NEXT GENERATION SEQUENCING FOR DIAGNOSIS IN MONOGENIC PEDIATRIC STROKE .. from NGS panel to Whole Exome Sequencing

Background and Purpose: Pediatric arterial ischemic stroke (AIS) may underlie an as yet undiagnosed syndrome often characterized by simple Mendelian inheritance. We aimed to establish and validate a targeted gene panel for AIS associated with monogenic disorders, and to determine its diagnostic yield and clinical utility. Methods: Clinical and neuro-radiological data were collected for every patient enrolled in the study. DNA samples were tested by means of a customized gene panel including 15 genes associated with known genetic diseases related with AIS. Results: Thirty-eight patients (23 males, mean age 6.5 years) were selected with heterogeneous AIS phenotypes, mostly multiple and asynchronous and secondary to vasculopathy. Ten out of 38 resulted to carry rare potentially causative mutations in at least one of the 15 genes analyzed. In 4 cases the analyses led to a definite genetic diagnosis while results were either controversial or null in the remaining patients. Conclusions: While the complexity of the different clinical phenotypes associated with AIS is not fully accounted for by the genes tested in the present study, the achieved diagnoses had a great beneficial impact on patient management. A wider gene panel or an unbiased genome wide approach would be better suited to explain a greater proportion of pediatric stroke events

17-Beta-Hydroxy-Steroid-Dehydrogenases in Hair Follicles of Normal and Bald Scalp: A Histochemical Study

Testosterone, dihydrotestosterone, and estradiol were used as substrates to evaluate 17-beta-hydroxy-steroid-dehydrogenases in the hair follicles of normal human scalp and scalps of patients affected by initial or advanced male pattern alopecia (MPA). Trace of strong reactions were found in follicles and moderate reactions were found in the dermal and hypodermal structures immediately surrounding the follicles. With testosterone and estradiol, the outer sheath of the late anagen follicles in initial MPA showed greater reactivity than did similar follicles of normal subjects. With dihydrotestosterone, reactivity in the outer sheath of normal late anagen follicles was strong and became progressively less strong in similar follicles from initial and advanced MPA subjects

A practical approach to the use of low molecular weight heparins in VTE treatment and prophylaxis in children and newborns.

Low-molecular weight heparins are currently the most commonly used anticoagulants in children and newborns. However, since thrombotic complications rarely occur outside large children’s hospitals, physicians often encounter some practical problems inmanaging these treatments when a pediatric thrombosis specialist is not available. The drug of choice is enoxaparin, due to its favorable FXa/FIIa ratio and the availability of pharmacokinetic and pharmacodynamic data. The treatment of acute thrombosis should be started with two daily injections but when compliance is an issue, a single daily administration schedule could be chosen for secondary prophylaxis ensuring careful measurement of the post 24-hour anti-FXa activity. Furthermore, a subcutaneous device may be a useful tool and a topical dermal anesthetic could be effective in controlling pain without affecting anti-FXa levels. In neonate and toddlers, where mini doses are frequently needed, the dead space of syringes and needles could represent an issue and therefore the use of insulin syringes without dead space is advisable,while a dilution of the drug is useful with other syringes. This article derives froma nonsystematic review of the available literature, with special attention to recent international guidelines and expert recommendations, combined to authors’ clinical practice in large tertiary pediatric hospitals and will provide concise and practical information for the use of low-molecular weight heparin in childhood and infancy in a sort of “answering frequently asked questions.

N-4 Alkyl Cytosine Derivatives Synthesis: A New Approach

The selective N-4 alkylation of cytosine plays a critical role in the synthesis of biologically active molecules. This work focuses on the development of practical reaction conditions toward a regioselective synthesis of N-4-alkyl cytosine derivatives. The sequence includes a direct and selective sulfonylation at the N-1 site of the cytosine, followed by the alkylation of the amino siteusing KHMDS in CH2Cl2/THF mixture, providing a fast and efficient approach consistent withpyrimidine-based drug design

Therapeutic potential of TRPM8 antagonists in prostate cancer.

Transient receptor potential melastatin-8 (TRPM8) represents an emerging target in prostate cancer, although its mechanism of action remains unclear. Here, we have characterized and investigated the effects of TRPM8 modulators in prostate cancer aggressiveness disclosing the molecular mechanism underlying their biological activity. Patch-clamp and calcium fluorometric assays were used to characterize the synthesized compounds. Androgen-stimulated prostate cancer-derived cells were challenged with the compounds and the DNA synthesis was investigated in a preliminary screening. The most effective compounds were then employed to inhibit the pro-metastatic behavior of in various PC-derived cells, at different degree of malignancy. The effect of the compounds was then assayed in prostate cancer cell-derived 3D model and the molecular targets of selected compounds were lastly identified using transcriptional and non-transcriptional reporter assays. TRPM8 antagonists inhibit the androgen-dependent prostate cancer cell proliferation, migration and invasiveness. They are highly effective in reverting the androgen-induced increase in prostate cancer cell spheroid size. The compounds also revert the proliferation of castrate-resistant prostate cancer cells, provided they express the androgen receptor. In contrast, no effects were recorded in prostate cancer cells devoid of the receptor. Selected antagonists interfere in non-genomic androgen action and abolish the androgen-induced androgen receptor/TRPM8 complex assembly as well as the increase in intracellular calcium levels in prostate cancer cells. Our results shed light in the processes controlling prostate cancer progression and make the transient receptor potential melastatin-8 as a ‘druggable’ target in the androgen receptor-expressing prostate cancers

Squaring the circle: lessons from the role-playing exercises on S3 regional and multi-level governance

Whilst there is widespread agreement on the importance of developing an appropriate governance structure for S3, there is little empirical evidence on the challenges this entails. The JRC, as part of its activities of Targeted Support, has developed a working group to address these issues. The working group operated in the first half of 2018, built upon an extensive background research as well as two role-playing exercises. The latter aimed at understanding and addressing key policy challenges, by enacting realistic policy scenarios. The paper explores both policy and methodological questions. As for the former, the paper investigates the main bottlenecks inhibiting the effectiveness of multi-stakeholder policy development, identifying the administrative and technical needs of those in charge of S3 governance. As for the latter, the paper provides an opportunity to reflect on role-playing as a tool for policy analysis in the context of Cohesion policy.JRC.B.3-Territorial Developmen

Characterization of New TRPM8 Modulators in Pain Perception

Background: Transient Receptor Potential Melastatin-8 (TRPM8) is a non-selective cation channel activated by cold temperature and by cooling agents. Several studies have proved that this channel is involved in pain perception. Although some studies indicate that TRPM8 inhibition is necessary to reduce acute and chronic pain, it is also reported that TRPM8 activation produces analgesia. These conflicting results could be explained by extracellular Ca2+-dependent desensitization that is induced by an excessive activation. Likely, this effect is due to phosphatidylinositol 4,5-bisphosphate (PIP2) depletion that leads to modification of TRPM8 channel activity, shifting voltage dependence towards more positive potentials. This phenomenon needs further evaluation and confirmation that would allow us to understand better the role of this channel and to develop new therapeutic strategies for controlling pain. Experimental approach: To understand the role of TRPM8 in pain perception, we tested two specific TRPM8-modulating compounds, an antagonist (IGM-18) and an agonist (IGM-5), in either acute or chronic animal pain models using male Sprague-Dawley rats or CD1 mice, after systemic or topical routes of administration. Results: IGM-18 and IGM-5 were fully characterized in vivo. The wet-dog shake test and the body temperature measurements highlighted the antagonist activity of IGM-18 on TRPM8 channels. Moreover, IGM-18 exerted an analgesic effect on formalin-induced orofacial pain and chronic constriction injury-induced neuropathic pain, demonstrating the involvement of TRPM8 channels in these two pain models. Finally, the results were consistent with TRPM8 downregulation by agonist IGM-5, due to its excessive activation. Conclusions: TRPM8 channels are strongly involved in pain modulation, and their selective antagonist is able to reduce both acute and chronic pain

No evidence of sars-cov-2 circulation in rome (Italy) during the pre-pandemic period. Results of a retrospective surveillance

In March 2020, the World Health Organization (WHO) declared that the COVID-19 outbreak recorded over the previous months could be characterized as a pandemic. The first known Italian SARS-CoV-2 positive case was reported on 21 February. In some countries, cases of suspected “COVID-19-like pneumonia” had been reported earlier than those officially accepted by health authorities. This has led many investigators to check preserved biological or environmental samples to see whether the virus was detectable on dates prior to those officially stated. With regard to Italy, the results of a microbiological screening in sewage samples collected between the end of February and the beginning of April 2020 from wastewaters in Milan (Northern Italy) and Rome (Central Italy) showed presence of SARS-CoV-2. In the present study, we evaluated, by means of a standardized diagnostic method, the SARS-CoV-2 infection prevalence amongst patients affected by severe acute respiratory syndrome (SARI) in an academic hospital located in Central Italy during the period of 1 November 2019–1 March 2020. Overall, the number of emergency room (ER) visits during the investigated period was 13,843. Of these, 1208 had an influenza-like syndrome, but only 166 matched the definition of SARI as stated in the study protocol. A total of 52 SARI cases were laboratory confirmed as influenza: 26 as a type B virus, 25 as a type A, and 1 as both viruses. Although about 17% of the total sample had laboratory or radiological data compatible with COVID-19, all the nasopharyngeal swabs stored underwent SARS-CoV-2 RT-PCR and tested negative. Based on our result, it is confirmed that the COVID-19 pandemic spread did not start prior to the “official” onset in central Italy. Routine monitoring of SARI causative agents at the local level is critical for reporting epidemiologic and etiologic trends that may differ from one country to another and also among different influenza seasons. This has a practical impact on prevention and control strategies