model to infer kinetic parameters with confidence intervals. We find that the effective chromatin-to-cytoplasm export rate is gene-specific, varying 100-fold: for some genes, less than 5% of synthesized transcripts arrive in the cytoplasm as mature mRNAs, while others show high export efficiency. Interestingly, effective export rates do not determine temporal gene responsiveness, but complement the wide range of mRNA decay rates; this ensures similar abundances of short- and long-lived mRNAs, which form successive innate immune response expression waves.

Laryngotracheal Resection in Thyroid Cancer - Experience from a Single Centre Series of 22 Cases.

Involvement of the aerodigestive tract is reported in one-third of patients with locally invasive thyroid cancer. It is associated with significant morbidity and mortality, with airway obstruction being the immediate cause of death in 50% of patients who die of thyroid cancer. Management is challenging and includes the risks of extensive surgery as well as decisions regarding the type of surgery and adjuvant therapy. Retrospective cohort study, reporting institutional experience with patients who underwent laryngotracheal resection for invasive thyroid cancer over the past 10 years. Twenty-two patients were included in the study. All patients had Shin stage 4 disease. The median follow-up was 18 months. Five patients had systemic metastasis at diagnosis. Nineteen patients underwent tracheal resection and end to end anastomosis, and 3 underwent laryngectomy. The mean length of the resected trachea was 2.94 cm. Tracheal releasing manoeuvres were utilized in 11 patients. Three patients required a tracheostomy postoperatively. Other complications included a temporary vocal cord palsy in 5 patients, temporary hypocalcemia in 6 and permanent hypocalcemia in 1 patient. Adjuvant radiotherapy was utilized in 9 patients and I-131 therapy in 13 patients. Three patients died during follow-up. Two patients developed thyroid bed recurrence, two patients developed systemic metastasis on follow-up. Most patients survived for a prolonged period with only biochemical evidence of disease persistence and three with no evidence of disease. Laryngotracheal resection with primary anastomosis is a safe and effective option, providing adequate symptomatic relief as well as prolonged survival in carefully selected patients with invasive Shin stage 4 disease.

The utility of indocyanine green (ICG) for the identification and assessment of viability of the parathyroid glands during thyroidectomy.

We conducted this study to evaluate the ability of indocyanine green (ICG) and near infra-red fluorescence (NIRF) camera to aid in the identification and assessment of viability of parathyroid glands during thyroid surgery. A prospective observational study was conducted between May and October 2020 among 50 consecutive patients who underwent total thyroidectomy at a single institution. Parathyroid glands were identified under white light during thyroidectomy following which reconstituted ICG was injected through a peripheral vein and the location of parathyroid glands was confirmed. The perfusion to the parathyroid gland was assessed by documenting the fluorescence intensity score (FIS) and the parathyroid angiogram score (PAS). There was no difference in the number of parathyroid glands seen on visual inspection 147 (73.5%) when compared to under NIRF camera, 146 (73%). Though the rate of postoperative hypoparathyroidism was lower in the cohort with FIS 3 (14.2%) compared to score 2 and 1 (28.5% and 100%, respectively), this was not significant (p = 0.35). A significant correlation was noted between a delayed flow on PAS and the development of post-thyroidectomy hypoparathyroidism (p = 0.01). PAS had a sensitivity of 100%, specificity of 88.6%, NPV of 100% and PPV of 55.6% to predict the development of post-thyroidectomy hypoparathyroidism. In this study, there was no additional benefit of ICG and NIRF camera in the identification of parathyroid glands. However, ICG angiogram seems to be a good adjunct for the intraoperative assessment of the viability of the parathyroid glands and accurately predicts the development of postoperative hypoparathyroidism.

Hepatocyte Deletion of IGF2 Prevents DNA Damage and Tumor Formation in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. Serine-arginine rich splicing factor 3 (SRSF3) plays a critical role in hepatocyte function and its loss in mice promotes chronic liver damage and leads to HCC. Hepatocyte-specific SRSF3 knockout mice (SKO mice) also overexpress insulin-like growth factor 2 (IGF2). In the present study, double deletion of Igf2 and Srsf3 (DKO mice) prevents hepatic fibrosis and inflammation, and completely prevents tumor formation, and is associated with decreased proliferation, apoptosis and DNA damage, and restored DNA repair enzyme expression. This is confirmed in vitro, where IGF2 treatment of HepG2 hepatoma cells decreases DNA repair enzyme expression and causes DNA damage. Tumors from the SKO mice also show mutational signatures consistent with homologous recombination and mismatch repair defects. Analysis of frozen human samples shows that SRSF3 protein is decreased sixfold in HCC compared to normal liver tissue but SRSF3 mRNA is increased. Looking at public TCGA data, HCC patients having high SRSF3 mRNA expression show poor survival, as do patients with alterations in known SRSF3-dependent splicing events. The results indicate that IGF2 overexpression in conjunction with reduced SRSF3 splicing activity could be a major cause of DNA damage and driver of liver cancer.

Muscleblind-like 1 (Mbnl1) promotes insulin receptor exon 11 inclusion via binding to a downstream evolutionarily conserved intronic enhancer.

The insulin receptor exists as two isoforms, IR-A and IR-B, which result from alternative splicing of exon 11 in the primary transcript. These two isoforms show a cell-specific distribution, and their relative proportions also vary during development, aging, and in different disease states. We have previously demonstrated that both intron 10 and the alternatively spliced exon 11 contain regulatory sequences that affect insulin receptor splicing both positively and negatively and that these sequences bind the serine/arginine-rich (SR) proteins SRp20 and SF2/ASF and the CELF protein CUG-BP1. In this study, we describe a new intronic splicing element within intron 11 that is highly conserved across species. Using minigenes carrying deletion mutations within intron 11, we demonstrated that this sequence functions as an intronic splicing enhancer. We subsequently used RNA affinity chromatography to identify Mbnl1 as a splicing factor that recognizes this enhancer. By ribonucleoprotein immunoprecipitation, we also established that Mbnl1 binds specifically to the INSR (insulin receptor gene) RNA. Overexpression or knockdown of Mbnl1 in hepatoma and embryonic kidney cells altered the levels of exon 11 inclusion. Finally, we showed that deletion of the intronic enhancer eliminates the ability of Mbnl1 to promote exon inclusion. Collectively, these findings demonstrate a role for Mbnl1 in controlling insulin receptor exon 11 inclusion via binding to a downstream intronic enhancer element.

Sentinel Lymph Node Biopsy in Early Breast Cancer Using Methylene Blue Dye Alone: a Safe, Simple, and Cost-Effective Procedure in Resource-Constrained Settings.

Sentinel lymph node biopsy (SLNB) is done by different techniques in clinically node-negative patients with early breast cancer. In this study, we aim to estimate the identification rates, positivity rates, cost-effectiveness, and outcomes for patients who underwent sentinel node biopsy using methylene blue dye alone. This was a retrospective review of 172 patients with early breast cancer (cT1-3, N0) who underwent SLNB using methylene blue dye alone between January 2014 and December 2018 including their follow-up details until December 2019. The mean age was 51 ± 10.3 (range: 28 to 76) years. There were 63 (36.6%) patients with cT1 tumor, 108 (62.7%) with cT2, and only 1 patient with cT3 tumor. Breast conservation surgery was performed in 62 (36%) while the remaining 110 (64%) underwent simple mastectomy. Sentinel nodes were successfully identified in 165 (95.9%) with a positivity rate of 23.6%. There was no dye-related adverse reactions intra-operatively. The mean duration of follow-up was 26.68 ± 15.9 months (range: 1-60). Chronic arm pain was present in 7 (4%) while none of the patients had lymphedema or restriction of shoulder joint motion. There were no documented axillary nodal recurrences in this cohort. Eight (4.65%) patients were detected to have systemic metastasis. One patient died of brain metastasis from bilateral breast cancer. The mean disease-free survival was 57 months (95% CI: 55-59). Sentinel lymph node biopsy using methylene dye alone is a safe, simple, and cost-effective alternative to isosulfan blue or radio isotope technique in surgical centers with resource constraints.

An unedited 1.1 kb mitochondrial orfB gene transcript in the wild abortive cytoplasmic male sterility (WA-CMS) system of Oryza sativa L. subsp. indica.

BACKGROUND: The application of hybrid rice technology has significantly increased global rice production during the last three decades. Approximately 90% of the commercially cultivated rice hybrids have been derived through three-line breeding involving the use of WA-CMS lines. It is believed that during the 21st century, hybrid rice technology will make significant contributions to ensure global food security. This study examined the poorly understood molecular basis of the WA-CMS system in rice. RESULTS: RFLPs were detected for atp6 and orfB genes in sterile and fertile rice lines, with one copy of each in the mt-genome. The RNA profile was identical in both lines for atp6, but an additional longer orfB transcript was identified in sterile lines. 5' RACE analysis of the long orfB transcript revealed it was 370 bp longer than the normal transcript, with no indication it was chimeric when compared to the genomic DNA sequence. cDNA clones of the longer orfB transcript in sterile lines were sequenced and the transcript was determined unedited. Sterile lines were crossed with the restorer and maintainer lines, and fertile and sterile F1 hybrids were respectively generated. Both hybrids contained two types of orfB transcripts. However, the long transcript underwent editing in the fertile F1 hybrids and remained unedited in the sterile lines. Additionally, the editing of the 1.1 kb orfB transcript co-segregated with fertility restoring alleles in a segregating population of F2 progeny; and the presence of unedited long orfB transcripts was detected in the sterile plants from the F2 segregating population. CONCLUSION: This study helped to assign plausible operative factors responsible for male-sterility in the WA cytoplasm of rice. A new point of departure to dissect the mechanisms governing the CMS-WA system in rice has been identified, which can be applied to further harness the opportunities afforded by hybrid vigor in rice.

Gene Regulatory Strategies that Decode the Duration of NFκB Dynamics Contribute to LPS- versus TNF-Specific Gene Expression.

Pathogen-derived lipopolysaccharide (LPS) and cytokine tumor necrosis factor (TNF) activate NFκB with distinct duration dynamics, but how immune response genes decode NFκB duration to produce stimulus-specific expression remains unclear. Here, detailed transcriptomic profiling of combinatorial and temporal control mutants identified 81 genes that depend on stimulus-specific NFκB duration for their stimulus-specificity. Combining quantitative experimentation with mathematical modeling, we found that for some genes a long mRNA half-life allowed effective decoding, but for many genes this was insufficient to account for the data; instead, we found that chromatin mechanisms, such as a slow transition rate between inactive and RelA-bound enhancer states, could also decode NFκB dynamics. Chromatin-mediated decoding is favored by genes acting as immune effectors (e.g., tissue remodelers and T cell recruiters) rather than immune regulators (e.g., signaling proteins and monocyte recruiters). Overall, our results delineate two gene regulatory strategies that decode stimulus-specific NFκB dynamics and determine distinct biological functions.

Sequential conditioning-stimulation reveals distinct gene- and stimulus-specific effects of Type I and II IFN on human macrophage functions.

Macrophages orchestrate immune responses by sensing and responding to pathogen-associated molecules. These responses are modulated by prior conditioning with cytokines such as interferons (IFNs). Type I and II IFN have opposing functions in many biological scenarios, yet macrophages directly stimulated with Type I or II IFN activate highly overlapping gene expression programs. We hypothesized that a sequential conditioning-stimulation approach would reveal with greater specificity the differential effects of Type I and II IFN on human macrophages. By first conditioning with IFN then stimulating with toll-like receptor ligands and cytokines, followed by genome-wide RNA-seq analysis, we identified 713 genes whose expression was unaffected by IFN alone but showed potentiated or diminished responses to a stimulus after conditioning. For example, responses to the cytokine TNF were restricted by Type II IFN conditioning but potentiated by Type I IFN conditioning. We observed that the effects of IFN were not uniformly pro- or anti-inflammatory, but highly gene-specific and stimulus-specific. By assessing expression levels of key signal transducers and characterizing chromatin accessibility by ATAC-seq, we identify the likely molecular mechanisms underlying Type I and Type II-specific effects, distinguishing between modulation of cytoplasmic signaling networks and the nuclear epigenome that synergistically regulate macrophage immune responses.

Focused Parathyroidectomy Under Local Anesthesia - A Feasibility Study.

PURPOSE: We conducted this study to evaluate the feasibility, patient satisfaction, and cost of performing focused parathyroidectomy under local anesthesia (LA) and mild sedation, administered and monitored by a surgeon. MATERIALS AND METHODS: This was a prospective observational study of 30 patients with primary hyperparathyroidism (PHPT) undergoing a focused parathyroidectomy under LA and mild sedation at a single institution. The clinical features, gland weight, operating time, procedure time, postoperative pain scores, overall patient satisfaction, postoperative nausea and vomiting, analgesic requirements, complications, cost, and cure rates were documented. Data were analyzed using SPSS software version 17.0. RESULTS: In two patients (6.7%), the procedure had to be completed under general anesthesia (GA). Postoperative temporary hypocalcemia was witnessed in 14 of 30 (46.7%), but only 1 required intravenous calcium infusion. About 21 of 30 (75%) were completely satisfied with LA, whereas 25 of 30 (89%) were completely satisfied with surgical procedure. Furthermore, all patients were keen to recommend this procedure under LA to their friends and family. Comparing the cost between performing the procedure under LA with that under GA, a significant difference was witnessed (P = 0.001). Among the 26 patients reviewed at 6 months, all had a normal serum calcium and parathyroid hormone levels indicating 100% cure rate. CONCLUSION: Performing focused parathyroidectomy under LA is feasible; additionally, this method can significantly reduce the cost of the procedure (P = 0.001).

Dermatofibrosarcoma Protuberans of the Breast-a Rare Entity.

Dermatofibrosarcoma protuberans (DFSP) represents about 1% of soft-tissue sarcomas with an estimated incidence of 0.8 to 5.0 cases per million per year. This lesion may occur anywhere in the body but more than 50% occur on the trunk, 20% on the head and neck and 30% on the extremities. DFSP of the breast is an extremely uncommon site of presentation. Data regarding DFSP of the breast is limited and mostly in the form of case reports. Clinical presentation is not uniform and may mimic benign skin lesions [1]. However, it typically presents as a nodular cutaneous mass in early or mid-adult life. We herein report a case of DFSP of the breast in a 33-year-old lady who was managed successfully in our institute and review the literature associated with it.

Acute suppurative thyroiditis secondary to urinary tract infection by E. coli: a rare clinical scenario.

A 60-year-old woman with diabetes and symptomatic urinary tract infection presented to us with a painful neck swelling for 2 weeks. We discuss diagnostic and management issues in acute suppurative thyroiditis caused by Escherichia coli.

Splicing factor SRSF3 is crucial for hepatocyte differentiation and metabolic function.

SR family RNA binding proteins regulate splicing of nascent RNAs in vitro but their physiological role in vivo is largely unexplored, as genetic deletion of many SR protein genes results in embryonic lethality. Here we show that SRSF3HKO mice carrying a hepatocyte-specific deletion of Srsf3 (homologous to human SRSF3/SRp20) have a disrupted hepatic architecture and show pre- and postnatal growth retardation. SRSF3HKO mice exhibit impaired hepatocyte maturation with alterations in glucose and lipid homeostasis characterized by reduced glycogen storage, fasting hypoglycemia, increased insulin sensitivity and reduced cholesterol synthesis. We identify various splicing alterations in the SRSF3HKO liver that explain the in vivo phenotype. In particular, loss of SRSF3 causes aberrant splicing of Hnf1α, Ern1, Hmgcs1, Dhcr7 and Scap genes, which are critical regulators of glucose and lipid metabolism. Our study provides the first evidence for a SRSF3-driven genetic programme required for morphological and functional differentiation of hepatocytes that may have relevance for human liver disease and metabolic dysregulation.

Innovation in global collaborations: from student placement to mutually beneficial exchanges.

Five years ago, an academic department in the United States and the Ann Sullivan Center of Peru (CASP) initiated an international partnership to foster research collaborations and reciprocal consultation, and to create an advanced clinical placement for occupational therapy doctoral students. CASP is a globally recognized hub for community-based research, demonstration and training for people with disabilities (most of whom are from low-income families). CASP has provided occupational therapy students and faculty with a rich cultural environment in which to learn and collaborate as well as opportunities for developing research collaborations. The purpose of this manuscript is to discuss an innovative model of international collaboration highlighting specific areas of exchange and reciprocal learning. First, we will describe the collaboration and CASP's rich learning opportunities. Second, we will discuss a model of collaboration that includes three main phases: planning and preparation, developing and sustaining the partnership, and evaluating and celebrating outcomes and benefits. We illustrate the partnership with a case example and describe exchanges between CASP and a local community agency with whom faculty have collaborated for 20 years. Finally, we discuss implications of our innovative model towards developing and sustaining global partnerships. .

hnRNP A1 and hnRNP F modulate the alternative splicing of exon 11 of the insulin receptor gene.

Exon 11 of the insulin receptor gene (INSR) is alternatively spliced in a developmentally and tissue-specific manner. Linker scanning mutations in a 5' GA-rich enhancer in intron 10 identified AGGGA sequences that are important for enhancer function. Using RNA-affinity purification and mass spectrometry, we identified hnRNP F and hnRNP A1 binding to these AGGGA sites and also to similar motifs at the 3' end of the intron. The hnRNPs have opposite functional effects with hnRNP F promoting and hnRNP A1 inhibiting exon 11 inclusion, and deletion of the GA-rich elements eliminates both effects. We also observed specific binding of hnRNP A1 to the 5' splice site of intron 11. The SR protein SRSF1 (SF2/ASF) co-purified on the GA-rich enhancer and, interestingly, also competes with hnRNP A1 for binding to the splice site. A point mutation -3U→C decreases hnRNP A1 binding, increases SRSF1 binding and renders the exon constitutive. Lastly, our data point to a functional interaction between hnRNP F and SRSF1 as a mutant that eliminates SRSF1 binding to exon 11, or a SRSF1 knockdown, which prevents the stimulatory effect of hnRNP F over expression.