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In diploid organisms, biallelic gene expression enables the production of adequate levels of mRNA1,2. This is essential for haploinsufficient genes, which require biallelic expression for optimal function to prevent the onset of developmental disorders1,3. Whether and how a biallelic or monoallelic state is determined in a cell-type-specific manner at individual loci remains unclear. MSL2 is known for dosage compensation of the male X chromosome in flies. Here we identify a role of MSL2 in regulating allelic expression in mammals. Allele-specific bulk and single-cell analyses in mouse neural progenitor cells revealed that, in addition to the targets showing biallelic downregulation, a class of genes transitions from biallelic to monoallelic expression after MSL2 loss. Many of these genes are haploinsufficient. In the absence of MSL2, one allele remains active, retaining active histone modifications and transcription factor binding, whereas the other allele is silenced, exhibiting loss of promoter–enhancer contacts and the acquisition of DNA methylation. Msl2-knockout mice show perinatal lethality and heterogeneous phenotypes during embryonic development, supporting a role for MSL2 in regulating gene dosage. The role of MSL2 in preserving biallelic expression of specific dosage-sensitive genes sets the stage for further investigation of other factors that are involved in allelic dosage compensation in mammalian cells, with considerable implications for human disease.

The Anophelesmosquito is one of thousands of species in which sex differences play a central part in their biology, as only females need a blood meal to produce eggs. Sex differentiation is regulated by sex chromosomes, but their presence creates a dosage imbalance between males (XY) and females (XX). Dosage compensation (DC) can re-equilibrate the expression of sex chromosomal genes. However, because DC mechanisms have only been fully characterized in a few model organisms, key questions about its evolutionary diversity and functional necessity remain unresolved1. Here we report the discovery of a previously uncharacterized gene (sex chromosome activation(SOA)) as a master regulator of DC in the malaria mosquito Anopheles gambiae. Sex-specific alternative splicing prevents functional SOA protein expression in females. The male isoform encodes a DNA-binding protein that binds the promoters of active X chromosomal genes. Expressing male SOA is sufficient to induce DC in female cells. Male mosquitoes lacking SOA or female mosquitoes ectopically expressing the male isoform exhibit X chromosome misregulation, which is compatible with viability but causes developmental delay. Thus, our molecular analyses of a DC master regulator in a non-model organism elucidates the evolutionary steps that lead to the establishment of a chromosome-specific fine-tuning mechanism.

In mammals, X-chromosomal genes are expressed from a single copy since males (XY) possess a single X chromosome, while females (XX) undergo X inactivation. To compensate for this reduction in dosage compared with two active copies of autosomes, it has been proposed that genes from the active X chromosome exhibit dosage compensation. However, the existence and mechanisms of X-to-autosome dosage compensation are still under debate. Here we show that X-chromosomal transcripts have fewer m6A modifications and are more stable than their autosomal counterparts. Acute depletion of m6A selectively stabilizes autosomal transcripts, resulting in perturbed dosage compensation in mouse embryonic stem cells. We propose that higher stability of X-chromosomal transcripts is directed by lower levels of m6A, indicating that mammalian dosage compensation is partly regulated by epitranscriptomic RNA modifications.

The design of multitarget drugs represents a promising strategy in medicinal chemistry and seems particularly suitable for the discovery of anti-inflammatory drugs. Here, we describe the identification of an indoline-based compound inhibiting both 5-lipoxygenase (5-LOX) and soluble epoxide hydrolase (sEH). In silicoanalysis of an in-house library identified nine compounds as potential 5-LOX inhibitors. Enzymatic and cellular assays revealed the indoline derivative 43as a notable 5-LOX inhibitor, guiding the design of new analogues. These compounds underwent extensive in vitroinvestigation revealing dual 5-LOX/sEH inhibitors, with 73showing the most promising activity (IC50s of 0.41 ± 0.01 and 0.43 ± 0.10 μM for 5-LOX and sEH, respectively). When challenged in vivo in zymosan-induced peritonitis and experimental asthma in mice, compound 73showed remarkable anti-inflammatory efficacy. These results pave the way for the rational design of 5-LOX/sEH dual inhibitors and for further investigation of their potential use as anti-inflammatory agents.

Neuronal Kv7 channels represent important pharmacological targets for hyperexcitability disorders including epilepsy. Retigabine is the prototype Kv7 activator clinically approved for seizure treatment; however, severe side effects associated with long-term use have led to its market discontinuation. Building upon the recently described cryoEM structure of Kv7.2 complexed with retigabine and on previous structure–activity relationship studies, a small library of retigabine analogues has been designed, synthesized, and characterized for their Kv7 opening ability using both fluorescence- and electrophysiology-based assays. Among all tested compounds, 60emerged as a potent and photochemically stable neuronal Kv7 channel activator. Compared to retigabine, compound 60displayed a higher brain/plasma distribution ratio, a longer elimination half-life, and more potent and effective anticonvulsant effects in an acute seizure model in mice. Collectively, these data highlight compound 60as a promising lead compound for the development of novel Kv7 activators for the treatment of hyperexcitability diseases.

Inactivation of one of the two female X chromosomes involves condensing it into a repressive subnuclear territory, which is depleted of transcriptional components and undergoes late-stage DNA replication. Two new studies unravel how compartmentalization of the inactive mammalian X chromosome affects transcription and DNA replication.

Confinement of the X chromosome to a territory for dosage compensation is a prime example of how subnuclear compartmentalization is used to regulate transcription at the megabase scale. In Drosophila melanogaster, two sex-specific non-coding RNAs (roX1 and roX2) are transcribed from the X chromosome. They associate with the male-specific lethal (MSL) complex1, which acetylates histone H4 lysine 16 and thereby induces an approximately twofold increase in expression of male X-linked genes2,3. Current models suggest that X-over-autosome specificity is achieved by the recognition of cis-regulatory DNA high-affinity sites (HAS) by the MSL2 subunit4,5. However, HAS motifs are also found on autosomes, indicating that additional factors must stabilize the association of the MSL complex with the X chromosome. Here we show that the low-complexity C-terminal domain (CTD) of MSL2 renders its recruitment to the X chromosome sensitive to roX non-coding RNAs. roX non-coding RNAs and the MSL2 CTD form a stably condensed state, and functional analyses in Drosophilaand mammalian cells show that their interactions are crucial for dosage compensation in vivo. Replacing the CTD of mammalian MSL2 with that from Drosophilaand expressing roX in cisis sufficient to nucleate ectopic dosage compensation in mammalian cells. Thus, the condensing nature of roX–MSL2CTDis the primary determinant for specific compartmentalization of the X chromosome in Drosophila.

Transient receptor potential melastatin 8 (TRPM8) ion channel represents a valuable pharmacological option for several therapeutic areas. Here, a series of conformationally restricted derivatives of the previously described TRPM8 antagonist N,N′-dibenzyl tryptophan 4were prepared and characterized in vitro by Ca2+-imaging and patch-clamp electrophysiology assays. Molecular modeling studies led to identification of a broad and well-defined interaction network of these derivatives inside the TRPM8 binding site, underlying their antagonist activity. The (5R,11aS)-5-(4-chlorophenyl)-2-(4-fluorobenzyl)-5,6,11,11a-tetrahydro-1H-imidazo[1′,5′:1,6]pyrido[3,4-b]indole-1,3(2H)-dione (31a) emerged as a potent (IC50= 4.10 ± 1.2 nM), selective, and metabolically stable TRPM8 antagonist. In vivo, 31ashowed significant target coverage in an icilin-induced WDS (at 11.5 mg/kg ip), an oxaliplatin-induced cold allodynia (at 10–30 μg sc), and CCI-induced thermal hyperalgesia (at 11.5 mg/kg ip) mice models. These results confirm the tryptophan moiety as a solid pharmacophore template for the design of highly potent modulators of TRPM8-mediated activities.

Kv7 K+channels represent attractive pharmacological targets for the treatment of different neurological disorders, including epilepsy. In this paper, 42 conformationally restricted analogues of the prototypical Kv7 activator retigabine have been synthesized and tested by electrophysiological patch-clamp experiments as Kv7 agonists. When compared to retigabine (0.93 ± 0.43 μM), the EC50s for Kv7.2 current enhancements by compound 23a(0.08 ± 0.04 μM) were lower, whereas no change in potency was observed for 24a(0.63 ± 0.07 μM). In addition, compared to retigabine, 23aand 24ashowed also higher potency in activating heteromeric Kv7.2/Kv7.3 and homomeric Kv7.4 channels. Molecular modeling studies provided new insights into the chemical features required for optimal interaction at the binding site. Stability studies evidenced improved chemical stability of 23aand 24ain comparison with retigabine. Overall, the present results highlight that the N5-alkylamidoindole moiety provides a suitable pharmacophoric scaffold for the design of chemically stable, highly potent and selective Kv7 agonists.

Haspin is an emerging, but rather unexplored, divergent kinase involved in tumor growth by regulating the mitotic phase. In this paper, the in-silicodesign, synthesis, and biological characterization of a new series of substituted indoles acting as potent Haspin inhibitors are reported. The synthesized derivatives have been evaluated by FRET analysis, showing very potent Haspin inhibition. Then, a comprehensive in-cell investigation highlighted compounds 47and 60as the most promising inhibitors. These compounds were challenged for their synergic activity with paclitaxel in 2D and 3D cellular models, demonstrating a twofold improvement of the paclitaxel antitumor activity. Compound 60also showed remarkable selectivity when tested in a panel of 70 diverse kinases. Finally, in-silicostudies provided new insight about the chemical requirements useful to develop new Haspin inhibitors. Biological results, together with the drug-likeness profile of 47and 60, make these derivatives deserving further studies.

Gain-of-function (GoF) variants in KCNT1 channels cause severe, drug-resistant forms of epilepsy. Quinidine is a known KCNT1 blocker, but its clinical use is limited due to severe drawbacks. To identify novel KCNT1 blockers, a homology model of human KCNT1 was built and used to screen an in-house library of compounds. Among the 20 molecules selected, five (CPK4, 13, 16, 18, and 20) showed strong KCNT1-blocking ability in an in vitrofluorescence-based assay. Patch-clamp experiments confirmed a higher KCNT1-blocking potency of these compounds when compared to quinidine, and their selectivity for KCNT1 over hERG and Kv7.2 channels. Among identified molecules, CPK20displayed the highest metabolic stability; this compound also blocked KCNT2 currents, although with a lower potency, and counteracted GoF effects prompted by 2 recurrent epilepsy-causing KCNT1 variants (G288S and A934T). The present results provide solid rational basis for future design of novel compounds to counteract KCNT1-related neurological disorders.

The etiological spectrum of ultra-rare developmental disorders remains to be fully defined. Chromatin regulatory mechanisms maintain cellular identity and function, where misregulation may lead to developmental defects. Here, we report pathogenic variations in MSL3, which encodes a member of the chromatin-associated male-specific lethal (MSL) complex responsible for bulk histone H4 lysine 16 acetylation (H4K16ac) in flies and mammals. These variants cause an X-linked syndrome affecting both sexes. Clinical features of the syndrome include global developmental delay, progressive gait disturbance, and recognizable facial dysmorphism. MSL3 mutations affect MSL complex assembly and activity, accompanied by a pronounced loss of H4K16ac levels in vivo. Patient-derived cells display global transcriptome alterations of pathways involved in morphogenesis and cell migration. Finally, we use histone deacetylase inhibitors to rebalance acetylation levels, alleviating some of the molecular and cellular phenotypes of patient cells. Taken together, we characterize a syndrome that allowed us to decipher the developmental importance of MSL3 in humans.

A long-term psychiatric 40 years-old male patient was found dead at 9:00 a.m. in the clinic where he lived. Death was caused by traumatic injuries, which the sanitary staff imputed to a fall. Nurses declared that the patient refused having breakfast, whereas at autopsy the stomach contained 350 g of whitish semifluid material. Using both shotgun and gel-based proteomics, we demonstrated that the chyme contained partly digested milk- and bread-derived proteins, eaten during a recent breakfast. The conflict between evidence and assertions of the attending sanitary staff prompted the Legal Authority to undertake detailed investigations to ascertain facts and possible responsibilities. The herein characterization provides insights in the in vivo mechanisms of gastric breakdown of food proteins in a real meal. β-lactoglobulin was partially resistant to gastric digestion as confirmed by Western blot analysis, in contrast to caseins and wheat gluten proteins, which had been degraded by gastric fluids. In addition to a complex pattern of gastric proteins (e.g., mucin-5AC, pepsin A-3, pepsinogen C, gastric lipase, gastrokine-2, trefoil factors), chyme contained intact proteins and variably sized food-derived polypeptides arising from peptic and nonpeptic proteolytic cleavage as well as heterodimeric disulfide-cross-linked peptides. These findings suggest that the current analytical workflows offer only a partial picture of the real complexity of the human “digestome”.

Acute pancreatitis (AP) is a potentially life-threatening illness characterized by an exacerbated inflammatory response with limited options for pharmacological treatment. Here, we describe the rational development of a library of soluble epoxide hydrolase (sEH) inhibitors for the treatment of AP. Synthesized compounds were screened in vitrofor their sEH inhibitory potency and selectivity, and the results were rationalized by means of molecular modeling studies. The most potent compounds were studied in vitrofor their pharmacokinetic profile, where compound 28emerged as a promising lead. In fact, compound 28demonstrated a remarkable in vivoefficacy in reducing the inflammatory damage in cerulein-induced AP in mice. Targeted metabololipidomic analysis further substantiated sEH inhibition as a molecular mechanism of the compound underlying anti-AP activity in vivo. Finally, pharmacokinetic assessment demonstrated a suitable profile of 28in vivo. Collectively, compound 28displays strong effectiveness as sEH inhibitor with potential for pharmacological AP treatment.

The aim of the present work was to optimize the performance of the Non-Thermal Plasma (NTP) technology coupled with a structured catalyst for the degradation of organic and recalcitrant pollutants in aqueous solution. Specifically, the catalyst consists of CeO2supported onto ɣ-Al2O3spheres, synthesized by the wet-impregnation method, and characterized through Raman spectroscopy, specific surface area (BET) by N2adsorption at − 196 °C and Scanning Electron Microscopy (SEM). All experiments were carried out in a Dielectric Barrier Discharge (DBD) reactor. The first sets of tests were accomplished to optimize the degradation of Acid Orange 7 (AO7) azo dye in aqueous solution. The effect of catalyst formulation and oxygen flow rate in the reactor, as well as the reusability of the catalyst, were studied in detail. Moreover, the effect of radical scavengers was examined to suggest a plausible degradation mechanism, and an HPLC analysis was performed to understand to which extent degradation proceeds with the formation of intermediates. The best performance of the system in terms of total degradation and mineralization efficiencies is found for 15 min run time using 2.9 wt% CeO2/ɣ-Al2O3, with a voltage equal to 12 kV and 0.045 NL/min oxygen flow rate. The catalyst showed reusability properties after 5 runs and no intermediates were found by HPLC analysis, suggesting that AO7 degradation takes place with complete mineralization. Scavenger analysis showed that ozone is the most important reactive species responsible for degradation since its decomposition leads to the generation of effective secondary oxygen reactive species. Furthermore, given the semiconductor features of CeO2, it may also be excited by the plasma-generated UV radiation, suggesting that electron-hole pairs were also involved in the degradation mechanism. The remarkable efficiency of the proposed catalytic NTP treatment shows the potential of the system for the decontamination of aqueous solutions containing organic and recalcitrant pollutants. To confirm the potentiality of the system, the degradation of glyphosate (GLY) was also assessed under the optimal conditions set up for AO7 degradation, obtaining 84% degradation efficiency after 30 min run time.

Tomato cultivation farms of Southern Italy were investigated in order to evaluate the general working conditions and the levels of exposure of farm workers to pesticides, during the mixing/loading and the application of pesticides on fields. Information on working modalities, personal protective equipment, etc. was collected using a questionnaire. Inhaling and cutaneous exposure levels were measured, and the estimated pesticide total absorbed dose was compared with Admissible Daily Intakes (ADIs). Field treatments were mainly carried out by using sprayers with open cab tractors, and, in 57.9% of cases, the pesticide mixture was manually prepared by mixing pesticides in a pail, often without using gloves (59.5%). The estimated pesticides absorbed doses varied in the range 0.56–2630.31 mg (mean value, 46.9 mg), and 20% of the measured absorbed doses exceeded ADIs. The findings obtained in the 18 examined farms show a worrying situation, suggesting the investigation of many more farms, so that a statistically significant picture of tomato cultivations in Southern Italy could be formed. Besides, the planning of training courses aimed to increase workers consciousness about health risks and how they can be prevented is advisable.

Evaluation of Occupational Exposure to Antiblastic Drugs in an Italian Hospital Oncological Department: Loredana Castiglia, et al. Department of Public Medicine and Social Health, University of Naples “Federico II”, Italy—The determination of the current antiblastic drug contamination levels in an Italian hospital oncology ward was carried out. Statistical evaluation of data aiming to identify potential exposure causes was performed. Cyclophosphamide (CP), ifosfamide (IF) and 5‐fluorouracil (5‐FU) were determined by wipe tests, extracted with diatomaceous earths and quantified by GC/MSMS or HPLC/UV. Data were analysed with respect to the potential contamination levels of sampled surfaces, and various amounts of handled analyte. χ2tests and Spearman correlation coefficients were calculated. Median concentration levels of 0.086, 0.071, 2.363 µg/dm2were obtained, (CP, IF, 5‐FU, respectively). 3.8 and 13.5% of investigated surfaces showed CP and IF concentrations higher than 1 µg/dm2(up to 26.96 µg/ dm2) and 13.4% of samples contained 5‐FU concentrations in the range 20–208.9 µg/dm2. Analytes' concentration levels were dependent on sampling sites, with significant correlations showing a progressive contamination decrement going from workbenches, floor, hood planes and other surfaces. A diffuse contamination (traces of all the three analytes) was found on all investigated surfaces, even when analytes had not been used during the sampling days. A significant correlation (ρs=0.303, p=0.001) between the measured analyte concentration and the analyte handled amount was found only in the case of IF. The risk management strategy should be improved, as suggested by the measured and widespread levels of contamination. Since contamination also depends on other factors attributable to working modalities and cleaning procedures, the obtained results suggest that performance of specific training courses as well as scheduling environmental monitoring plans to achieve an actual decrement of the observed contamination levels should be implemented.

Evaluation of Occupational Exposure to Antiblastic Drugs in an Italian Hospital Oncological Department: Loredana Castiglia, et al. Department of Public Medicine and Social Health, University of Naples “Federico II”, Italy—The determination of the current antiblastic drug contamination levels in an Italian hospital oncology ward was carried out. Statistical evaluation of data aiming to identify potential exposure causes was performed. Cyclophosphamide (CP), ifosfamide (IF) and 5-fluorouracil (5-FU) were determined by wipe tests, extracted with diatomaceous earths and quantified by GC/MSMS or HPLC/UV. Data were analysed with respect to the potential contamination levels of sampled surfaces, and various amounts of handled analyte. ?2tests and Spearman correlation coefficients were calculated. Median concentration levels of 0.086, 0.071, 2.363 µg/dm2were obtained, (CP, IF, 5-FU, respectively). 3.8 and 13.5% of investigated surfaces showed CP and IF concentrations higher than 1 µg/dm2(up to 26.96 µg/ dm2) and 13.4% of samples contained 5-FU concentrations in the range 20–208.9 µg/dm2. Analytes’ concentration levels were dependent on sampling sites, with significant correlations showing a progressive contamination decrement going from workbenches, floor, hood planes and other surfaces. A diffuse contamination (traces of all the three analytes) was found on all investigated surfaces, even when analytes had not been used during the sampling days. A significant correlation (?s=0.303, p=0.001) between the measured analyte concentration and the analyte handled amount was found only in the case of IF. The risk management strategy should be improved, as suggested by the measured and widespread levels of contamination. Since contamination also depends on other factors attributable to working modalities and cleaning procedures, the obtained results suggest that performance of specific training courses as well as scheduling environmental monitoring plans to achieve an actual decrement of the observed contamination levels should be implemented.

The ongoing H5N1 Asian epidemic is currently affecting a number of avian species including ducks. These birds are an important part of the poultry industry in the affected countries, and it is likely that they are acting as a reservoir of infection. Ten Pekin ducks were challenged with 100 μl containing 10750% egg infective dose of the highly pathogenic avian influenza virus (HPAIV) A/Duck/Vietnam/12/05 (H5N1), administered by an intra-nasal and oral route. Clinical symptoms were recorded twice a day up to 14 days postinfection (dpi). Clinical signs were first noted at 2 dpi, with conjunctivitis and slight depression, and progressed over a period of 1–3 days to severe neurologic signs consisting of torticollis, incoordination, tremors, and seizures. Survival times varied from 3 to 7 dpi. On postmortem examination, hemorrhages were observed in the duodenum, ceca, proventriculus, ventriculus, trachea, pancreas, and brain. Histologic lesions, as well as immunohistochemistry positivity, were recorded in the pancreas and brain. In situ hybridization revealed viral antigen associated with acinar pancreatic cells, bronchial epithelial cells, and with cells of the central nervous system as well as neurons of the submucosal plexus of the duodenum. Our experimental findings agree with those previously observed in ducks naturally infected with HPAIV H5N1 viruses, confirming the acquired viral neurotropism and pancreatotropism, as previously noted in other avian species, as well as in humans.

Introduction. Laparoscopic Roux-en-Y gastric bypass (LRYGBP) is a common procedure in obesity surgery. The aim of an antecolic approach is to reduce the rate of internal herniation. Our aim is to make bariatric surgeons aware of another possible complication of antecolic LRYGBP. Methods and Results. We present a case report of omental torsion 24 months after antecolic LRYGBP presenting as an acute abdomen, suggesting appendicitis. During diagnostic laparoscopy, omental infarction due to torsion was observed. Resection of the avital omentum was performed. Discussion. Omental torsion after antecolic LRYGBP is a rare complication. When appearing in the early postoperative phase, it may mimic an anastomotic leakage. It may also occur as late complication, presenting with acute abdomen as an appendicitis.

Lipoblastomas are benign tumors of the embryonic lipoid cells mainly occurring in infancy and early childhood. They are clinicopathologically distinguished in two forms: the well-circumscribed and localized type and the diffuse, irregularly confined type with infiltrative growth pattern, also called lipoblastomatosis. We report two pediatric cases of a mesentery localized and cervical diffuse lipoblastomas investigated both with ultrasound and computed tomography examinations.