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Abstract Background Antenatal steroid therapy for fetal lung maturation is routinely administered to women at risk of preterm delivery. There is strong evidence to demonstrate benefit from antenatal steroids in terms of survival and respiratory disease, notably in infants delivered at or below 32 weeks’ gestation. However, dosing remains unoptimized and lung benefits are highly variable. Current treatment regimens generate high-concentration, pulsatile fetal steroid exposures now associated with increased risk of childhood neurodevelopmental diseases. We hypothesized that damage-associated changes in the fetal hippocampal transcriptome would be independent of preterm lung function. Methods Date-mated ewes carrying a single fetus at 122 ± 2dGA (term = 150dGA) were randomized into 4 groups: (i) Saline Control Group, 4×2ml maternal saline intramuscular(IM) injections at 12hr intervals (n = 11); or (ii) Dex High Group, 2×12mg maternal IM dexamethasone phosphate injections at 12hr intervals followed by 2×2ml IM saline injections at 12hr intervals (n = 12; representing a clinical regimen used in Singapore); or (iii) Dex Low Group, 4×1.5mg maternal IM dexamethasone phosphate injections 12hr intervals (n = 12); or (iv) Beta-Acetate Group, 1×0.125mg/kg maternal IM betamethasone acetate injection followed by 3×2ml IM sterile normal saline injections 12hr intervals (n = 8). Lambs were surgically delivered 48hr after first maternal injection at 122–125dGA, ventilated for 30min to establish lung function, and euthanised for necropsy and tissue collection. Results Preterm lambs from the Dex Low and Beta-Acetate Groups had statistically and biologically significant lung function improvements (measured by gas exchange, lung compliance). Compared to the Saline Control Group, hippocampal transcriptomic data identified 879 differentially significant expressed genes (at least 1.5-fold change and FDR

The discovery of gene fusions involving Neuregulin-1 (NRG1) within solid tumors has important therapeutic implications, as they are being actively explored as targets for emerging ERBB/ERBB2/ERBB3-directed anti-cancer agents. NRG1 fusions are very uncommon across all tumor types and are infrequently documented in the medical literature. We report a female patient presenting with widespread peritoneal carcinomatosis diagnosed as high grade serous fallopian tube carcinoma, which harbored a previously undescribed MYH10::NRG1 fusion. Moreover, we queried the whole transcriptome sequencing results of neoplasms analyzed by a commercial laboratory (Caris Life Sciences) to determine the overall incidence of NRG1 fusions in carcinomas of the ovary, fallopian tube, and peritoneum (0.18%). Twenty-five additional tumors were found to demonstrate NRG1 fusions, including 20 new genes partners that had not been previously identified in gynecologic carcinomas. Overall, NRG1 fusion events are rare in ovarian, fallopian tube, and primary peritoneal carcinomas, but they may carry diagnostic significance in the context of borderline/low grade serous tumors, which demonstrated exclusively CLU::NRG1 fusions, and could have important predictive implications for response to ERBB/ERBB2/ERBB3-directed therapies.

IntroductionPreclinical studies suggest that cannabinoid receptor type 2 (CB2R) activation has a therapeutic effect in animal models on chronic inflammation and vascular permeability, which are key pathological features of diabetic retinopathy (DR). A novel CB2R agonist, triazolopyrimidine RG7774, was generated through lead optimization of a high-throughput screening hit. The aim of this study was to characterize the pharmacology, absorption, distribution, metabolism, elimination, and toxicity (ADMET) profile of RG7774, and to explore its potential for managing the key pathological features associated with retinal disease in rodents.MethodsThe in vitro pharmacology of RG7774 was investigated for CB2R binding and receptor activation using recombinant human and mouse CB2R expression in Chinese hamster ovary cells, and endogenous CB2R expression in human Jurkat cells, and rat and mouse spleen cells. The ADMET profile was evaluated and the effects of RG7774 on retinal permeability, leukocyte adhesion, and choroidal neovascularization (CNV) were investigated in rodent models of retinal disease. Pharmacokinetic (PK) parameters and the exposure-response relationship were characterized in healthy animals and in animals with laser-induced CNV.ResultsRG7774 was found to be a potent (EC50: 2.8 nM and Ki: 51.3 nM), selective, and full CB2R agonist with no signs of cannabinoid receptor type 1 (CB1R) binding or activation. The ligand showed a favorable ADMET profile and exhibited systemic and ocular exposure after oral delivery. Functional potency in vitro translated from recombinant to endogenous expression systems. In vivo, orally administered RG7774 reduced retinal permeability and leukocyte adhesion in rodents with lipopolysaccharide (LPS)-induced uveitis and streptozotocin (STZ)-induced DR, and reduced lesion areas in rats with laser-induced CNV with an ED50 of 0.32 mg/kg. Anatomically, RG7774 reduced the migration of retinal microglia to retinal lesions.DiscussionRG7774 is a novel, highly selective, and orally bioavailable CB2R agonist, with an acceptable systemic and ocular PK profile, and beneficial effects on retinal vascular permeability, leukocyte adhesion, and ocular inflammation in rodent animal models. Results support the development of RG7774 as a potential treatment for retinal diseases with similar pathophysiologies as addressed by the animal models.

Chronic myelomonocytic leukemia (CMML) is a myelodysplastic/myeloproliferative neoplasm characterized by peripheral blood monocytosis and bone marrow dysplasia. In approximately one-fourth of cases, CMML can demonstrate progression to acute myeloid leukemia (AML), referred to as AML ex CMML. We present a 58-year-old woman with a past medical history of idiopathic thrombocytopenic purpura (ITP) who demonstrated 24% bone marrow blasts on a repeat biopsy obtained two years after being diagnosed with CMML. By the flow cytometric analysis, the blasts expressed partial CD34, CD13, CD117, partial MPO, and partial CD123 with coexpression of the T-lymphoid markers CD2, CD5, CD7, partial CD4, cytoplasmic CD3, partial cytoplasmic TDT, and CD38, suggestive of AML with rare mixed myeloid/T-cell phenotype. Treatment with various agents including decitabine, cytarabine, daunorubicin, etoposide, and venetoclax, and two experimental bromodomain and extraterminal (BET) inhibitors did not produce sustained remissions, and the patient eventually succumbed to her disease. T-cell phenotype is an exceedingly rare feature of AML ex CMML, and whether this unique differentiation pathway contributed to the aggressive disease course remains unclear.

Low-dose interleukin-2 is showing promise in the treatment of several autoimmune inflammatory diseases. Here authors map the trajectory of cellular and transcriptional changes in type 1 diabetes patients receiving an interval dosing interleukin-2 regimen, which shows an anti-inflammatory gene expression signature shared by all immune cell types analysed, persisting for at least a month after ending treatment.

Soft tissue sarcomas harboring EWSR1::CREM fusion are rare and challenging to treat. Pazopanib, a multi-tyrosine kinase inhibitor, is FDA-approved for advanced soft tissue sarcomas, but predictive biomarkers for its efficacy remain unidentified. We conducted a study on > 240,000 neoplasms submitted to Caris Life Sciences (Phoenix, AZ) to detect rearrangements using whole transcriptome sequencing. Two sarcoma-experienced, board-certified pathologists performed histological reviews, and treatment/outcome information was collected. Among the identified cases (n = 18), we observed a diverse range of sarcoma and other cancers, including an intracranial myxoid mesenchymal tumor, mesothelioma, hyalinizing clear cell carcinomas of the head and neck, clear cell sarcomas, and undifferentiated round cell sarcomas, as well as histologically malignant tumors with epithelioid morphology. Notably, two undifferentiated, metastatic, abdominal round cell sarcoma cases treated with pazopanib demonstrated significant sustained partial response and clinical benefit. To explore the genetic factors associated with the efficacy of pazopanib in these cases, next-generation sequencing and fluorescence in situ hybridization were analyzed for alterations in the tumors. The genomic analysis provided compelling evidence confirming the presence of EWSR1::CREM fusion in both cases, with no other pathogenic gene variants or copy number alterations detected. These cases demonstrate the potential of Pazopanib as a promising therapeutic option for patients with EWSR1::CREM fusion-positive soft tissue sarcomas, including metastatic undifferentiated round cell sarcomas. The sustained clinical benefit and partial responses observed in these cases warrant further research to validate these findings and explore the wider utility of Pazopanib in this rare and challenging subset of soft tissue sarcomas. Case studies: Case 1: A 49-year-old man presented with abdominal pain, weight loss, and chronic cough. A computed tomography (CT) of the chest, abdomen, and pelvis showed multiple lung nodules and masses and a right rectus mass that was biopsied and revealed an undifferentiated round cell sarcoma with a rare fusion EWSR1-CREM. No additional pathogenic gene variants or copy number alterations were detected. He received neoadjuvant chemotherapy with three cycles of Vincristine, Adriamycin, and Ifosfamide (VAI) and seven cycles of Vincristine/Irinotecan and Temodar (VIT). After cycle 7 of VIT, he had surgical resection of the abdominal mass and received radiation for lung metastasis. He completed 13 cycles of VIT after which he presented with progression of disease and switched to monotherapy with Pazopanib. At the time of this analysis he had stable disease for 28 months. Case 2: A 75-year-old woman presented with pelvic pain and new onset constipation. CT abdomen showed a large pelvic mass and intraperitoneal tumor spread. Exploratory laparotomy revealed a ruptured pelvic mass and a small bowel tumor. Both tumors were proved to be high-grade, poorly differentiated sarcoma. Genomic analysis demonstrated an EWSR1::CREM fusion but no other pathogenic gene variants or copy number alterations. She was treated initially for a primitive neuroectodermal tumor (PNET) with four cycles of Vincristine/Adriamycin/Cytoxan/Olaratumab but declined additional chemotherapy after progression. Two years later, she presented with recurrent abdominal mass and received one cycle of Temodar/Irinotecan, then she began Pozapanib and underwent palliative radiation to the entire pelvis. She has been on Pazopanib for 23 months with stable disease.

Summary: DISC1 is a genetic risk factor for multiple psychiatric disorders. Compared to the dozens of murine Disc1 models, there is a paucity of zebrafish disc1 models—an organism amenable to high-throughput experimentation. We conducted the longitudinal neurobehavioral analysis of disc1 mutant zebrafish across key stages of life. During early developmental stages, disc1 mutants exhibited abrogated behavioral responses to sensory stimuli across multiple testing platforms. Moreover, during exposure to an acoustic sensory stimulus, loss of disc1 resulted in the abnormal activation of neurons in the pallium, cerebellum, and tectum—anatomical sites involved in the integration of sensory perception and motor control. In adulthood, disc1 mutants exhibited sexually dimorphic reduction in anxiogenic behavior in novel paradigms. Together, these findings implicate disc1 in sensorimotor processes and the genesis of anxiogenic behaviors, which could be exploited for the development of novel treatments in addition to investigating the biology of sensorimotor transformation in the context of disc1 deletion.

Individuals with Down syndrome are at decreased risk of developing most types of solid tumors, including central nervous system malignancies. Several mechanisms have been proposed to explain how additional genetic material on chromosome 21 may confer this increased protection. Only two individuals with Down syndrome and meningioma have been described in the medical literature, whose tumors were both World Health Organization (WHO) grade 1. Here, we report the first individual with Down syndrome to our knowledge who developed an atypical meningioma, WHO grade 2. We also provide a hypothesis for how this tumor could have arisen in the setting of trisomy 21.

Abstract Microenvironmental and molecular factors mediating the progression of Breast Ductal Carcinoma In Situ (DCIS) are not well understood, impeding the development of prevention strategies and the safe testing of treatment de-escalation. We addressed methodological barriers and characterized the mutational, transcriptional, histological, and microenvironmental landscape across 85 multiple microdissected regions from 39 cases. Most somatic alterations, including whole-genome duplications, were clonal, but genetic divergence increased with physical distance. Phenotypic and subtype heterogeneity was frequently associated with underlying genetic heterogeneity and regions with low-risk features preceded those with high-risk features according to the inferred phylogeny. B- and T-lymphocytes spatial analysis identified three immune states, including an epithelial excluded state located preferentially at DCIS regions, and characterized by histological and molecular features of immune escape, independently from molecular subtypes. Such breast pre-cancer atlas with uniquely integrated observations will help scope future expansion studies and build finer models of outcomes and progression risk.

The tumor microenvironment is a complex mixture of cell types that bi-directionally interact and influence tumor initiation, progression, recurrence, and patient survival. Mesenchymal stromal cells (MSCs) of the tumor microenvironment engage in crosstalk with cancer cells to mediate epigenetic control of gene expression. We identified CD90+ MSCs residing in the tumor microenvironment of patients with invasive breast cancer that exhibit a unique gene expression signature. Single-cell transcriptional analysis of these MSCs in tumor-associated stroma identified a distinct subpopulation characterized by increased expression of genes functionally related to extracellular matrix signaling. Blocking the TGFβ pathway reveals that these cells directly contribute to cancer cell proliferation. Our findings provide novel insight into communication between breast cancer cells and MSCs that are consistent with an epithelial to mesenchymal transition and acquisition of competency for compromised control of proliferation, mobility, motility, and phenotype.

Collagenous gastritis has been reported as a rare cause of nausea, diarrhea, weight changes, and early satiety in female patients. Here, we describe two women aged 43 and 71 years who presented with similar symptoms. Gastric biopsies from both individuals showed thickened, irregular subepithelial collagen bands (>10 μm). The pathogenesis of collagenous gastritis is poorly understood, but it may be the presenting symptom for many underlying autoimmune conditions. In particular, there is a well-established connection between collagenous disorders of the gastrointestinal tract and celiac sprue, Sjögren syndrome, and lymphocytic colitis; however, none of these conditions had been diagnosed in our patients. The older woman had incidentally discovered hypogammaglobinemia and IgA deficiency, whereas the younger woman suffered from fibromyalgia. Although a gluten-free diet and budesonide have been effective in some cases, there is no standardized therapy for collagenous gastritis. Our patients trialed diet modification and have required no additional medical interventions.

Omega-3 long-chain polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are poorly synthesised in the human body and are substantially lower in Western diets compared with their shorter-chain omega-3 essential fatty acid precursors, α-linolenic acid (18:3n-3; ALA). We assessed their intake among 111 (20.45 years old; 78 females) De Montfort University (DMU, England) students. The dietary intakes of total fat (100.55 vs. 81.72; p-value = 0.032), PUFA (14.61 vs. 12.91; NS), linoleic acid (LA; 3.893 vs. 2.787; p-value = 0.0019), ALA (0.925 vs. 0.613; p-value = 0.00008), arachidonic acid (AA; 0.109 vs. 0.082; p-value = 0.0303), EPA (0.088 vs. 0.075; NS), DHA (0.153 vs. 0.121; NS), and docosapentaenoic acid (DPA; 0.043 vs. 0.032 all in g/day; NS) were significantly higher or higher in male participants, respectively. The dietary intakes of DHA + EPA in the whole group monitored (0.130 + 0.079 = 0.209 g/day) were lower than the RDI of 0.5 g/day, which considers the intake of one to two portions of fish per week. Our results highlight that some DMU students did not meet the nutritional goals for ALA, EPA, and DHA. DMU students should specifically enhance the intake of oily fish (12.422, 13.406, and 10.054 g/day for the overall, female, and male population, respectively), as these intakes only provide around 0.228, 0.246, and 0.184 g of DHA + EPA/day. Education would be required to increase awareness of the importance of consuming more fish among these young adults. Another option would be to encourage the intake of dietary fish oil supplements or the enrichment of food items largely consumed by young British adults with these long-chain PUFAs.

The aim was two-fold: to characterise the risks of lead (Pb) in Agaricus bitorquis collected in the city of Leicester (England), and to evaluate its presence in urban topsoils. Pb was monitored by ICP-MS in twenty-two homogenised mushroom samples (caps and stipes) mineralised with HNO3/H2O2 [LoD = 0.872 mg/kg dry weight (dw)]. Moreover, 450 topsoil samples were collected from 18 urban parks across Leicester; Pb was also measured by ICP-MS after appropriate digestion (LoD = 0.698 mg/kg). Levels were significantly higher in the mushroom caps (p-value = 3 × 10−5); median and ranges are provided in mg/kg dw: 2.461 (1.806–6.664) vs. 1.579 (0.988–4.223). Concentrations were much higher than those reported in sixteen A. bisporus (median < 1.0 mg/kg DW) specifically cultivated in high-traffic areas in the inner city of Berlin, suggesting some contamination by Pb. All caps monitored exceeded the established maximum concentration limit for Pb in cultivated mushrooms in the European Union (3 mg/kg dw), in line with the high accumulative metal capability described in the literature for Agaricus spp. Although non-carcinogenic risks characterised for Pb were negligible in the monitored mushrooms, a high consumption of wild green edibles in Leicester’s city should be limited as there are multiple additional sources of Pb and other metals, and they should be substituted by cultivated edibles where possible.

The aims were: (a) to biomonitor vanadium (V) in wild edible mushrooms collected from urban/rural areas across Leicestershire (England); (b) to characterise the risk to humans caused by its content in topsoils. Thirty-four mushrooms were collected: twenty-two Agaricus bitorquis from a green area close to a busy traffic area; four Marasmius oreades from the NE; and eight Coprinus atramentarius from Bradgate Park, which is northwest of Leicester city. Moreover, 850 topsoil samples were collected and processed as composite samples (eighteen urban and eight rural), which were further processed in duplicate. The level of V was measured twice in each of the 52 composite samples and in the cleaned, dried and homogenised mushrooms by ICP-MS. Significant higher levels of V were found in C. atramentarius (0.856 (0.175–4.338)) than in the edible mushrooms collected in the urban areas (M. oreades, 0.305 (A. bitorquis, 0.078 (

Summary: The protein tau has been implicated in many brain disorders. In animal models, tau reduction suppresses epileptogenesis of diverse causes and ameliorates synaptic and behavioral abnormalities in various conditions associated with excessive excitation-inhibition (E/I) ratios. However, the underlying mechanisms are unknown. Global genetic ablation of tau in mice reduces the action potential (AP) firing and E/I ratio of pyramidal cells in acute cortical slices without affecting the excitability of these cells. Tau ablation reduces the excitatory inputs to inhibitory neurons, increases the excitability of these cells, and structurally alters their axon initial segments (AISs). In primary neuronal cultures subjected to prolonged overstimulation, tau ablation diminishes the homeostatic response of AISs in inhibitory neurons, promotes inhibition, and suppresses hypersynchrony. Together, these differential alterations in excitatory and inhibitory neurons help explain how tau reduction prevents network hypersynchrony and counteracts brain disorders causing abnormally increased E/I ratios.

Abstract The maintenance of genomic integrity is essential for normal cellular functions. However, it is difficult to maintain over a lifetime in postmitotic cells such as neurons, in which DNA damage increases with age and is exacerbated by multiple neurological disorders, including Alzheimer’s disease (AD). Here we used immunohistochemical staining to detect DNA double strand breaks (DSBs), the most severe form of DNA damage, in postmortem brain tissues from patients with mild cognitive impairment (MCI) or AD and from cognitively unimpaired controls. Immunostaining for γH2AX—a post-translational histone modification that is widely used as a marker of DSBs—revealed increased proportions of γH2AX-labeled neurons and astrocytes in the hippocampus and frontal cortex of MCI and AD patients, as compared to age-matched controls. In contrast to the focal pattern associated with DSBs, some neurons and glia in humans and mice showed diffuse pan-nuclear patterns of γH2AX immunoreactivity. In mouse brains and primary neuronal cultures, such pan-nuclear γH2AX labeling could be elicited by increasing neuronal activity. To assess whether pan-nuclear γH2AX represents DSBs, we used a recently developed technology, DNA damage in situ ligation followed by proximity ligation assay, to detect close associations between γH2AX sites and free DSB ends. This assay revealed no evidence of DSBs in neurons or astrocytes with prominent pan-nuclear γH2AX labeling. These findings suggest that focal, but not pan-nuclear, increases in γH2AX immunoreactivity are associated with DSBs in brain tissue and that these distinct patterns of γH2AX formation may have different causes and consequences. We conclude that AD is associated with an accumulation of DSBs in vulnerable neuronal and glial cell populations from early stages onward. Because of the severe adverse effects this type of DNA damage can have on gene expression, chromatin stability and cellular functions, DSBs could be an important causal driver of neurodegeneration and cognitive decline in this disease.

Oxidatively generated damage to DNA has been implicated in the pathogenesis of a wide variety of diseases. Increasingly, interest is also focusing upon the effects of damage to the other nucleic acids, RNA and the (2′-deoxy-)ribonucleotide pools, and evidence is growing that these too may have an important role in disease. LC-MS/MS has the ability to provide absolute quantification of specific biomarkers, such as 8-oxo-7,8-dihydro-2′-deoxyGuo (8-oxodG), in both nuclear and mitochondrial DNA, and 8-oxoGuo in RNA. However, significant quantities of tissue are needed, limiting its use in human biomonitoring studies. In contrast, the comet assay requires much less material, and as little as 5 μL of blood may be used, offering a minimally invasive means of assessing oxidative stress in vivo, but this is restricted to nuclear DNA damage only. Urine is an ideal matrix in which to non-invasively study nucleic acid-derived biomarkers of oxidative stress, and considerable progress has been made towards robustly validating these measurements, not least through the efforts of the European Standards Committee on Urinary (DNA) Lesion Analysis. For urine, LC-MS/MS is considered the gold standard approach, and although there have been improvements to the ELISA methodology, this is largely limited to 8-oxodG. Emerging DNA adductomics approaches, which either comprehensively assess the totality of adducts in DNA, or map DNA damage across the nuclear and mitochondrial genomes, offer the potential to considerably advance our understanding of the mechanistic role of oxidatively damaged nucleic acids in disease.

Primary bone marrow diffuse large B-cell lymphoma is an exceedingly rare form of non-Hodgkin lymphoma. It may demonstrate a leukemic presentation, and a proportion of cases have CD5 expression. The prognostic implications of this CD5-positivity remain unknown. Here, we present a 78-year-old man who presented with circulating peripheral blood lymphoma cells and a hypercellular marrow involved by diffuse large B-cell lymphoma, germinal center B-cell subtype. The patient responded favorably to six cycles of etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and rituximab (EPOCH-R) and intrathecal methotrexate. He unfortunately relapsed in several enlarged inguinal lymph nodes and succumbed to the lymphoma approximately one year after diagnosis, demonstrating the particularly aggressive clinical course of his disease.

A diversified energy portfolio may include marine energy in the form of current energy converters (CECs) such as tidal or in-river turbines. New technology development in the research stage typically requires monitoring for environmental effects. A significant environmental effect of concern for CECs is the risk of moving parts (e.g., turbine blades) colliding with animals such as fishes. CECs are installed in energetic locations in which it is difficult to operate sensors to fulfill monitoring requirements for informing collision risk. Collecting data (i.e., about blade strikes or near-misses) that inform interactions of fishes with CECs is usually attempted using active acoustic sensors or video cameras (VCs). Limitations of low-light conditions or water turbidity that preclude effective use of VCs are overcome by using high-resolution multibeam echosounders (or acoustic cameras (ACs)). We used an AC at two sites to test its ability to detect artificial and real fish targets and determine if strike, near-miss, and near-field behavior could be observed. Interactions with fish and artificial targets with turbines have been documented but strike confirmation with an AC is novel. The first site was in a tidal estuary with a 25 kW turbine and water clarity sufficient to allow VC data to be collected concurrently with AC data showing turbine blade strike on tethered artificial fish targets. The second site was a turbid, debris-laden river with a 5 kW turbine where only AC data were collected due to high water turbidity. Data collection at the second site coincided with downstream Pacific salmon (Oncorhynchus spp.) smolt migration. Physical fish capture downstream of the turbine was performed with an incline plane trap (IPT) to provide context for the AC observations, by comparing fish catches. Discrimination between debris and fishes in the AC data was not possible, because active movement of fishes was not discernable. Nineteen fishes were released upstream of the turbine to provide known times of possible fish/turbine interactions, but detection was difficult to confirm in the AC data. ACs have been used extensively in past studies to count large migratory fish such as Pacific salmon, but their application for small fish targets has been limited. The results from these two field campaigns demonstrate the ability of ACs to detect targets in turbid water and observe blade strikes, as well as their limitations such as the difficulty of distinguishing small fishes from debris in a high-energy turbid river. Recommendations are presented for future applications associated with CEC device testing.

Objective: Appropriate glucose levels are essential for survival; thus, the detection and correction of low blood glucose is of paramount importance. Hypoglycemia prompts an integrated response involving reduction in insulin release and secretion of key counter-regulatory hormones glucagon and epinephrine that together promote endogenous glucose production to restore normoglycemia. However, specifically how this response is orchestrated remains to be fully clarified. The low affinity hexokinase glucokinase is found in glucose-sensing cells involved in glucose homeostasis including pancreatic β-cells and in certain brain areas. Here, we aimed to examine the role of glucokinase in triggering counter-regulatory hormonal responses to hypoglycemia, hypothesizing that reduced glucokinase activity would lead to increased and/or earlier triggering of responses. Methods: Hyperinsulinemic glucose clamps were performed to examine counter-regulatory responses to controlled hypoglycemic challenges created in humans with monogenic diabetes resulting from heterozygous glucokinase mutations (GCK-MODY). To examine the relative importance of glucokinase in different sensing areas, we then examined responses to clamped hypoglycemia in mice with molecularly defined disruption of whole body and/or brain glucokinase. Results: GCK-MODY patients displayed increased and earlier glucagon responses during hypoglycemia compared with a group of glycemia-matched patients with type 2 diabetes. Consistent with this, glucagon responses to hypoglycemia were also increased in I366F mice with mutated glucokinase and in streptozotocin-treated β-cell ablated diabetic I366F mice. Glucagon responses were normal in conditional brain glucokinase-knockout mice, suggesting that glucagon release during hypoglycemia is controlled by glucokinase-mediated glucose sensing outside the brain but not in β-cells. For epinephrine, we found increased responses in GCK-MODY patients, in β-cell ablated diabetic I366F mice and in conditional (nestin lineage) brain glucokinase-knockout mice, supporting a role for brain glucokinase in triggering epinephrine release. Conclusions: Our data suggest that glucokinase in brain and other non β-cell peripheral hypoglycemia sensors is important in glucose homeostasis, allowing the body to detect and respond to a falling blood glucose. Keywords: Glucokinase, Hypoglycemia, Counter-regulation, Glucagon, Epinephrine, Insulin clamp, Maturity onset diabetes of young (MODY), β-cells

Abstract Background Targeted therapies are based on exploiting cancer-cell-specific genetic features or phenotypic traits to selectively kill cancer cells while leaving normal cells unaffected. Oxidative stress is a cancer hallmark phenotype. Given that free nucleotide pools are particularly vulnerable to oxidation, the nucleotide pool sanitising enzyme, MTH1, is potentially conditionally essential in cancer cells. However, findings from previous MTH1 studies have been contradictory, meaning the relevance of MTH1 in cancer is still to be determined. Here we ascertained the role of MTH1 specifically in lung cancer cell maintenance, and the potential of MTH1 inhibition as a targeted therapy strategy to improve lung cancer treatments. Methods Using siRNA-mediated knockdown or small-molecule inhibition, we tested the genotoxic and cytotoxic effects of MTH1 deficiency on H23 (p53-mutated), H522 (p53-mutated) and A549 (wildtype p53) non-small cell lung cancer cell lines relative to normal MRC-5 lung fibroblasts. We also assessed if MTH1 inhibition augments current therapies. Results MTH1 knockdown increased levels of oxidatively damaged DNA and DNA damage signaling alterations in all lung cancer cell lines but not normal fibroblasts, despite no detectable differences in reactive oxygen species levels between any cell lines. Furthermore, MTH1 knockdown reduced H23 cell proliferation. However, unexpectedly, it did not induce apoptosis in any cell line or enhance the effects of gemcitabine, cisplatin or radiation in combination treatments. Contrastingly, TH287 and TH588 MTH1 inhibitors induced apoptosis in H23 and H522 cells, but only increased oxidative DNA damage levels in H23, indicating that they kill cells independently of DNA oxidation and seemingly via MTH1-distinct mechanisms. Conclusions MTH1 has a NSCLC-specific p53-independent role for suppressing DNA oxidation and genomic instability, though surprisingly the basis of this may not be reactive-oxygen-species-associated oxidative stress. Despite this, overall our cell viability data indicates that targeting MTH1 will likely not be an across-the-board effective NSCLC therapeutic strategy; rather it induces non-cytotoxic DNA damage that could promote cancer heterogeneity and evolution.

Objective: Dietary proteins are sensed by hypothalamic neurons and strongly influence multiple aspects of metabolic health, including appetite, weight gain, and adiposity. However, little is known about the mechanisms by which hypothalamic neural circuits controlling behavior and metabolism sense protein availability. The aim of this study is to characterize how neurons from the mediobasal hypothalamus respond to a signal of protein availability: the amino acid l-leucine. Methods: We used primary cultures of post-weaning murine mediobasal hypothalamic neurons, hypothalamic neurons derived from human induced pluripotent stem cells, and calcium imaging to characterize rapid neuronal responses to physiological changes in extracellular l-Leucine concentration. Results: A neurochemically diverse subset of both mouse and human hypothalamic neurons responded rapidly to l-leucine. Consistent with l-leucine's anorexigenic role, we found that 25% of mouse MBH POMC neurons were activated by l-leucine. 10% of MBH NPY neurons were inhibited by l-leucine, and leucine rapidly reduced AGRP secretion, providing a mechanism for the rapid leucine-induced inhibition of foraging behavior in rodents. Surprisingly, none of the candidate mechanisms previously implicated in hypothalamic leucine sensing (KATP channels, mTORC1 signaling, amino-acid decarboxylation) were involved in the acute activity changes produced by l-leucine. Instead, our data indicate that leucine-induced neuronal activation involves a plasma membrane Ca2+ channel, whereas leucine-induced neuronal inhibition is mediated by inhibition of a store-operated Ca2+ current. Conclusions: A subset of neurons in the mediobasal hypothalamus rapidly respond to physiological changes in extracellular leucine concentration. Leucine can produce both increases and decreases in neuronal Ca2+ concentrations in a neurochemically-diverse group of neurons, including some POMC and NPY/AGRP neurons. Our data reveal that leucine can signal through novel mechanisms to rapidly affect neuronal activity. Keywords: l-leucine sensing, Hypothalamus, Calcium imaging, Pluripotent, Metabolism

Objective: The increasing prevalence of type 2 diabetes (T2D) and associated morbidity and mortality emphasizes the need for a more complete understanding of the mechanisms mediating glucose homeostasis to accelerate the identification of new medications. Recent reports indicate that the obesity medication lorcaserin, a 5-hydroxytryptamine (5-HT, serotonin) 2C receptor (5-HT2CR) agonist, improves glycemic control in association with weight loss in obese patients with T2D. Here we evaluate whether lorcaserin has an effect on glycemia without body weight loss and how this effect is achieved. Methods: Murine models of common and genetic T2D were utilized to probe the direct effect of lorcaserin on glycemic control. Results: Lorcaserin dose-dependently improves glycemic control in mouse models of T2D in the absence of reductions in food intake or body weight. Examining the mechanism of this effect, we reveal a necessary and sufficient neurochemical mediator of lorcaserin's glucoregulatory effects, brain pro-opiomelanocortin (POMC) peptides. To clarify further lorcaserin's therapeutic brain circuit, we examined the receptor target of POMC peptides. We demonstrate that lorcaserin requires functional melanocortin4 receptors on cholinergic preganglionic neurons (MC4RChAT) to exert its effects on glucose homeostasis. In contrast, MC4RChAT signaling did not impact lorcaserin's effects on feeding, indicating a divergence in the neurocircuitry underpinning lorcaserin's therapeutic glycemic and anorectic effects. Hyperinsulinemic-euglycemic clamp studies reveal that lorcaserin reduces hepatic glucose production, increases glucose disposal and improves insulin sensitivity. Conclusions: These data suggest that lorcaserin's action within the brain represents a mechanistically novel treatment for T2D: findings of significance to a prevalent global disease.

Epithelial ovarian cancer (EOC) is a leading cause of cancer mortality among women but unfortunately is usually not diagnosed until advanced stage. Early detection of EOC is of paramount importance to improve outcomes. Liquid biopsy of circulating tumor cells (CTCs) is emerging as one of the promising biomarkers for early detection of solid tumors. However, discrepancies in terms of oncogenomics (i.e., different genetic defects detected) between the germline, primary tumor, and liquid biopsy are a serious concern and may adversely affect downstream cancer management. Here, we illustrate the potential and pitfalls of CTCs by presenting two patients of Stage I EOC. We successfully isolated and recovered CTCs by a silicon-based nanostructured microfluidics system, the automated Cell RevealTM. We examined the genomics of CTCs as well as the primary tumor and germline control (peripheral blood mononuclear cells) by whole exome sequencing. Different signatures were then investigated by comparisons of identified mutation loci distinguishing those that may only arise in the primary tumor or CTCs. A novel model is proposed to test if the highly variable allele frequencies, between primary tumor and CTCs results, are due to allele dropout in plural CTCs or tumor heterogeneity. This proof-of-principle study provides a strategy to elucidate the possible cause of genomic discrepancy between the germline, primary tumor, and CTCs, which is helpful for further large-scale use of such technology to be integrated into clinical management protocols.

A false negative can happen in many kinds of medical tests, regardless of whether they are screening or diagnostic in nature. However, it inevitably poses serious concerns especially in a prenatal setting because its sequelae can mark the birth of an affected child beyond expectation. False negatives are not a new thing because of emerging new tests in the field of reproductive, especially prenatal, genetics but has occurred throughout the evolution of prenatal screening and diagnosis programs. In this paper we aim to discuss the basic differences between screening and diagnosis, the trade-offs and the choices, and also shed light on the crucial points clinicians need to know and be aware of so that a quality service can be provided in a coherent and sensible way to patients so that vital issues related to a false negative result can be appropriately comprehended by all parties.

Objective: Obesity is one of the primary healthcare challenges of the 21st century. Signals relaying information regarding energy needs are integrated within the brain to influence body weight. Central among these integration nodes are the brain pro-opiomelanocortin (POMC) peptides, perturbations of which disrupt energy balance and promote severe obesity. However, POMC neurons are neurochemically diverse and the crucial source of POMC peptides that regulate energy homeostasis and body weight remains to be fully clarified. Methods: Given that a 5-hydroxytryptamine 2c receptor (5-HT2CR) agonist is a current obesity medication and 5-HT2CR agonist's effects on appetite are primarily mediated via POMC neurons, we hypothesized that a critical source of POMC regulating food intake and body weight is specifically synthesized in cells containing 5-HT2CRs. To exclusively manipulate Pomc synthesis only within 5-HT2CR containing cells, we generated a novel 5-HT2CRCRE mouse line and intercrossed it with Cre recombinase-dependent and hypothalamic specific reactivatable PomcNEO mice to restrict Pomc synthesis to the subset of hypothalamic cells containing 5-HT2CRs. This provided a means to clarify the specific contribution of a defined subgroup of POMC peptides in energy balance and body weight. Results: Here we transform genetically programed obese and hyperinsulinemic male mice lacking hypothalamic Pomc with increased appetite, reduced physical activity and compromised brown adipose tissue (BAT) into lean, healthy mice via targeted restoration of Pomc function only within 5-HT2CR expressing cells. Remarkably, the same metabolic transformation does not occur in females, who despite corrected feeding behavior and normalized insulin levels remain physically inactive, have lower energy expenditure, compromised BAT and develop obesity. Conclusions: These data provide support for the functional heterogeneity of hypothalamic POMC neurons, revealing that Pomc expression within 5-HT2CR expressing neurons is sufficient to regulate energy intake and insulin sensitivity in male and female mice. However, an unexpected sex difference in the function of this subset of POMC neurons was identified with regard to energy expenditure. We reveal that a large sex difference in physical activity, energy expenditure and the development of obesity is driven by this subpopulation, which constitutes approximately 40% of all POMC neurons in the hypothalamic arcuate nucleus. This may have broad implications for strategies utilized to combat obesity, which at present largely ignore the sex of the obese individual. Author Video: Author Video Watch what authors say about their articles Keywords: Pro-opiomelanocortin (Pomc), 5-HT2c receptor, Obesity, Energy expenditure, Brown adipose tissue, Hyperinsulinemia, Sexual dimorphism, Hypothalamus