Forensic species identification: practical guide for animal and plant DNA analysis.

The importance of non-human DNA in the forensic field has increased greatly in recent years, together with the type of applications. The molecular species identification of animal and botanical material may be crucial both for wildlife trafficking and crime scene investigation. However, especially for forensic botany, several challenges slow down the implementation of the discipline in the routine.Although the importance of molecular analysis of animal origin samples is widely recognized and the same value is acknowledged to the botanical counterpart, the latter does not find the same degree of application.The availability of molecular methods, especially useful in cases where the material is fragmented, scarce or spoiled preventing the morphological identification, is not well known. This work is intended to reaffirm the relevance of non-human forensic genetics (NHFG), highlighting differences, benefits and pitfalls of the current most common molecular analysis workflow for animal and botanical samples, giving a practical guide. A flowchart describing the analysis paths, divided in three major working areas (inspection and sampling, molecular analysis, data processing and interpretation), is provided. More real casework examples of the utility of non-human evidence in forensic investigations should be shared by the scientific community, especially for plants. Moreover, concrete efforts to encourage initiatives in order to promote quality and standardization in the NHFG field are also needed.

High prevalence of morphometric vertebral fractures opportunistically detected on thoracic radiograms in patients with non-functioning pituitary adenoma.

PURPOSE: Vertebral fractures (VFs), the hallmark of skeletal fragility, have been reported as an emerging complication in patients with pituitary diseases associated with hormonal excess and/or deficiency, independently from bone mineral density. Non-functioning pituitary adenoma (NFPA) is amongst the most frequent pituitary adenomas; however, skeletal health in this context has never been investigated. We aimed at assessing the prevalence and the determinants of morphometric VFs in patients with NFPA. METHODS: We enrolled 156 patients (79 M/77F, mean age 55.75 ± 12.94 years) at admission in Neurosurgery Unit before trans-sphenoidal surgery and compared them with an age and sex-matched control group of subjects with neither history/risk factors for secondary osteoporosis nor pituitary disorders. We performed a vertebral morphometric evaluation of the thoracic spine on pre-operative X-ray images (MTRx) and collected biochemical, demographic, and clinical data from the entire cohort. RESULTS: The prevalence of thoracic VFs in patients with NFPA was significantly higher than the control group (26.3% vs. 10.3%; p < 0.001). In the NFPA group, 20 patients (48.8% of the fractured patients) showed multiple VFs, 14 (34.1% of them) showed moderate/severe VFs. Patients with VFs were significantly older and had lower serum free triiodothyronine (fT3) levels than non-fractured ones (p = 0.002 and p = 0.004; respectively). The prevalence of secondary male hypogonadism was higher among men with VFs as compared to those with no VFs (72% vs. 48.1%; p = 0.047). Consistently, total testosterone levels in males were significantly lower in fractured patients than in non-fractured ones (p = 0.02). The prevalence of gonadotroph adenomas was significantly higher among patients with VFs (p = 0.02). In multiple logistic regression analysis, older age and lower serum fT3 levels were independent factors predicting the risk for VFs. CONCLUSIONS: For the first time, we reported a high prevalence of thoracic radiological VFs in patients with NFPAs. Our data should prompt clinicians to proceed with a clinical bone fragility evaluation already during the diagnostic work-up, particularly in those with concomitant hypogonadism, or in those with older age and/or with lower fT3.

Molecular Determinants of EphA2 and EphB2 Antagonism Enable the Design of Ligands with Improved Selectivity.

With the aim of identifying novel antagonists selective for the EphA receptor family, a combined experimental and computational approach was taken to investigate the molecular basis of the recognition between a prototypical Eph-ephrin antagonist (UniPR1447) and two representative receptors of the EphA and EphB subfamilies, namely, EphA2 and EphB2 receptors. The conformational free-energy surface (FES) of the binding state of UniPR1447 within the ligand binding domain of EphA2 and EphB2, reconstructed from molecular dynamics (MD) simulations performed on the microsecond time scale, was exploited to drive the design and synthesis of a novel antagonist selective for EphA2 over the EphB2 receptor. The availability of compounds with this pharmacological profile will help discriminate the importance of these two receptors in the insurgence and progression of cancer.

Being a technician during COVID-19: a qualitative cross-sectional survey on the experiences of clinical neurophysiology technicians.

BACKGROUND: During the Sars-CoV-2 virus pandemic, Italy faced an unrivaled health emergency. Its impact has been significant on the hospital system and personnel. Clinical neurophysiology technicians played a central role (but less visibly so compared to other healthcare workers) in managing the COVID-19 pandemic. This research aims to explore the experiences of clinical neurophysiology technicians during the pandemic and contribute to the debate on the well-being of healthcare workers on the front line. METHODS: We implemented a cross-sectional survey across Italy. It contained questions that were open-ended for participants to develop their answers and acquire a fuller perspective. The responses were analyzed according to the framework method. RESULTS: One hundred and thirty-one responses were valid, and the following themes were generated: technicians' experiences in their relationship with patients, technicians' relationship with their workgroup and directors, and technicians' relationship with the context outside of their work. The first theme included sub-themes: fear of infection, empathy, difficulty, a sense of obligation and responsibility, anger, and sadness. The second theme contained selfishness/solidarity in the workgroup, lack of protection/collaboration from superiors, stress, and distrust. The last theme included fear, stress/tiredness, serenity, sadness, and anger. CONCLUSION: This study contributes to building a humanized perspective for personnel management, bringing attention to the technical work of healthcare professionals in an emergency and the emotional and relational dimensions. These are the starting points to define proper, contextually adequate support.

"Somewhere Between an Actual Disease and a Disease": A Grounded Theory Study on Diagnosing Functional Neurological Disorders From a Multi-Informant Perspective.

Functional Neurological Disorders are characterized by sensory-motor or cognitive symptoms. Recent research has revealed their complex nature involving biological, psychological, and social factors. Care requires a multidisciplinary approach, which, to date, has yet to be considered. A Constructivist Grounded Theory study was conducted to understand the reasons behind this, exploring Functional Neurological Disorders diagnosis, communication, and understanding from multiple perspectives (patients and healthcare professionals). The core category was "negotiating Functional Neurological Disorders meanings and care amid a dissatisfying dichotomy," with sub-categories: i) seeking to "word" the disease, ii) exposing reductionism, and iii) a pluralist vision emerging. Diagnosing and communicating Functional Neurological Disorders is a process of negotiating meanings and care that hinges on participants' diverse ontological perspectives regarding the condition. Results highlight the difficulty in finding common ground and achieving mutual understanding among the various viewpoints, creating a challenge in establishing a unified approach to Functional Neurological Disorders care. In this context, only a few healthcare professionals emphasized the potential benefits of increased integration. A shift is required from a reductionist to an integrated biopsychosocial perspective to develop a more cohesive approach. Defining a medical paradigm through dialogue with teams and patients is essential in addressing Functional Neurological Disorders effectively. Furthermore, the required interdisciplinary approach holds the potential to mitigate the dissatisfaction arising from fragmented and compartmentalized care (the "dissatisfying dichotomy") experienced by our participants. It signifies a comprehensive strategy that could address the concerns of all involved parties and enhance the overall quality of care provided.

Long-term follow-up of a case of feline leishmaniosis treated with a combination of allopurinol and meglumine antimoniate.

A 9-year-old domestic cat, positive for antibodies to feline immunodeficiency virus (FIV), was brought to a veterinary clinic with alopecia, ulcerative skin lesions, and upper respiratory tract (URT) signs. This was after being treated for suspected allergic dermatitis, without clinical improvement, for 2 y. Biopsy of the skin and fine-needle aspirates of the spleen and of the lymph nodes were taken which detected the presence of Leishmania amastigotes. Leishmania infection was further confirmed by detection of a high titer of anti-Leishmania antibodies (≥ 3200) with an indirect fluorescent antibody technique (IFAT) serology. After the diagnosis of feline leishmaniosis (FeL) was made, allopurinol and meglumine antimoniate were started and led to quick and complete clinical improvement. After 7 mo, allopurinol administration was briefly interrupted but was resumed following relapse of the skin lesions. One month later, the cat was treated for suspected acute kidney injury, which prompted reduction of the total daily dose of allopurinol by 50%. The cat remained clinically well, with complete resolution of the cutaneous and URT signs, for nearly 24 mo after the diagnosis of FeL; at which point it was euthanized for worsening cardiac disease. To our knowledge, this represents a rare case of successful treatment of FeL with a suspected nephrotoxic effect associated with long-term use of allopurinol. Further studies are required to clarify the relationship, if any, between leishmaniosis and congestive heart failure in cats.

Suivi à long terme d'un cas de leishmaniose féline traité par une association d'allopurinol et d'antimoniate de méglumine. Un chat domestique de 9 ans, positif pour les anticorps contre le virus de l'immunodéficience féline (FIV), a été présenté dans une clinique vétérinaire avec une alopécie, des lésions cutanées ulcéreuses et des signes des voies respiratoires supérieures (URT). Ceci après avoir été traité pour une suspicion de dermatite allergique sans amélioration clinique, pendant 2 ans. Une biopsie de la peau et des ponctions à l'aiguille fine de la rate et des ganglions lymphatiques ont été réalisées et ont détecté la présence d'amastigotes de Leishmania. L'infection à Leishmania a été confirmée par la détection d'un titre élevé d'anticorps sériques anti-Leishmania (≥ 3200) par une technique d'immunofluorescence indirecte (IFAT). Après le diagnostic de leishmaniose féline (FeL), un traitement à l'allopurinol et l'antimoniate de méglumine a été instauré et a entraîné une amélioration clinique rapide et complète. Après 7 mois, l'administration d'allopurinol a été brièvement interrompue mais a été reprise après la rechute des lésions cutanées. Un mois plus tard, le chat a été traité pour une lésion rénale aiguë suspectée, ce qui a entraîné une réduction de 50 % de la dose quotidienne totale d'allopurinol. Le chat est resté cliniquement en bonne santé, avec une résolution complète des signes cutanés et urinaires, pendant près de 24 mois après le diagnostic de FeL; à quel point il a été euthanasié pour aggravation de la maladie cardiaque.À notre connaissance, ceci représente un cas rare de traitement réussi de FeL avec un effet néphrotoxique suspecté associé à une utilisation à long terme d'allopurinol. D'autres études sont nécessaires pour clarifier la relation, le cas échéant, entre la leishmaniose et l'insuffisance cardiaque congestive chez les chats.(Traduit par Dr Serge Messier).

Two caseworks for one gene: successful species identification from compromised bone materials with the 12S rRNA.

The availability of a reliable molecular assay in species recognition in forensic cases is of paramount importance when visual inspection or morphological methods are not exhaustive, especially from challenging samples. Here, two different caseworks involving bone samples founded during medico-legal outdoor investigations are presented. In order to exclude the human nature of the specimens and to determine the exact species they belong to, we proceeded with the molecular approach trying to generate sequences from the classical mtDNA markers cyt b and COI. However, they both gave critical results. For this reason, a short amplicon of ~ 150 bp of the 12S rRNA gene was used as an alternative.This short fragment was sufficient to identify the biological origin of the bone specimens with a high degree of certainty leading to the exclusion of their human nature. This work highlights the utility of the 12S rRNA and underlines the importance of deepen the choice of alternative shorter markers with respect to the classical ones, in order to achieve species identification even from challenging and degraded material in forensic criminal and wildlife caseworks.

Organic cation transporters (OCTs/OCTNs) in human primary alveolar epithelial cells.

Alveolar epithelium, besides exerting a key role in gas exchange and surfactant production, plays important functions in host defense and inflammation. Pathological conditions associated to alveolar dysfunction include Acute Respiratory Distress Syndrome (ARDS), asthma, chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). The use of predictive in vitro models of human alveolar epithelium is nowadays required for the study of disease mechanisms, as well as of pharmacokinetic parameters of pulmonary drugs delivery. Here, we employed a novel 3D model of human alveoli, namely EpiAlveolar™, consisting of primary alveolar epithelial cells, pulmonary endothelial cells and fibroblasts, that reflects properly the in vivo-like conditions. In EpiAlveolar™ we performed a characterization of Organic Cation Transporters (OCTs and OCTNs) expression and activity and we found that OCTN2, OCT1 and OCT3 are expressed on the basolateral membrane; instead, ATB0,+ transporter for cationic and neutral amino acids, which shares with OCTN2 the affinity for carnitine as substrate, is readily detectable and functional at the apical side. We also show that these transporters differentially interact with anticholinergic drugs. Overall, our findings reveal close similarities of EpiAlveolar™ with the tracheal/bronchial epithelium (EpiAirway™ model) and entrust this alveolar tissue as a potential tool for the screening of biopharmaceuticals molecules.

"Achieve the best while rushing against time": A grounded theory study on caring for low-income immigrant cancer patients at end of life.

BACKGROUND: Between 2000 and 2020, Europe experienced an annual net arrival of approximately 1.6 million immigrants per year. While having lower mortality rates, in the setting of severe diseases, immigrants bear a greater cancer-related burden due to linguistic and cultural barriers and socio-economic conditions. Professionals face a two-fold task: managing clinical conditions while considering the social, economic, cultural, and spiritual sphere of patients and their families. In this regard, little is known about the care provision to low-income immigrant cancer patients in real contexts. AIM: To investigate the perspective of professionals, family members, and stakeholders on the caring process of low-income immigrant cancer patients at the end of life. DESIGN: A Constructivist Grounded Theory study. SETTING/PARTICIPANTS: The study, conducted at a Hospital in Northern Italy, involved 27 participants among health professionals, family caregivers, and other stakeholders who had recently accompanied immigrant cancer patients in their terminal phase of illness. RESULTS: Findings evidenced that professionals feel they were not adequately trained to cope with immigrant cancer patients, nonetheless, they were highly committed in providing the best care they could, rushing against the (short) time the patients have left. Analyses evidenced four main categories: "providing and receiving hospitality," "understanding each other," "addressing diversity," and "around the patient," which we conceptualized under the core category "Achieve the best while rushing against time." CONCLUSIONS: The model reveals the activation of empathic and compassionate behavior by professionals. It evidences the need for empowering professionals with cultural competencies by employing interpreters and specific training programs.

HT-SuMD: making molecular dynamics simulations suitable for fragment-based screening. A comparative study with NMR.

Fragment-based lead discovery (FBLD) is one of the most efficient methods to develop new drugs. We present here a new computational protocol called High-Throughput Supervised Molecular Dynamics (HT-SuMD), which makes it possible to automatically screen up to thousands of fragments, representing therefore a new valuable resource to prioritise fragments in FBLD campaigns. The protocol was applied to Bcl-XL, an oncological protein target involved in the regulation of apoptosis through protein-protein interactions. Initially, HT-SuMD performances were validated against a robust NMR-based screening, using the same set of 100 fragments. These independent results showed a remarkable agreement between the two methods. Then, a virtual screening on a larger library of additional 300 fragments was carried out and the best hits were validated by NMR. Remarkably, all the in silico selected fragments were confirmed as Bcl-XL binders. This represents, to date, the largest computational fragments screening entirely based on MD.

y+LAT1 and y+LAT2 contribution to arginine uptake in different human cell models: Implications in the pathophysiology of Lysinuric Protein Intolerance.

y+LAT1 (encoded by SLC7A7), together with y+LAT2 (encoded by SLC7A6), is the alternative light subunits composing the heterodimeric transport system y+L for cationic and neutral amino acids. SLC7A7 mutations cause lysinuric protein intolerance (LPI), an inherited multisystem disease characterized by low plasma levels of arginine and lysine, protein-rich food intolerance, failure to thrive, hepatosplenomegaly, osteoporosis, lung involvement, kidney failure, haematologic and immunological disorders. The reason for the heterogeneity of LPI symptoms is thus far only poorly understood. Here, we aimed to quantitatively compare the expression of SLC7A7 and SLC7A6 among different human cell types and evaluate y+LAT1 and y+LAT2 contribution to arginine transport. We demonstrate that system y+L-mediated arginine transport is mainly accounted for by y+LAT1 in monocyte-derived macrophages (MDM) and y+LAT2 in fibroblasts. The kinetic analysis of arginine transport indicates that y+LAT1 and y+LAT2 share a comparable affinity for the substrate. Differences have been highlighted in the expression of SLC7A6 and SLC7A7 mRNA among different cell models: while SLC7A6 is almost equally expressed, SLC7A7 is particularly abundant in MDM, intestinal Caco-2 cells and human renal proximal tubular epithelial cells (HRPTEpC). The characterization of arginine uptake demonstrates that system y+L is operative in renal cells and in Caco-2 where, at the basolateral side, it mediates arginine efflux in exchange with leucine plus sodium. These findings explain the defective absorption/reabsorption of arginine in LPI. Moreover, y+LAT1 is the prevailing transporter in MDM sustaining a pivotal role in the pathogenesis of immunological complications associated with the disease.

Early life weight patterns and risk of obesity at 5 years: A population-based cohort study.

Childhood obesity is a major public health problem in industrialized countries. The aim of this study was to estimate the risk of obesity at age 5 based on BMI categories at age 3 and changes in BMI z-score from birth to 3 years of age. In this population-based study BMI data of 5173 children were collected at ages 3 and 5 and were linked to information relative to birth weight. The prevalence of obesity at age 5 was 3.8%. The risk of obesity for children born large for gestational age was 6.5%, while it was 18.6% for children overweight at age 3 and 62% for children who were obese at 3. An increase in BMI z-score from birth to 3 years increases the risk of obesity at age 5 (OR for increase of one standard deviation 2.8%; 95% CI: 2.46-3.20), but adjusting for BMI z-score at age 3, the effect of trajectory disappears (OR 1.08 95% CI: 0.9-1.29). In other words, if one targeted early preventive interventions to 3-year-olds affected by overweight/obesity (only 9.8% of the study cohort), one could possibly address 71% of children potentially affected by obesity at age 5.

Three-manifold quantum invariants and mock theta functions.

Mock modular forms have found applications in numerous branches of mathematical sciences since they were first introduced by Ramanujan nearly a century ago. In this proceeding, we highlight a new area where mock modular forms start to play an important role, namely the study of three-manifold invariants. For a certain class of Seifert three-manifolds, we describe a conjecture on the mock modular properties of a recently proposed quantum invariant. As an illustration, we include concrete computations for a specific three-manifold, the Brieskorn sphere Σ(2, 3, 7). This article is part of a discussion meeting issue 'Srinivasa Ramanujan: in celebration of the centenary of his election as FRS'.

Characterization of ABC Transporters in EpiAirway™, a Cellular Model of Normal Human Bronchial Epithelium.

The ATP-binding cassette (ABC) transporters P-glycoprotein (MDR1/ABCB1), multidrug resistance-associated protein 1 (MRP1/ABCC1), and breast cancer resistance protein (BCRP/ABCG2) play a crucial role in the translocation of a broad range of drugs; data about their expression and activity in lung tissue are controversial. Here, we address their expression, localization and function in EpiAirway™, a three-dimensional (3D)-model of human airways; Calu-3 cells, a representative in vitro model of bronchial epithelium, are used for comparison. Transporter expression has been evaluated with RT-qPCR and Western blot, the localization with immunocytochemistry, and the activity by measuring the apical-to-basolateral and basolateral-to-apical fluxes of specific substrates in the presence of inhibitors. EpiAirway™ and Calu-3 cells express high levels of MRP1 on the basolateral membrane, while they profoundly differ in terms of BCRP and MDR1: BCRP is detected in EpiAirway™, but not in Calu-3 cells, while MDR1 is expressed and functional only in fully-differentiated Calu-3; in EpiAirway™, MDR1 expression and activity are undetectable, consistently with the absence of the protein in specimens from human healthy bronchi. In summary, EpiAirway™ appears to be a promising tool to study the mechanisms of drug delivery in the bronchial epithelium and to clarify the role of ABC transporters in the modulation of the bioavailability of administered drugs.

Vascular and metabolic profiles in offspring born to pregnant mice with metabolic syndrome treated with inositols.

BACKGROUND: Inositols (INOs) supplementation during pregnancy, specifically the combination of myo-inositol (MI) and D-chiro-inositol (DCI), has been reported to improve vascular parameters in women with gestational diabetes mellitus. We demonstrated previously that offspring born to pregnant mice lacking the endothelial nitric oxide synthase (eNOS+/-) gene have hypertension (HTN) as adults and, when fed a high-fat diet (HFD), develop a metabolic syndrome (MS) phenotype. OBJECTIVE: Our aim was to evaluate whether INOs treatment in pregnancy complicated by MS improves the vascular and metabolic profile in mice offspring programmed in utero to develop HTN and MS. MATERIALS AND METHODS: Heterozygous eNOS+/- mice fed an HFD manifest a MS phenotype. Female eNOS+/- mice with MS were bred with a wild-type (WT) male. On gestational day 1, pregnant females were randomly allocated to receive either a mixture of INOs (MI/DCI: 7.2/0.18 mg/mL) or water as placebo until delivery. The female offspring obtained were genotyped and categorized as: WT (genetically normal, with eNOS gene) and eNOS+/- offspring (genetically modified, heterozygous for eNOS gene). Both offspring developed in an abnormal uterine environment due to maternal MS. At 9-10 weeks of age, the offspring underwent a glucose tolerance test (GTT) and systolic blood pressure (SBP) measurement. The mice were then sacrificed, and the carotid arteries were isolated for evaluation of vascular responses. Responses to phenylephrine (PE), in the presence and absence of a nonspecific nitric oxide inhibitor (N-nitro-L-arginine methyl ester [L-NAME]), the vasodilator acetylcholine (ACh), and sodium nitroprusside (SNP) were assessed. RESULTS: The GTT showed lower glucose levels in both eNOS+/-INOs (P = .03) and WT-INOs (P = .05) offspring born to MS dams on INOs supplementation compared to offspring born to untreated dams. SBP was higher in eNOS+/- offspring compared to WT (169 ± 7 vs 142 ± 9 mm Hg, respectively, P = .04) and INOs treatment decreased SBP in WT-INOs (110 ± 10 mm Hg, P = .01) but not in eNOS+/-INOs offspring. Maximal (%Max) contractile response to PE was higher in eNOS+/- offspring born to MS dams and was decreased in those born to MS dams treated with INOs (%Max, eNOS+/-, 123 ± 7 vs eNOS+/-INOs, 82 ± 11 mm Hg, P = .007). No differences were seen in PE contractile responses in WT offspring born to MS dams treated or not treated with INOs (WT, 92 ± 4 vs WT-INOs, 75 ± 7). The L-NAME response was decreased in eNOS+/-INOs and WT-INOs offspring compared to untreated ones. The ACh vasorelaxation was impaired in eNOS+/- and WT offspring born to MS dams, and maternal INOs treatment improved offspring vascular relaxation in both offspring (P = .01 and P = .03, respectively). No differences were seen in response to SNP. CONCLUSION: Inositols supplementation improved glucose tolerance, SBP, and vascular responses in adult eNOS+/- and WT offspring born to dams with MS. Interestingly, WT born to MS dams show an altered vascular profile similar to eNOS+/- offspring and exhibit an improved response to INOs treatment. Our findings suggest that the benefits of INOs treatment are more pronounced in offspring exposed to environmental factors in utero, and less likely in those due to genetic factors.

The preload force affects the perception threshold of muscle vibration-induced movement illusions.

The control and the execution of motor tasks are largely influenced by proprioceptive feedback, i.e. the information about the position and movement of the body. In 1972, it was discovered that a vibratory stimulation applied non-invasively to a muscle or a tendon induces a movement illusion consistent with the elongation of the vibrated muscle/tendon. Although this phenomenon was reported by several studies, it is still unclear how to reliably reproduce it because of the many different features of the stimulation altering the sensation (e.g. frequency, duration, location). By performing a psychophysical test, we analysed the effects of the stimulation point and the preload force on the minimum stimulation amplitude needed to elicit an illusion of movement. In particular, we stimulated two groups of healthy subjects on three target regions of the biceps brachii muscle (the distal tendon, the muscle belly and one of the proximal tendons) applying three preload force ranges (0.5-0.75N, 1-2N and 3-4N). Our results showed that the minimum stimulation amplitude eliciting a sensation is affected by the preload force. On the contrary, it did not change significantly among the three stimulated regions. Nevertheless, the reported vividness of the illusion of movement changed across the stimulated points decreasing while moving from the distal to the proximal tendons. Overall, these outcomes contribute to the scientific debate on the features that modulate the vibration-induced movement illusion proposing ways to increase the reliability of the procedure in basic and applied research studies.

Polymerase ε (POLE) ultra-mutation in uterine tumors correlates with T lymphocyte infiltration and increased resistance to platinum-based chemotherapy in vitro.

OBJECTIVE: Up to 12% of all endometrial-carcinomas (EC) harbor DNA-polymerase-ε-(POLE) mutations. It is currently unknown whether the favorable prognosis of POLE-mutated EC is derived from their low metastatic capability, extraordinary number of somatic mutations thus imparting immunogenicity, or a high sensitivity to chemotherapy. METHODS: Polymerase-chain-reaction-amplification and Sanger-sequencing were used to test for POLE exonuclease-domain-mutations (exons 9-14) 131 EC. Infiltration of CD4+ and CD8+ T-lymphocytes (TIL) and PD-1-expression in POLE-mutated vs POLE wild-type EC was studied by immunohistochemistry (IHC) and the correlations between survival and molecular features were investigated. Finally, primary POLE-mutated and POLE-wild-type EC cell lines were established and compared in-vitro for their sensitivity to chemotherapy. RESULTS: Eleven POLE-mutated EC (8.5%) were identified. POLE-mutated tumors were associated with improved progression-free-survival (P<0.05) and displayed increased numbers of CD4+ (44.5 vs 21.8; P=0.001) and CD8+ (32.8 vs 13.5; P<0.001) TILs when compared to wild-type POLE EC. PD-1 receptor was overexpressed in TILs from POLE-mutated vs wild-type-tumors (81% vs 28%; P<0.001). Primary POLE tumor cell lines were significantly more resistant to platinum-chemotherapy in-vitro when compared to POLE-wild-type tumors (P<0.004). CONCLUSIONS: POLE ultra-mutated EC are heavily infiltrated with CD4+/CD8+ TIL, overexpress PD-1 immune-check-point (i.e., features consistent with chronic antigen-exposure), and have a better prognosis when compared to other molecular subtypes of EC patients. POLE-mutated tumor-cell lines are resistant to platinum-chemotherapy in-vitro suggesting that the better prognosis of POLE-patients is not secondary to a higher sensitivity to chemotherapy but likely linked to enhanced immunogenicity.

Evaluation of quantitative fFn test in predicting the risk of preterm birth.

OBJECTIVE: To evaluate diagnostic accuracy of quantitative fetal fibronectin (qfFN) test in predicting preterm birth (PTB) risk <34 weeks' gestation or within 14 days from testing. We explored the predictive potential of the test in five-predefined PTB risk categories based on predefined qfFN thresholds (<10, 10-49, 50-199, 200-499 and ≥500 ng/mL). METHODS: Measurement of cervicovaginal qfFN with Rapid fFN 10Q System (Hologic) in 126 women with singleton pregnancy (23-33 weeks' gestation) reporting signs and symptoms indicative of preterm labour (PTL). RESULTS: For PTB prediction risk <34 weeks' gestation, sensitivity decreased from 100% to 41.7% and specificity increased from 0% to 99.1% with increasing fFN thresholds. Positive predictive value (PPV) increased from 9.5% to 83.3% with increasing qfFN thresholds, while negative predictive value (NPV) was higher than 90% among the fFN-predefined categories. Diagnostic accuracy results showed an area under a receiving operator characteristic (ROC) curve of 84.5% (95% CI, 0.770-0.903). For delivery prediction within 14 days from the testing, sensitivity decreased from 100% to 42.8% and specificity increased from 0% to 100% with increasing fFN thresholds. Diagnostic accuracy determined by the ROC curve was 66.1% (95% CI, 0.330-0.902). CONCLUSIONS: The QfFN thresholds of tests are a useful tool to distinguish pregnant women for PTB prediction risk <34 weeks' gestation.

Dual CCNE1/PIK3CA targeting is synergistic in CCNE1-amplified/PIK3CA-mutated uterine serous carcinomas in vitro and in vivo.

BACKGROUND: Clinical options for patients harbouring advanced/recurrent uterine serous carcinoma (USC), an aggressive variant of endometrial tumour, are very limited. Next-generation sequencing (NGS) data recently demonstrated that cyclin E1 (CCNE1) gene amplification and pik3ca driver mutations are common in USC and may therefore represent ideal therapeutic targets. METHODS: Cyclin E1 expression was evaluated by immunohistochemistry (IHC) on 95 USCs. The efficacy of the cyclin-dependent kinase 2/9 inhibitor CYC065 was assessed on multiple primary USC cell lines with or without CCNE1 amplification. Cell-cycle analyses and knockdown experiments were performed to assess CYC065 targeting specificity. Finally, the in vitro and in vivo activity of CYC065, Taselisib (a PIK3CA inhibitor) and their combinations was tested on USC xenografts derived from CCNE1-amplified/pik3ca-mutated USCs. RESULTS: We found that 89.5% of the USCs expressed CCNE1. CYC065 blocked cells in the G1 phase of the cell cycle and inhibited cell growth specifically in CCNE1-overexpressing USCs. Cyclin E1 knockdown conferred increased resistance to CYC065, whereas CYC065 treatment of xenografts derived from CCNE1-amplified USCs significantly reduced tumour growth. The combination of CYC065 and Taselisib demonstrated synergistic effect in vitro and was significantly more effective than single-agent treatment in decreasing tumour growth in xenografts of CCNE1-amplified/pik3ca-mutated USCs. CONCLUSIONS: Dual CCNE1/PIK3CA blockade may represent a novel therapeutic option for USC patients harbouring recurrent CCNE1-amplified/pi3kca-mutated tumours.

The effect of combined inositol supplementation on maternal metabolic profile in pregnancies complicated by metabolic syndrome and obesity.

BACKGROUND: Myoinositol and D-chiroinositol improve insulin resistance in women with obesity and gestational diabetes and in postmenopausal women with metabolic syndrome. We previously reported that offspring born to hypertensive dams lacking endothelial nitric oxide synthase and fed a high-fat diet develop metabolic-like syndrome phenotype. OBJECTIVE: The objective of the study was to investigate the effect of a mixture of myoinositol/D-chiroinositol supplementation during pregnancy on the maternal metabolic profile in pregnancies complicated by the metabolic-like syndrome and obesity using a pregnant mouse model. STUDY DESIGN: Female heterozygous endothelial nitric oxide synthase(-/+) mice with moderate hypertension were placed on a high-fat diet for 4 weeks to induce a metabolic-like syndrome phenotype. Similarly, wild-type C57BL/6 mice were placed on a high-fat diet for 4 weeks to induce a murine obesity model. Mice were then bred with wild-type males. On gestational day 1, dams were randomly allocated to receive either a mixture of myoinositol/D-chiroinositol in water (7.2/0.18 mg/mL, respectively) or water as control (placebo). At term (gestational day 18), maternal weights, systolic blood pressure, and a glucose tolerance test were obtained. Dams were then killed; pups and placentas were weighed and maternal blood collected. Serum levels of metabolic biomarkers relevant to diabetes and obesity (ghrelin, gastric inhibitory peptide, glucagon-like peptide 1, glucagon, insulin, leptin, resistin) were measured by a multiplex enzyme-linked immunosorbent assay. Analysis was done comparing metabolic-like syndrome-myoinositol/D-chiroinositol-treated vs metabolic-like syndrome-nontreated mice and obese-myoinositol/D-chiroinositol-treated vs obese nontreated mice. RESULTS: Mean systolic blood pressure was lower in metabolic-like syndrome pregnant mice treated with myoinositol/D-chiroinositol compared with placebo (P = .04), whereas there was no difference in systolic blood pressure between treated and placebo-treated obese pregnant mice. Pregnant metabolic-like syndrome mice treated with myoinositol/D-chiroinositol showed lower glucose values during the glucose tolerance test and in the area under the curve (myoinositol/D-chiroinositol: 17512.5 ± 3984.4 vs placebo: 29687.14 ± 8258.7; P = .003), but no differences were seen in the obese pregnant mice. Leptin serum levels were lower in the metabolic-like syndrome-myoinositol/D-chiroinositol-treated mice compared with the placebo group (myoinositol/D-chiroinositol: 16985 ± 976.4 pg/dL vs placebo: 24181.9 ± 3128.2 pg/dL, P = .045). No other differences were seen in any of the remaining serum metabolic biomarkers studied in metabolic-like syndrome and in obese pregnant mice. Maternal weight gain was not different in the pregnant metabolic-like syndrome dams, whereas it was lower in the obese myoinositol/D-chiroinositol-treated dams compared with the placebo group (myoinositol/D-chiroinositol: 10.9 ± 0.5 g vs 12.6 ± 0.6 g, P = .04). Fetal and placental weights did not differ between myoinositol/D-chiroinositol-treated and nontreated pregnant dams with metabolic-like syndrome and obesity. CONCLUSION: Combined inositol treatment during pregnancy improves blood pressure, glucose levels at the glucose tolerance test, and leptin levels in pregnant dams with metabolic-like syndrome phenotype but not in obese pregnant dams. In addition, inositol treatment was associated with lower gestational weight gain in the obese but not in the metabolic-like syndrome pregnant dams.