Abundant Intra-Subtype Reassortment Revealed in H13N8 Influenza Viruses.

Influenza A viruses (IAVs) pose a serious threat to global health. On the one hand, these viruses cause seasonal flu outbreaks in humans. On the other hand, they are a zoonotic infection that has the potential to cause a pandemic. The most important natural reservoir of IAVs are waterfowl. In this study, we investigated the occurrence of IAV in birds in the Republic of Buryatia (region in Russia). In 2020, a total of 3018 fecal samples were collected from wild migratory birds near Lake Baikal. Of these samples, 11 were found to be positive for the H13N8 subtype and whole-genome sequencing was performed on them. All samples contained the same virus with the designation A/Unknown/Buryatia/Arangatui-1/2020. To our knowledge, virus A/Unknown/Buryatia/Arangatui-1/2020 is the first representative of the H13N8 subtype collected on the territory of Russia, the sequence of which is available in the GenBank database. An analysis of reassortments based on the genome sequences of other known viruses has shown that A/Unknown/Buryatia/Arangatui-1/2020 arose as a result of reassortment. In addition, a reassortment most likely occurred several decades ago between the ancestors of the viruses recently collected in China, the Netherlands, the United States and Chile. The presence of such reassortment emphasizes the ongoing evolution of the H13N8 viruses distributed in Europe, North and East Asia, North and South America and Australia. This study underscores the importance of the continued surveillance and research of less-studied influenza subtypes.

A resveratrol derivative modulates TRH and TRH-like peptide expression throughout the brain and peripheral tissues of male rats.

INTRODUCTION: Resveratrol and related polyphenols have therapeutic effects ranging from treatment of depression, Alzheimer's and Parkinson's disease, obesity, diabetes, neurodegeneration and ageing. TRH and TRH-like peptides, with the structure pGlu-X-Pro-NH2 , where 'X can be any amino acid reside, have reproductive, caloric-restriction-like, anti-ageing, pancreatic-ß cell-enhancing, cardiovascular and neuroprotective effects. We hypothesize that TRH and TRH-like peptides are mediators of the therapeutic actions of the resveratrol derivative pterostilbene (PT). METHODS: Sixteen young adult male Sprague-Dawley rats were divided into four groups. Control group remained on ad libitum chow and water for 10 days. Acute group received ad libitum chow and water for 9 days and then 0.9 g PT/250 g rat chow for 24 h. Chronic animals received PT in chow for 10 days. Withdrawal rats received PT chow for 8 days and then normal chow for 2 days. TRH and TRH-like peptide levels were measured in medulla oblongata (MED), frontal cortex (FCX), hypothalamus (HY), amygdala (AY), hippocampus (HC), piriform cortex (PIR), nucleus accumbens (NA), entorhinal cortex (ENT), striatum (STR), cerebellum (CBL), anterior cingulate (ACNG), posterior cingulate (PCNG), prostate (PR), liver (L), testis (T), heart (H), pancreas (PAN), adrenals (AD) and epididymis (EP). RESULTS: Significant changes in the levels of TRH and TRH-like peptides occurred throughout the brain and peripheral tissues in response to PT treatment. CONCLUSION: The high responsiveness of PIR, CBL, HY, STR, PCNG, MED, FCX, NA, ACNG and AY in brain and EP and PR is consistent with TRH and TRH-like peptides participating in the therapeutic effects of PT.

Rifaximin modulates TRH and TRH-like peptide expression throughout the brain and peripheral tissues of male rats.

BACKGROUND: The TRH/TRH-R1 receptor signaling pathway within the neurons of the dorsal vagal complex is an important mediator of the brain-gut axis. Mental health and protection from a variety of neuropathologies, such as autism, Attention Deficit Hyperactivity Disorder, Alzheimer's and Parkinson's disease, major depression, migraine and epilepsy are influenced by the gut microbiome and is mediated by the vagus nerve. The antibiotic rifaximin (RF) does not cross the gut-blood barrier. It changes the composition of the gut microbiome resulting in therapeutic benefits for traveler's diarrhea, hepatic encephalopathy, and prostatitis. TRH and TRH-like peptides, with the structure pGlu-X-Pro-NH2, where "X" can be any amino acid residue, have reproduction-enhancing, caloric-restriction-like, anti-aging, pancreatic-ß cell-, cardiovascular-, and neuroprotective effects. TRH and TRH-like peptides occur not only throughout the CNS but also in peripheral tissues. To elucidate the involvement of TRH-like peptides in brain-gut-reproductive system interactions 16 male Sprague-Dawley rats, 203 ± 6 g, were divided into 4 groups (n = 4/group): the control (CON) group remained on ad libitum Purina rodent chow and water for 10 days until decapitation, acute (AC) group receiving 150 mg RF/kg powdered rodent chow for 24 h providing 150 mg RF/kg body weight for 200 g rats, chronic (CHR) animals receiving RF for 10 days; withdrawal (WD) rats receiving RF for 8 days and then normal chow for 2 days. RESULTS: Significant changes in the levels of TRH and TRH-like peptides occurred throughout the brain and peripheral tissues in response to RF. The number of significant changes in TRH and TRH-like peptide levels in brain resulting from RF treatment, in descending order were: medulla (16), piriform cortex (8), nucleus accumbens (7), frontal cortex (5), striatum (3), amygdala (3), entorhinal cortex (3), anterior (2), and posterior cingulate (2), hippocampus (1), hypothalamus (0) and cerebellum (0). The corresponding ranking for peripheral tissues were: prostate (6), adrenals (4), pancreas (3), liver (2), testis (1), heart (0). CONCLUSIONS: The sensitivity of TRH and TRH-like peptide expression to RF treatment, particularly in the medulla oblongata and prostate, is consistent with the participation of these peptides in the therapeutic effects of RF.

DNA repair pathway activation features in follicular and papillary thyroid tumors, interrogated using 95 experimental RNA sequencing profiles.

DNA repair can prevent mutations and cancer development, but it can also restore damaged tumor cells after chemo and radiation therapy. We performed RNA sequencing on 95 human pathological thyroid biosamples including 17 follicular adenomas, 23 follicular cancers, 3 medullar cancers, 51 papillary cancers and 1 poorly differentiated cancer. The gene expression profiles are annotated here with the clinical and histological diagnoses and, for papillary cancers, with BRAF gene V600E mutation status. DNA repair molecular pathway analysis showed strongly upregulated pathway activation levels for most of the differential pathways in the papillary cancer and moderately upregulated pattern in the follicular cancer, when compared to the follicular adenomas. This was observed for the BRCA1, ATM, p53, excision repair, and mismatch repair pathways. This finding was validated using independent thyroid tumor expression dataset PRJEB11591. We also analyzed gene expression patterns linked with the radioiodine resistant thyroid tumors (n = 13) and identified 871 differential genes that according to Gene Ontology analysis formed two functional groups: (i) response to topologically incorrect protein and (ii) aldo-keto reductase (NADP) activity. We also found RNA sequencing reads for two hybrid transcripts: one in-frame fusion for well-known NCOA4-RET translocation, and another frameshift fusion of ALK oncogene with a new partner ARHGAP12. The latter could probably support increased expression of truncated ALK downstream from 4th exon out of 28. Both fusions were found in papillary thyroid cancers of follicular histologic subtype with node metastases, one of them (NCOA4-RET) for the radioactive iodine resistant tumor. The differences in DNA repair activation patterns may help to improve therapy of different thyroid cancer types under investigation and the data communicated may serve for finding additional markers of radioiodine resistance.

Transcriptomic and Genomic Testing to Guide Individualized Treatment in Chemoresistant Gastric Cancer Case.

Gastric cancer is globally the fifth leading cause of cancer death. We present a case report describing the unique genomic characteristics of an Epstein-Barr virus-negative gastric cancer with esophageal invasion and regional lymph node metastasis. Genomic tests were performed first with the stomach biopsy using platforms FoundationOne, OncoDNA, and Oncopanel at Dana Farber Institute. Following neoadjuvant chemotherapy, residual tumor was resected and the stomach and esophageal residual tumor samples were compared with the initial biopsy by whole exome sequencing and molecular pathway analysis platform Oncobox. Copy number variation profiling perfectly matched the whole exome sequencing results. A moderate agreement was seen between the diagnostic platforms in finding mutations in the initial biopsy. Final data indicate somatic activating mutation Q546K in PIK3CA gene, somatic frameshifts in PIH1D1 and FBXW7 genes, stop-gain in TP53BP1, and a few somatic mutations of unknown significance. RNA sequencing analysis revealed upregulated expressions of MMP7, MMP9, BIRC5, and PD-L1 genes and strongly differential regulation of several molecular pathways linked with the mutations identified. According to test results, the patient received immunotherapy with anti-PD1 therapy and is now free of disease for 2 years. Our data suggest that matched tumor and normal tissue analyses have a considerable advantage over tumor biopsy-only genomic tests in stomach cancer.

Intratumoral Heterogeneity and Longitudinal Changes in Gene Expression Predict Differential Drug Sensitivity in Newly Diagnosed and Recurrent Glioblastoma.

Background: Inevitable recurrence after radiochemotherapy is the major problem in the treatment of glioblastoma, the most prevalent type of adult brain malignancy. Glioblastomas are notorious for a high degree of intratumor heterogeneity manifest through a diversity of cell types and molecular patterns. The current paradigm of understanding glioblastoma recurrence is that cytotoxic therapy fails to target effectively glioma stem cells. Recent advances indicate that therapy-driven molecular evolution is a fundamental trait associated with glioblastoma recurrence. There is a growing body of evidence indicating that intratumor heterogeneity, longitudinal changes in molecular biomarkers and specific impacts of glioma stem cells need to be taken into consideration in order to increase the accuracy of molecular diagnostics still relying on readouts obtained from a single tumor specimen. Methods: This study integrates a multisampling strategy, longitudinal approach and complementary transcriptomic investigations in order to identify transcriptomic traits of recurrent glioblastoma in whole-tissue specimens of glioblastoma or glioblastoma stem cells. In this study, 128 tissue samples of 44 tumors including 23 first diagnosed, 19 recurrent and 2 secondary recurrent glioblastomas were analyzed along with 27 primary cultures of glioblastoma stem cells by RNA sequencing. A novel algorithm was used to quantify longitudinal changes in pathway activities and model efficacy of anti-cancer drugs based on gene expression data. Results: Our study reveals that intratumor heterogeneity of gene expression patterns is a fundamental characteristic of not only newly diagnosed but also recurrent glioblastomas. Evidence is provided that glioblastoma stem cells recapitulate intratumor heterogeneity, longitudinal transcriptomic changes and drug sensitivity patterns associated with the state of recurrence. Conclusions: Our results provide a transcriptional rationale for the lack of significant therapeutic benefit from temozolomide in patients with recurrent glioblastoma. Our findings imply that the spectrum of potentially effective drugs is likely to differ between newly diagnosed and recurrent glioblastomas and underscore the merits of glioblastoma stem cells as prognostic models for identifying alternative drugs and predicting drug response in recurrent glioblastoma. With the majority of recurrent glioblastomas being inoperable, glioblastoma stem cell models provide the means of compensating for the limited availability of recurrent glioblastoma specimens.

Personalized prescription of tyrosine kinase inhibitors in unresectable metastatic cholangiocarcinoma.

BACKGROUND: Cholangiocarcinoma is an aggressive tumor with poor prognosis. Most of the cases are not available for surgery at the stage of the diagnosis and the best clinical practice chemotherapy results in about 12-month median survival. Several tyrosine kinase inhibitors (TKIs) are currently under investigation as an alternative treatment option for cholangiocarcinoma. Thus, the report of personalized selection of effective inhibitor and case outcome are of clinical interest. CASE PRESENTATION: Here we report a case of aggressive metastatic cholangiocarcinoma (MCC) in 72-year-old man, sequentially treated with two targeted chemotherapies. Initially disease quickly progressed during best clinical practice care (gemcitabine in combination with cisplatin or capecitabine), which was accompanied by significant decrease of life quality. Monotherapy with TKI sorafenib was prescribed to the patient, which resulted in stabilization of tumor growth and elimination of pain. The choice of the inhibitor was made based on high-throughput screening of gene expression in the patient's tumor biopsy, utilized by Oncobox platform to build a personalized rating of potentially effective target therapies. However, time to progression after start of sorafenib administration did not exceed 6 months and the regimen was changed to monotherapy with Pazopanib, another TKI predicted to be effective for this patient according to the same molecular test. It resulted in disease progression according to RECIST with simultaneous elimination of sorafenib side effects such as rash and hand-foot syndrome. After 2 years from the diagnosis of MCC the patient was alive and physically active, which is substantially longer than median survival for standard therapy. CONCLUSION: This case evidences that sequential personalized prescription of different TKIs may show promising efficacy in terms of survival and quality of life in MCC.

Direct control of somatic stem cell proliferation factors by the Drosophila testis stem cell niche.

Niches have traditionally been characterised as signalling microenvironments that allow stem cells to maintain their fate. This definition implicitly assumes that the various niche signals are integrated towards a binary fate decision between stemness and differentiation. However, observations in multiple systems have demonstrated that stem cell properties, such as proliferation and self-renewal, can be uncoupled at the level of niche signalling input, which is incompatible with this simplified view. We have studied the role of the transcriptional regulator Zfh1, a shared target of the Hedgehog and Jak/Stat niche signalling pathways, in the somatic stem cells of the Drosophila testis. We found that Zfh1 binds and downregulates salvador and kibra, two tumour suppressor genes of the Hippo/Wts/Yki pathway, thereby restricting Yki activation and proliferation to the Zfh1+ stem cells. These observations provide an unbroken link from niche signal input to an individual aspect of stem cell behaviour that does not, at any step, involve a fate decision. We discuss the relevance of these findings for an overall concept of stemness and niche function.

Nox4 and Duox1/2 Mediate Redox Activation of Mesenchymal Cell Migration by PDGF.

Platelet derived growth factor (PDGF) orchestrates wound healing and tissue regeneration by regulating recruitment of the precursor mesenchymal stromal cells (MSC) and fibroblasts. PDGF stimulates generation of hydrogen peroxide that is required for cell migration, but the sources and intracellular targets of H2O2 remain obscure. Here we demonstrate sustained live responses of H2O2 to PDGF and identify PKB/Akt, but not Erk1/2, as the target for redox regulation in cultured 3T3 fibroblasts and MSC. Apocynin, cell-permeable catalase and LY294002 inhibited PDGF-induced migration and mitotic activity of these cells indicating involvement of PI3-kinase pathway and H2O2. Real-time PCR revealed Nox4 and Duox1/2 as the potential sources of H2O2. Silencing of Duox1/2 in fibroblasts or Nox4 in MSC reduced PDGF-stimulated intracellular H2O2, PKB/Akt phosphorylation and migration, but had no such effect on Erk1/2. In contrast to PDGF, EGF failed to increase cytoplasmic H2O2, phosphorylation of PKB/Akt and migration of fibroblasts and MSC, confirming the critical impact of redox signaling. We conclude that PDGF-induced migration of mesenchymal cells requires Nox4 and Duox1/2 enzymes, which mediate redox-sensitive activation of PI3-kinase pathway and PKB/Akt.

Rapid modulation of TRH and TRH-like peptide release in rat brain and peripheral tissues by prazosin.

Hyperresponsiveness to norepinephrine contributes to post-traumatic stress disorder (PTSD). Prazosin, a brain-active blocker of α(1)-adrenoceptors, originally used for the treatment of hypertension, has been reported to alleviate trauma nightmares, sleep disturbance and improve global clinical status in war veterans with PTSD. Thyrotropin-releasing hormone (TRH, pGlu-His-Pro-NH(2)) may play a role in the pathophysiology and treatment of neuropsychiatric disorders such as major depression, and PTSD (an anxiety disorder). To investigate whether TRH or TRH-like peptides (pGlu-X-Pro-NH(2), where "X" can be any amino acid residue) participate in the therapeutic effects of prazosin, male rats were injected with prazosin and these peptides then measured in brain and endocrine tissues. Prazosin stimulated TRH and TRH-like peptide release in those tissues with high α(1)-adrenoceptor levels suggesting that these peptides may play a role in the therapeutic effects of prazosin.

Rapid modulation of TRH and TRH-like peptide release in rat brain, pancreas, and testis by a GSK-3beta inhibitor.

Antidepressants have been shown to be neuroprotective and able to reverse damage to glia and neurons. Thyrotropin-releasing hormone (TRH) is an endogenous antidepressant-like neuropeptide that reduces the expression of glycogen synthase kinase-3beta (GSK-3beta), an enzyme that hyperphosphorylates tau and is implicated in bipolar disorder, diabetes and Alzheimer's disease. In order to understand the potential role of GSK-3beta in the modulation of depression by TRH and TRH-like peptides and the therapeutic potential of GSK-3beta inhibitors for neuropsychiatric and metabolic diseases, young adult male Sprague-Dawley (SD) rats were (a) injected ip with 1.8mg/kg of GSK-3beta inhibitor VIII (GSKI) and sacrificed 0, 2, 4, 6, and 8h later or (b) injected with 0, 0.018, 0.18 or 1.8mg/kg GSKI and bled 4h later. Levels of TRH and TRH-like peptides were measured in various brain regions involved in mood regulation, pancreas and reproductive tissues. Large, 3-15-fold, increases of TRH and TRH-like peptide levels in cerebellum, for example, as well as other brain regions were noted at 2 and 4h. In contrast, a nearly complete loss of TRH and TRH-like peptides from testis within 2h and pancreas by 4h following GSKI injection was observed. We have previously reported similar acute effects of corticosterone in brain and peripheral tissues. Incubation of a decapsulated rat testis with either GSKI or corticosterone accelerated release of TRH, and TRH-like peptides. Glucocorticoids, via inhibition of GSK3-beta activity, may thus be involved in the inhibition of TRH and TRH-like peptide release in brain, thereby contributing to the depressogenic effect of this class of steroids. Corticosterone-induced acceleration of release of these peptides from testis may contribute to the decline in reproductive function and redirection of energy needed during life-threatening emergencies. These contrasting effects of glucocorticoid on peptide release appear to be mediated by GSK-3beta.

TRH and TRH-like peptide expression in rat following episodic or continuous corticosterone.

Sustained abnormalities of glucocorticoid levels have been associated with neuropsychiatric illnesses such as major depression, posttraumatic stress disorder (PTSD), panic disorder, and obsessive compulsive disorder. The pathophysiological effects of glucocorticoids may depend not only on the amount of glucocorticoid exposure but also on its temporal pattern, since it is well established that hormone receptors are down-regulated by continuously elevated cognate hormones. We have previously reported that TRH (pGlu-His-Pro-NH2) and TRH-like peptides (pGlu-X-Pro-NH2) have endogenous antidepressant-like properties and mediate or modulate the acute effects of a single i.p. injection of high dose corticosterone (CORT) in rats. For these reasons, two accepted methods for inducing chronic hyperglucocorticoidemia have been compared for their effects on brain and peripheral tissue levels of TRH and TRH-like peptides in male, 250 g, Sprague-Dawley rats: (1) the dosing effect of CORT hemisuccinate in drinking water, and (2) s.c. slow-release pellets. Overall, there were 93% more significant changes in TRH and TRH-like peptide levels in brain and 111% more in peripheral tissues of those rats ingesting various doses of CORT in drinking water compared to those with 1-3 s.c. pellets. We conclude that providing rats with CORT in drinking water is a convenient model for the pathophysiological effects of hyperglucocorticoidemia in rodents.

Lipopolysaccharide modulation of thyrotropin-releasing hormone (TRH) and TRH-like peptide levels in rat brain and endocrine organs.

Lipopolysaccharide (LPS) is a proinflammatory and depressogenic agent whereas thyrotropin-releasing hormone (TRH; pGlu-His-Pro-NH2) is an endogenous antidepressant and neuroprotective peptide. LPS and TRH also have opposing effects on K+ channel conductivity. We hypothesized that LPS can modulate the expression and release of not only TRH but also TRH-like peptides with the general structure pGlu-X-Pro-NH2, where "X" can be any amino acid residue. The response might be "homeostatic," that is, LPS might increase TRH and TRH-like peptide release, thereby moderating the cell damaging effects of this bacterial cell wall constituent. On the other hand, LPS might impair the synthesis and release of these neuropeptides, thus facilitating the induction of early response genes, cytokines, and other downstream biochemical changes that contribute to the "sickness syndrome." Sprague-Dawley rats (300 g) received a single intraperitoneal injection of 100 microg/kg LPS. Animals were then decapitated 0, 2, 4, 8, and 24 h later. Serum cytokines and corticosterone peaked 2 h after intraperitoneal LPS along with a transient decrease in serum T3. TRH and TRH-like peptides were measured by a combination of high-performance liquid chromatography and radioimmunoassay. TRH declined in the nucleus accumbens and amygdala in a manner consistent with LPS-accelerated release and degradation. Various TRH-like peptide levels increased at 2 h in the anterior cingulate, hippocampus, striatum, entorhinal cortex, posterior cingulate, and cerebellum, indicating decreased release and clearance of these peptides. These brain regions are part of a neuroimmunomodulatory system that coordinates the behavioral, endocrine, and immune responses to the stresses of sickness, injury, and danger. A sustained rise in TRH levels in pancreatic beta-cells accompanied LPS-impaired insulin secretion. TRH and Leu-TRH in prostate and TRH in epididymis remained elevated 2-24 h after intraperitoneal LPS. We conclude that these endogenous neuroprotective and antidepressant-like peptides both mediate and moderate some of the behavioral and toxic effects of LPS.

Circadian rhythms of TRH-like peptide levels in rat brain.

This is the first report of diurnal variations in the levels of thyrotropin-releasing hormone-like peptides (pGlu-X-Pro-NH(2), where "X" can be any amino acid residue) in brain regions involved in mood regulation. These peptides have neuroprotective and antidepressant-like properties that may help stabilize chronobiologic systems that are often abnormal in neuropsychiatric disease. We hypothesized that diurnal fluctuations in the levels of these neuropeptides are components of the chronobiologic regulation of autonomic, behavioral and emotional states. Optimal use of these potentially therapeutic agents will benefit from an understanding of their response to, and effect on, normal vegetative, activity and sleep patterns, and the corresponding disordered patterns of mental illness. For these reasons, 16 male, 200 g, Sprague-Dawley rats were maintained for 4 weeks in a stable 12 h lights on, 12 h lights off photoperiod. Levels of TRH and TRH-like peptides were measured at 3.0 h, 10.5 h, 13.5 h and 21.0 h, where the subjective midnight was 0.0 h, by a combination of HPLC and RIA. Highly significant changes in TRH-like peptide levels were observed in the striatum, posterior cingulate, cerebellum, pyriform cortex, nucleus accumbens and medulla oblongata. TRH-like peptide levels, in general, were highly correlated with changes in TRH concentration, within and between brain regions, and may be colocalized in large glutamatergic neurons innervating the rat limbic system. We conclude that TRH-like peptides may be important components of chronobiologic systems involved in maintaining autonomic, behavioral and mood equilibria.

The effects of prostomium and proventriculus removal on sex determination and gametogenesis inTyposyllis pulchra (Polychaeta: Syllidae).

The prostomium alone or the prostomium and proventriculus of reproductiveTyposyllis pulchra were periodically removed at known stages of oogenesis and the gametes were examined by transmission electron microscopy. If the proventriculus and prostomium were simultaneously removed prior to day 3 of the stolonization sequence, before gonial differentiation, the time reruired for stolon formation and concomitant gametogenesis was shortened; the animals, all of which had previously reproduced as females, produced only ultrastructurally normal sperm. Spermatogenesis in these induced males began earlier in the stolonization period than in normal males. However, the cytological events of spermatogenesis were not accelerated. When the same operation was performed after differentiated oocytes were present, gamete cytodifferentiation and development time did not appear to be affected and the animals remained female. Removal of the prostomium alone, formerly thought to have no effect, caused high mortality and if removed prior to day 3 appeared to prevent both stolonization and gametogenesis. Ultrastructural investigation of these animals shows that gonads are maintained, but that gonial cells fail to differentiate or produce gametes. The subsequent removal of the proventriculus and regenerating prostomium from these animals allows them to mature as induced males. This suggests a prostomial role in regulating the endocrine activity of the proventriculus during the reproductive cycle.