RNA m6A reader YTHDF2 facilitates precursor miR-126 maturation to promote acute myeloid leukemia progression.

As the most common internal modification of mRNA, N6-methyladenosine (m6A) and its regulators modulate gene expression and play critical roles in various biological and pathological processes including tumorigenesis. It was reported previously that m6A methyltransferase (writer), methyltransferase-like 3 (METTL3) adds m6A in primary microRNAs (pri-miRNAs) and facilitates its processing into precursor miRNAs (pre-miRNAs). However, it is unknown whether m6A modification also plays a role in the maturation process of pre-miRNAs and (if so) whether such a function contributes to tumorigenesis. Here, we found that YTHDF2 is aberrantly overexpressed in acute myeloid leukemia (AML) patients, especially in relapsed patients, and plays an oncogenic role in AML. Moreover, YTHDF2 promotes expression of miR-126-3p (also known as miR-126, as it is the main product of precursor miR-126 (pre-miR-126)), a miRNA that was reported as an oncomiRNA in AML, through facilitating the processing of pre-miR-126 into mature miR-126. Mechanistically, YTHDF2 recognizes m6A modification in pre-miR-126 and recruits AGO2, a regulator of pre-miRNA processing, to promote the maturation of pre-miR-126. YTHDF2 positively and negatively correlates with miR-126 and miR-126's downstream target genes, respectively, in AML patients, and forced expression of miR-126 could largely rescue YTHDF2/Ythdf2 depletion-mediated suppression on AML cell growth/proliferation and leukemogenesis, indicating that miR-126 is a functionally important target of YTHDF2 in AML. Overall, our studies not only reveal a previously unappreciated YTHDF2/miR-126 axis in AML and highlight the therapeutic potential of targeting this axis for AML treatment, but also suggest that m6A plays a role in pre-miRNA processing that contributes to tumorigenesis.

Osteopontin modulates microglial activation states and attenuates inflammatory responses after subarachnoid hemorrhage in rats.

AIMS: Osteopontin (OPN) has demonstrated neuroprotective effects in various stroke models. Its role in neuroinflammation after brain injury remains to be elucidated. This study aims to clarify the effect of OPN on neuroinflammation, particularly on the functional states of microglia after subarachnoid hemorrhage (SAH). METHODS: 77 rats were randomly divided into the following groups: Sham, SAH 24 h, SAH + rOPN, SAH + Vehicle (PBS), SAH + OPN siRNA, and SAH + Scr siRNA, SAH + rOPN+Fib-14 and SAH + rOPN+DMSO. Modified Garcia and beam balance tests were used to evaluate neurobehavioral outcomes. Semi-quantitative immunofluorescence staining was performed to measure expression of myeloperoxidase (MPO) and microglia activation state markers CD16, CD206 after SAH and recombinant OPN treatment. The quantification of microglia activation and functional markers CD16, CD206, TNF-α and IL-10 were further evaluated using Western-blotting. RESULTS: Nasal administration of rOPN improved neurological dysfunction, attenuated neutrophil infiltration, and decreased expression of phenotypic and functional markers of pro-inflammatory microglia CD16 and TNF-α. It also promoted an anti-inflammatory microglial state, as evidenced by increased expression of CD206 and IL-10. Furthermore, after blocking the phosphorylation of FAK signaling, the effects of rOPN on microglial activation states were partially reversed. The downstream pathways of STAT3 and NF-κB also exhibited consistent changes, suggesting the involvement of the STAT3 and NF-κB pathways in OPN's modulation of microglial activation via integrin-FAK signaling. CONCLUSION: OPN attenuates inflammatory responses after SAH by promoting an anti-inflammatory microglial state, potentially mediated through the integrin-FAK-STAT3 and NF-κB signaling pathways.

Association of Metformin, Dipeptidyl Dipeptidase-4 Inhibitors, and Insulin with Coronavirus Disease 2019-Related Hospital Outcomes in Patients with Type 2 Diabetes.

OBJECTIVE: The effects of diabetes medications on COVID-19 hospitalization outcomes have not been consistent. We sought to determine the effect of metformin, dipeptidyl peptidase-4 inhibitors (DPP-4i), and insulin on admission to the intensive care unit (ICU), need for assisted ventilation, development of renal insufficiency, and mortality in patients admitted with COVID-19 infection after controlling for clinical variables and other relevant diabetes-related medications in patients with type 2 diabetes mellitus (DM). METHODS: This was a retrospective study of patients hospitalized with COVID-19 from a single hospital system. Univariate and multivariate analyses were performed that included demographic data, glycated hemoglobin, kidney function, smoking status, insurance, Charlson comorbidity index, number of diabetes medications, and use of angiotensin-converting enzyme inhibitors and statin prior to admission and glucocorticoids during admission. RESULTS: A total of 529 patients with type 2 DM were included in our final analysis. Neither metformin nor DPP4i prescription was associated with ICU admission, need for assisted ventilation, or mortality. Insulin prescription was associated with increased ICU admission but not with need for assisted ventilation or mortality. There was no association of any of these medications with development of renal insufficiency. CONCLUSIONS: In this population, limited to type 2 DM and controlled for multiple variables that have not been consistently studied (such as a measure of general health, glycated hemoglobin, and insurance status), insulin prescription was associated with increased ICU admission. Metformin and DPP4i prescriptions did not have an association with the outcomes.

QKI shuttles internal m7G-modified transcripts into stress granules and modulates mRNA metabolism.

N7-methylguanosine (m7G) modification, routinely occurring at mRNA 5' cap or within tRNAs/rRNAs, also exists internally in messenger RNAs (mRNAs). Although m7G-cap is essential for pre-mRNA processing and protein synthesis, the exact role of mRNA internal m7G modification remains elusive. Here, we report that mRNA internal m7G is selectively recognized by Quaking proteins (QKIs). By transcriptome-wide profiling/mapping of internal m7G methylome and QKI-binding sites, we identified more than 1,000 high-confidence m7G-modified and QKI-bound mRNA targets with a conserved "GANGAN (N = A/C/U/G)" motif. Strikingly, QKI7 interacts (via C terminus) with the stress granule (SG) core protein G3BP1 and shuttles internal m7G-modified transcripts into SGs to regulate mRNA stability and translation under stress conditions. Specifically, QKI7 attenuates the translation efficiency of essential genes in Hippo signaling pathways to sensitize cancer cells to chemotherapy. Collectively, we characterized QKIs as mRNA internal m7G-binding proteins that modulate target mRNA metabolism and cellular drug resistance.

Phosphorylation stabilized TET1 acts as an oncoprotein and therapeutic target in B cell acute lymphoblastic leukemia.

Although the overall survival rate of B cell acute lymphoblastic leukemia (B-ALL) in childhood is more than 80%, it is merely 30% in refractory/relapsed and adult patients with B-ALL. This demonstrates a need for improved therapy targeting this subgroup of B-ALL. Here, we show that the ten-eleven translocation 1 (TET1) protein, a dioxygenase involved in DNA demethylation, is overexpressed and plays a crucial oncogenic role independent of its catalytic activity in B-ALL. Consistent with its oncogenic role in B-ALL, overexpression of TET1 alone in normal precursor B cells is sufficient to transform the cells and cause B-ALL in mice within 3 to 4 months. We found that TET1 protein is stabilized and overexpressed because of its phosphorylation mediated by protein kinase C epsilon (PRKCE) and ATM serine/threonine kinase (ATM), which are also overexpressed in B-ALL. Mechanistically, TET1 recruits STAT5B to the promoters of CD72 and JCHAIN and promotes their transcription, which in turn promotes B-ALL development. Destabilization of TET1 protein by treatment with PKC or ATM inhibitors (staurosporine or AZD0156; both tested in clinical trials), or by pharmacological targeting of STAT5B, greatly decreases B-ALL cell viability and inhibits B-ALL progression in vitro and in vivo. The combination of AZD0156 with staurosporine or vincristine exhibits a synergistic effect on inhibition of refractory/relapsed B-ALL cell survival and leukemia progression in PDX models. Collectively, our study reveals an oncogenic role of the phosphorylated TET1 protein in B-ALL independent of its catalytic activity and highlights the therapeutic potential of targeting TET1 signaling for the treatment of refractory/relapsed B-ALL.

Prevalence and clinical determinants of non-alcoholic fatty liver disease by liver scores in adults with type 1 diabetes.

AIMS: To investigate the prevalence and clinical risk factors for non-alcoholic fatty liver disease (NAFLD) in type 1 diabetes (T1DM) by liver scores. METHODS: A retrospective, unicenter, cross-sectional analysis was performed of adults with T1DM from 2015 to 2018. Steatosis scores (hepatic steatosis index-HSI, Framingham steatosis index-FSI) and fibrosis scores (FIB-4 index, AST-to-platelet ratio index-APRI) were associated with clinical parameters. RESULTS: We identified 447 patients, 38 ± 14.5 yrs, 54 % female, BMI 28 ± 5.9 kg/m2. Liver steatosis was prevalent at 61 % by HSI ≥ 36 and 52 % by FSI ≥ 23. A majority of these individuals had normal liver transaminase levels. The presence of advanced fibrosis was 4 % by APRI > 0.7 and 4 % by FIB-4 > 2.67. BMI ≥ 25 kg/m2 correlated with steatosis scores (P < 0.001) but not fibrosis scores. Older age (≥40 yrs), hypertension, dyslipidemia, and history of cardiovascular disease were associated with steatosis markers. Only 21 % had any abdominal imaging, 2 % had hepatology referral and 1 % had a liver biopsy. Glucagon-like peptide-1 agonist was prescribed in 5 % and thiazolidinedione in 4 %. CONCLUSION: Liver scores indicating steatosis but not fibrosis is common in adults with T1DM with obesity and/or metabolic syndrome, and is associated with older age, hypertension, and dyslipidemia. NAFLD is under-diagnosed and under-investigated; a minority of patients have had any liver evaluation or treatment.

ChIPBase v3.0: the encyclopedia of transcriptional regulations of non-coding RNAs and protein-coding genes.

Non-coding RNAs (ncRNAs) are emerging as key regulators of various biological processes. Although thousands of ncRNAs have been discovered, the transcriptional mechanisms and networks of the majority of ncRNAs have not been fully investigated. In this study, we updated ChIPBase to version 3.0 (https://rnasysu.com/chipbase3/) to provide the most comprehensive transcriptional regulation atlas of ncRNAs and protein-coding genes (PCGs). ChIPBase has identified ∼151 187 000 regulatory relationships between ∼171 600 genes and ∼3000 regulators by analyzing ∼55 000 ChIP-seq datasets, which represent a 30-fold expansion. Moreover, we de novo identified ∼29 000 motif matrices of transcription factors. In addition, we constructed a novel 'Enhancer' module to predict ∼1 837 200 regulation regions functioning as poised, active or super enhancers under ∼1300 conditions. Importantly, we constructed exhaustive coexpression maps between regulators and their target genes by integrating expression profiles of ∼65 000 normal and ∼15 000 tumor samples. We built a 'Disease' module to obtain an atlas of the disease-associated variations in the regulation regions of genes. We also constructed an 'EpiInter' module to explore potential interactions between epitranscriptome and epigenome. Finally, we designed 'Network' module to provide extensive and gene-centred regulatory networks. ChIPBase will serve as a useful resource to facilitate integrative explorations and expand our understanding of transcriptional regulation.

The m6A reader IGF2BP2 regulates glutamine metabolism and represents a therapeutic target in acute myeloid leukemia.

N6-Methyladenosine (m6A) modification and its modulators play critical roles and show promise as therapeutic targets in human cancers, including acute myeloid leukemia (AML). IGF2BP2 was recently reported as an m6A binding protein that enhances mRNA stability and translation. However, its function in AML remains largely elusive. Here we report the oncogenic role and the therapeutic targeting of IGF2BP2 in AML. High expression of IGF2BP2 is observed in AML and associates with unfavorable prognosis. IGF2BP2 promotes AML development and self-renewal of leukemia stem/initiation cells by regulating expression of critical targets (e.g., MYC, GPT2, and SLC1A5) in the glutamine metabolism pathways in an m6A-dependent manner. Inhibiting IGF2BP2 with our recently identified small-molecule compound (CWI1-2) shows promising anti-leukemia effects in vitro and in vivo. Collectively, our results reveal a role of IGF2BP2 and m6A modification in amino acid metabolism and highlight the potential of targeting IGF2BP2 as a promising therapeutic strategy in AML.

RNA N6 -methyladenosine reader YTHDC1 is essential for TGF-beta-mediated metastasis of triple negative breast cancer.

RNA N6 -methyladenosine (m6A) modification and its regulators fine tune gene expression and contribute to tumorigenesis. This study aims to uncover the essential role and the underlying molecular mechanism(s) of the m6A reader YTHDC1 in promoting triple negative breast cancer (TNBC) metastasis. METHODS: In vitro and in vivo models were employed to determine the pathological function of YTHDC1 in TNBC metastasis. To identify bona fide YTHDC1 target RNAs, we conducted RNA-seq, m6A-seq, and RIP-seq, followed by integrative data analysis and validation assays. RESULTS: By analyzing The Cancer Genome Atlas (TCGA) dataset, we found that elevated expression of YTHDC1 is positively correlated with poor prognosis in breast cancer patients. Using a mammary fat pad mouse model of TNBC, YTHDC1 significantly promoted lung metastasis of TNBC cells. Through multiple transcriptome-wide sequencing and integrative data analysis, we revealed dysregulation of metastasis-related pathways following YTHDC1 depletion and identified SMAD3 as a bona fide YTHDC1 target RNA. Depletion of YTHDC1 caused nuclear retention of SMAD3 mRNA, leading to lower SMAD3 protein levels. Loss of YTHDC1 led to impaired TGF-ß-induced gene expression, leading to inhibition of epithelial-mesenchymal transition (EMT) and suppressed TNBC cell migration and invasion. SMAD3 overexpression was able to restore the response to TGF-ß in YTHDC1 depleted TNBC cells. Furthermore, we demonstrated that the oncogenic role of YTHDC1 is mediated through its recognition of m6A as m6A-binding defective mutants of YTHDC1 were unable to rescue the impaired cell migration and invasion of YTHDC1 knockout TNBC cells. CONCLUSIONS: We show that YTHDC1 plays a critical oncogenic role in TNBC metastasis through promoting the nuclear export and expression of SMAD3 to augment the TGF-ß signaling cascade. Overall, our study demonstrates that YTHDC1 is vital for TNBC progression by enhancing TNBC cell survival and TGF-ß-mediated EMT via SMAD3 to enable the formation of distant metastasis and highlights the therapeutic potential of targeting the YTHDC1/m6A/SMAD3 axis for TNBC treatment.

Perioperative management of diabetes in patients undergoing bariatric and metabolic surgery: a narrative review and the Cleveland Clinic practical recommendations.

Bariatric and metabolic surgery is an effective treatment for patients with severe obesity and obesity-related diseases. In patients with type 2 diabetes, it provides marked improvement in glycemic control and even remission of diabetes. In patients with type 1 diabetes, bariatric surgery may offer improvement in insulin sensitivity and other cardiometabolic risk factors, as well as amelioration of the mechanical complications of obesity. Because of these positive outcomes, there are increasing numbers of patients with diabetes who undergo bariatric surgical procedures each year. Prior to surgery, efforts should be made to optimize glycemic control. However, there is no need to delay or withhold bariatric surgery until a specific glycosylated hemoglobin target is reached. Instead, treatment should focus on avoidance of early postoperative hyperglycemia. In general, oral glucose-lowering medications and noninsulin injectables are not favored to control hyperglycemia in the inpatient setting. Hyperglycemia in the hospital is managed with insulin, aiming for perioperative blood glucose concentrations between 80 and 180 mg/dL. Following surgery, substantial changes of the antidiabetic medication regimens are common. Patients should have a clear understanding of the modifications made to their treatment and should be followed closely thereafter. In this review article, we describe practical recommendations for the perioperative management of diabetes in patients with type 2 or type 1 diabetes undergoing bariatric surgery. Specific recommendations are delineated based on the different treatments that are currently available for glycemic control, including oral glucose-lowering medications, noninsulin injectables, and a variety of insulin regimens.

TP53-inducible putative long noncoding RNAs encode functional polypeptides that suppress cell proliferation.

Polypeptides encoded by long noncoding RNAs (lncRNAs) are a novel class of functional molecules. However, whether these hidden polypeptides participate in the TP53 pathway and play a significant biological role is still unclear. Here, we discover that TP53-regulated lncRNAs can encode peptides, two of which are functional in various human cell lines. Using ribosome profiling and RNA-seq approaches in HepG2 cells, we systematically identified more than 300 novel TP53-regulated lncRNAs and further confirmed that 15 of these TP53-regulated lncRNAs encode peptides. Furthermore, several peptides were validated by mass spectrometry. Ten of the novel translational lncRNAs are directly inducible by TP53 in response to DNA damage. We show that the TP53-inducible peptides TP53LC02 and TP53LC04, but not their lncRNAs, can suppress cell proliferation. TP53LC04 peptide also has a function associated with cell proliferation by regulating the cell cycle in response to DNA damage. This study shows that TP53-regulated lncRNAs can encode new functional peptides, leading to the expansion of the TP53 tumor-suppressor network and providing novel potential targets for cancer therapy.

Biochemical assessment of adrenal insufficiency after adrenalectomy for non-cortisol secreting tumors: clinical correlation and recommendations.

BACKGROUND: Data regarding changes in cortisol axis after adrenalectomy for non-cortisol secreting tumors and their correlation with adrenal insufficiency are limited. Our aim was to analyze these changes and their clinical correlations to guide management after adrenalectomy for non-Cushing's tumors. METHODS: Following IRB approval, postoperative cortisol axis changes were analyzed in patients who underwent unilateral adrenalectomy for non-Cushing's tumors. A morning serum cortisol of ≥ 10 µg/dl was accepted as a sufficient adrenal response. RESULTS: 223 adrenalectomies were analyzed. In 63% of patients, POD1 serum cortisol was ≥ 10 µg/dl and in 37% < 10 µg/dl. No patient with a POD1 cortisol ≥ 10 µg/dl developed AI symptoms, whereas symptoms of AI were observed in 4% of those with < 10 µg/dl. In patients with a POD1 cortisol of < 10 µg/dl, the rate of steroid replacement therapy initiation was 100%, 8%, and 25% when the decision was based on serum cortisol, clinical symptoms, and serum cortisol plus ACTH stimulation test results, respectively. In 90% of asymptomatic patients, hypocortisolemia resolved uneventfully within a week on repeat morning cortisol testing. 75% of patients with hypocortisolemia on POD1 demonstrated an adequate cortisol response to ACTH stimulation test. CONCLUSION: Although postoperative hypocortisolemia was observed in 37% of patients undergoing unilateral adrenalectomy for non-cortisol secreting tumors, majority did not develop symptoms of adrenal insufficiency. All three steroid initiation approaches appeared safe, with management based on clinical symptoms or selective ACTH stimulation testing sparing more patients from steroids compared to steroid initiation based on POD 1 cortisol levels alone.

METTL16 exerts an m6A-independent function to facilitate translation and tumorigenesis.

METTL16 has recently been identified as an RNA methyltransferase responsible for the deposition of N6-methyladenosine (m6A) in a few transcripts. Whether METTL16 methylates a large set of transcripts, similar to METTL3 and METTL14, remains unclear. Here we show that METTL16 exerts both methyltransferase activity-dependent and -independent functions in gene regulation. In the cell nucleus, METTL16 functions as an m6A writer to deposit m6A into hundreds of its specific messenger RNA targets. In the cytosol, METTL16 promotes translation in an m6A-independent manner. More specifically, METTL16 directly interacts with the eukaryotic initiation factors 3a and -b as well as ribosomal RNA through its Mtase domain, thereby facilitating the assembly of the translation-initiation complex and promoting the translation of over 4,000 mRNA transcripts. Moreover, we demonstrate that METTL16 is critical for the tumorigenesis of hepatocellular carcinoma. Collectively, our studies reveal previously unappreciated dual functions of METTL16 as an m6A writer and a translation-initiation facilitator, which together contribute to its essential function in tumorigenesis.

Prevalence and Clinical Determinants of Obesity in Adults With Type 1 Diabetes Mellitus: A Single-Center Retrospective Observational Study.

OBJECTIVE: To determine the prevalence of obesity and assess the cardiometabolic risk profile and treatments associated with obesity management in the type 1 diabetes mellitus adult population. METHODS: We reviewed the records of all patients with type 1 diabetes mellitus seen in our institution's outpatient endocrinology clinic between 2015 and 2018. We stratified the patients into 4 weight categories on the basis of body mass index (BMI) (normal, overweight, obesity class I, and combined obesity class II and III) and evaluated their associated clinical characteristics and relevant medications. RESULTS: Of 451 patients, 64% had a BMI of >25 kg/m2, and 25% had a BMI of ≥30 kg/m2. Over 40% of patients with a BMI of >30 kg/m2 had a history of cardiovascular disease. The off-label use of the glucagon-like peptide 1 receptor agonist was 12% and the sodium glucose cotransporter 2 inhibitor use was 5% in those with obesity. Only 2 patients were prescribed phentermine and 3 had undergone bariatric surgery. Hemoglobin A1C and low-density lipoprotein did not significantly differ between the normal weight and obesity groups. The obesity groups had significantly higher levels of median triglycerides and lower high-density lipoprotein than the normal weight group. CONCLUSION: Obesity was prevalent in a population of patients with type 1 diabetes mellitus seen in a specialty clinic. Those with obesity had a higher prevalence of cardiovascular disease than their normal weight counterparts. The use of weight loss medications was scarce. Studies exploring the safety and efficacy of obesity-targeted therapy in the type 1 diabetes mellitus population are needed.

Serum thyroid stimulating hormone level for predicting utility of thyroid uptake and scan.

BACKGROUND: Thyroid uptake and scan (TUS) is a clinical tool used for differentiation of thyrotoxicosis etiologies. Although guidelines recommend ordering a TUS for evaluation of low TSH levels, no specific value is defined. This study aimed to determine a TSH cutoff at which TUSs yield a greater likelihood of successful determination of etiology to avoid unnecessary testing. METHODS: This was a retrospective study on 137 patients seen by an endocrinologist who underwent TUS for evaluation of low TSH (<0.4 µU/mL). A receiver operating curve analysis was performed to determine the TSH cutoff with maximal sensitivity and specificity for prediction of diagnostic utility. RESULTS: Ninety percent of TUSs (n = 123) led to a diagnosis, while 10% (n = 14) were inconclusive or normal. Diagnoses included Graves' diseases (52%), toxic multinodular goiter (19%), thyroiditis (12%), and solitary toxic adenoma (7%). The median TSH value was 0.008 µU/mL (IQR 0.005, 0.011), and the median free T4 value was 1.7 µU/mL (IQR 1.3, 2.8). The ROC analysis produced an area under the curve of 0.86. The optimal TSH cutoff value was 0.02 µU/mL (sensitivity 80%, specificity 93%) for prediction of diagnostic yield. CONCLUSION: This study demonstrates that TSH is a useful predictor of the utility of TUS in yielding an etiology of thyrotoxicosis. Our analysis showed that TUS had a greater likelihood of determining an etiology when TSH was ≤0.02 µU/mL. This information can help clinicians avoid unnecessary cost and patient time burden when TUS is unlikely to aid in determining the etiology of thyrotoxicosis.

Hormonal Outcomes Following Hemithyroidectomy.

OBJECTIVE: To assess hormonal outcomes and thyroid hormone (TH) replacement after hemithyroidectomy (HT). STUDY DESIGN: Retrospective chart review. SETTING: Quaternary care hospital system. METHODS: A retrospective analysis was performed on patients who had an HT at Cleveland Clinic between 2000 and 2010 with outcomes assessed up to 5 years post-HT. Patients with overt hypothyroidism (OH; thyroid-stimulating hormone [TSH] >10 mIU/L, TSH >4.2 mIU/L on thyroid hormone [TH]), subclinical hypothyroidism (SH; TSH >4.2-10 mIU/L, no TH), or euthyroidism (EU; TSH 0.4-4.2 mIU/L, no TH) were compared. Patients with SH who returned to EU were compared to those who continued to have SH. For immediate start on TH, a receiver operating characteristic analysis was performed to determine dosage of TH above which suppression of TSH <0.4 mIU/L was predicted. RESULTS: We identified 335 patients (average age 51 years, 78% female, median follow-up of 50 months). Of the 210 not immediately started on TH, 32.4% were OH, 13.3% were SH, and 54.3% were EU. EU patients were younger (48 years), had more remaining gland, were less likely to have lymphocytic infiltrate, and had a lower preoperative TSH (1.2 mIU/L). In the SH group, 58.3% of patients normalized their TSH. With immediate TH start, 45% developed suppressed TSH. Those on LT4 >1.05 mcg/kg/d were more likely to suppress (sensitivity 89%). CONCLUSION: Most patients post-HT will remain EU, and immediate start of TH may lead to TSH suppression. Those with SH may ultimately normalize TSH. These findings together suggest that observation may be a better option than TH replacement after HT.

Pituitary Adenylate Cyclase-Activating Polypeptide Attenuates Brain Edema by Protecting Blood-Brain Barrier and Glymphatic System After Subarachnoid Hemorrhage in Rats.

Brain edema is a vital contributor to early brain injury after subarachnoid hemorrhage (SAH), which is responsible for prolonged hospitalization and poor outcomes. Pharmacological therapeutic targets on edema formation have been the focus of research for decades. Pituitary adenylate cyclase-activating polypeptide (PACAP) has been shown to participate in neural development and brain injury. Here, we used PACAP knockout CRISPR to demonstrate that endogenous PACAP plays an endogenous neuroprotective role against brain edema formation after SAH in rats. The exogenous PACAP treatment provided both short- and long-term neurological benefits by preserving the function of the blood-brain barrier and glymphatic system after SAH. Pretreatment of inhibitors of PACAP receptors showed that the PACAP-involved anti-edema effect and neuroprotection after SAH was facilitated by the selective PACAP receptor (PAC1). Further administration of adenylyl cyclase (AC) inhibitor and sulfonylurea receptor 1 (SUR1) CRISPR activator suggested that the AC-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) axis participated in PACAP signaling after SAH, which inhibited the expression of edema-related proteins, SUR1 and aquaporin-4 (AQP4), through SUR1 phosphorylation. Thus, PACAP may serve as a potential clinical treatment to alleviate brain edema in patients with SAH.

Recombinant OX40 attenuates neuronal apoptosis through OX40-OX40L/PI3K/AKT signaling pathway following subarachnoid hemorrhage in rats.

Subarachnoid hemorrhage (SAH) is the most devastating form of stroke. Reducing neuronal apoptosis is an important countermeasure against early brain injury (EBI) after SAH. Recent evidence indicates that OX40-OX40L coupling is critical for cell survival and proliferation. Current study was performed to detect the role of recombinant OX40 (ReOX40) against neuronal apoptosis after SAH. The endovascular perforation model of SAH was performed on Sprague-Dawley (SD) rats. ReOX40 was injected intracerebroventricularly (i.c.v) 1 h after SAH induction and the following methods were employed: neurological function evaluation, immunofluorescence staining, fluoro-Jade C staining, and western blot. To study the underlying precise molecular mechanism, small interfering ribonucleic acid (siRNA) for OX40L and a specific inhibitor of PI3K, LY294002, were injected i.c.v. into SAH + ReOX40 rats before induction of SAH. When compared with sham rats, the expression of OX40 and OX40L was seen to decrease in the brain at 24 h after SAH induction. Administration of ReOX40 (5 µg/kg) increased expression of the OX40L, reduced the neuronal apoptosis, and improved short and long-term neurological function deficits. Furthermore, ReOx40 heightened activation of OX40L/PI3K/AKT axis, increased the downstream anti-apoptotic protein (Bcl2, Bcl-XL), and depressed the apoptotic protein (cleaved caspase 3, Bax). However, the protective effects of ReOX40 were abolished by the administration of OX40L siRNA and LY294002, respectively. These results demonstrate that ReOX40 attenuates neuronal apoptosis through OX40-OX40L/PI3K/AKT pathway in EBI after SAH.

Osteopontin attenuates early brain injury through regulating autophagy-apoptosis interaction after subarachnoid hemorrhage in rats.

AIM: To determine the effect of osteopontin (OPN) on autophagy and autophagy-apoptosis interactions after SAH. METHODS: The endovascular perforation model of SAH or sham surgery was performed in a total of 86 Sprague-Dawley male rats. The temporal expressions of endogenous OPN and autophagy-related proteins (Beclin 1, ATG5, LC3 II to I ratio) were measured in sham and SAH rats at different time points (3, 6, 12, 24, and 72 hours). Rats were randomly divided into three groups: Sham, SAH + Vehicle (PBS, phosphate-buffered saline), and SAH + rOPN (5 µg/rat recombinant OPN). Neurobehavioral tests were performed 24 hours after SAH, followed by the collection of brain samples for assessment of autophagy and apoptosis proteins. These tests assessed whether an autophagy-apoptosis relationship existed on the histological level in the brain. RESULTS: Endogenous OPN and autophagy-related proteins all increased after SAH. rOPN administration improved neurological dysfunction, increased the expression of autophagy-related proteins (Beclin 1, ATG5, LC3 II to I ratio) and antiapoptotic protein Bcl-2, while decreasing the expression of proapoptotic proteins (cleaved Caspase-3 and Bax). rOPN also regulated autophagy-apoptosis interactions 24 hours after SAH. CONCLUSION: rOPN attenuates early brain injury and inhibits neuronal apoptosis by activating autophagy and regulating autophagy-apoptosis interactions.

Histone H3 trimethylation at lysine 36 guides m6A RNA modification co-transcriptionally.

DNA and histone modifications have notable effects on gene expression1. Being the most prevalent internal modification in mRNA, the N6-methyladenosine (m6A) mRNA modification is as an important post-transcriptional mechanism of gene regulation2-4 and has crucial roles in various normal and pathological processes5-12. However, it is unclear how m6A is specifically and dynamically deposited in the transcriptome. Here we report that histone H3 trimethylation at Lys36 (H3K36me3), a marker for transcription elongation, guides m6A deposition globally. We show that m6A modifications are enriched in the vicinity of H3K36me3 peaks, and are reduced globally when cellular H3K36me3 is depleted. Mechanistically, H3K36me3 is recognized and bound directly by METTL14, a crucial component of the m6A methyltransferase complex (MTC), which in turn facilitates the binding of the m6A MTC to adjacent RNA polymerase II, thereby delivering the m6A MTC to actively transcribed nascent RNAs to deposit m6A co-transcriptionally. In mouse embryonic stem cells, phenocopying METTL14 knockdown, H3K36me3 depletion also markedly reduces m6A abundance transcriptome-wide and in pluripotency transcripts, resulting in increased cell stemness. Collectively, our studies reveal the important roles of H3K36me3 and METTL14 in determining specific and dynamic deposition of m6A in mRNA, and uncover another layer of gene expression regulation that involves crosstalk between histone modification and RNA methylation.