A novel bioinformatic method for the identification of antimicrobial peptides in metagenomes.

AIMS: This study aimed to develop a new bioinformatic approach for the identification of novel antimicrobial peptides (AMPs), which did not depend on sequence similarity to known AMPs held within databases, but on structural mimicry of another antimicrobial compound, in this case an ultrashort, synthetic, cationic lipopeptide (C12-OOWW-NH2). METHODS AND RESULTS: When applied to a collection of metagenomic datasets, our outlined bioinformatic method successfully identified several short (8-10aa) functional AMPs, the activity of which was verified via disk diffusion and minimum inhibitory concentration assays against a panel of 12 bacterial strains. Some peptides had activity comparable to, or in some cases, greater than, those from published studies that identified AMPs using more conventional methods. We also explored the effects of modifications, including extension of the peptides, observing an activity peak at 9-12aa. Additionally, the inclusion of a C-terminal amide enhanced activity in most cases. Our most promising candidate (named PB2-10aa-NH2) was thermally stable, lipid-soluble, and possessed synergistic activity with ethanol but not with a conventional antibiotic (streptomycin). CONCLUSIONS: While several bioinformatic methods exist to predict AMPs, the approach outlined here is much simpler and can be used to quickly scan huge datasets. Searching for peptide sequences bearing structural similarity to other antimicrobial compounds may present a further opportunity to identify novel AMPs with clinical relevance, and provide a meaningful contribution to the pressing global issue of AMR.

A New Lung Ultrasound Protocol Able to Predict Worsening in Patients Affected by Severe Acute Respiratory Syndrome Coronavirus 2 Pneumonia.

OBJECTIVES: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can generate severe pneumonia associated with high mortality. A bedside lung ultrasound (LUS) examination has been shown to have a potential role in this setting. The purpose of this study was to evaluate the potential prognostic value of a new LUS protocol (evaluation of 14 anatomic landmarks, with graded scores of 0-3) in patients with SARS-CoV-2 pneumonia and the association of LUS patterns with clinical or laboratory findings. METHODS: A cohort of 52 consecutive patients with laboratory-confirmed SARS-CoV-2 underwent LUS examinations on admission in an internal medicine ward and before their discharge. A total LUS score as the sum of the scores at each explored area was computed. We investigated the association between the LUS score and clinical worsening, defined as a combination of high-flow oxygen support, intensive care unit admission, or 30-day mortality as the primary end point. RESULTS: Twenty (39%) patients showed a worse outcome during the observation period; the mean LUS scores ± SDs were 20.4 ± 8.5 and 29.2 ± 7.3 in patients without and with worsening, respectively (P < .001). In a multivariable analysis, adjusted for comorbidities (>2), age (>65 years), sex (male), and body mass index (≥25 kg/m2 ), the association between the LUS score and worsening (odds ratio, 1.17; 95% confidence interval, 1.05 to 1.29; P = .003) was confirmed, with good discrimination of the model (area under the receiver operating characteristic curve, 0.82). A median LUS score higher than 24 was associated with an almost 6-fold increase in the odds of worsening (odds ratio, 5.67; 95% confidence interval, 1.29 to 24.8; P = .021). CONCLUSIONS: Lung ultrasound can represent an effective tool for monitoring and stratifying the prognosis of patients with SARS-CoV-2 pulmonary involvement.

SMARCE1-related meningiomas: A clear example of cancer predisposing syndrome.

We report the case of a 16-year-old girl presenting with spinal clear-cell multiple meningiomas (CCMs). In view of this presentation, we sequenced a bioinformatic panel of genes associated with susceptibility to meningioma, identifying a germline heterozygous variant in SMARCE1. Somatic DNA investigations in the CCM demonstrated the deletion of the wild-type allele (loss of heterozygosity, LOH), supporting the causative role of this variant. Family segregation study detected the SMARCE1 variant in the asymptomatic father and in the asymptomatic sister who, nevertheless, presents 2 spinal lesions. Germline heterozygous loss-of-function (LoF) variants in SMARCE1, encoding a protein of the chromatin-remodeling complex SWI/SNF, have been described in few familial cases of susceptibility to meningioma, in particular the CCM subtype. Our case confirms the role of NGS in investigating predisposing genes for meningiomas (multiple or recurrent), with specific regard to SMARCE1 in case of pediatric CCM. In addition to the age of onset, the presence of familial clustering or the coexistence of multiple synchronous meningiomas also supports the role of a genetic predisposition that deserves a molecular assessment. Additionally, given the incomplete penetrance, it is of great importance to follow a specific screening or follow-up program for symptomatic and asymptomatic carriers of pathogenic variants in SMARCE1.

Ultrasound Patterns of Hepatocellular Carcinoma and Their Prognostic Impact: A Retrospective Study.

BACKGROUND: Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death. Abdominal ultrasound (US) is by far the most widely used first-level exam for the diagnosis of HCC. We aimed to assess whether different ultrasound patterns were related to tumor prognosis. METHODS: We retrospectively reviewed all patients with a new diagnosis of HCC (single nodule) and undergoing radiofrequency thermal ablation (RFTA) at our clinic between January 2009 and December 2021. Patients were classified according to four HCC ultrasound patterns: 1A, single capsulated nodule; 1B, well capsulated intra-node nodule; 1C, cluster consisting of capsulated nodules; and 2, non-capsulated nodule. RESULTS: 149 patients were analysed; median follow-up time was 43 months. US patterns 1A (32.9%) and 1B (61.1%) were the most commonly seen. Median overall survival (OS) and recurrence-free survival (RFS) from RFTA were 54 months (95% CI, 42-66) and 22 months (95% CI, 12-32), respectively. Pattern 1A showed the best OS. Compared to pattern 1A, 1B was independently associated with worse OS (51 months (95% CI, 34-68) vs. 46 months (95% CI, 18-62)) and RFS (34 months (95% CI, 27-41) vs. 18 months (95% CI, 12-24)). Patterns 1C and 2 were associated with worse RFS compared to 1A, while no difference was seen for OS. Among baseline clinical variables, pattern 1B exhibited higher histological grade (p = 0.048) and tumor dimension (p = 0.034) compared to pattern 1A. CONCLUSIONS: Our findings demonstrate that different US patterns correlate with different survival outcomes and tumor behavior in patients with HCC. Prospective studies are needed to confirm these results.

FOXI3 pathogenic variants cause one form of craniofacial microsomia.

Craniofacial microsomia (CFM; also known as Goldenhar syndrome), is a craniofacial developmental disorder of variable expressivity and severity with a recognizable set of abnormalities. These birth defects are associated with structures derived from the first and second pharyngeal arches, can occur unilaterally and include ear dysplasia, microtia, preauricular tags and pits, facial asymmetry and other malformations. The inheritance pattern is controversial, and the molecular etiology of this syndrome is largely unknown. A total of 670 patients belonging to unrelated pedigrees with European and Chinese ancestry with CFM, are investigated. We identify 18 likely pathogenic variants in 21 probands (3.1%) in FOXI3. Biochemical experiments on transcriptional activity and subcellular localization of the likely pathogenic FOXI3 variants, and knock-in mouse studies strongly support the involvement of FOXI3 in CFM. Our findings indicate autosomal dominant inheritance with reduced penetrance, and/or autosomal recessive inheritance. The phenotypic expression of the FOXI3 variants is variable. The penetrance of the likely pathogenic variants in the seemingly dominant form is reduced, since a considerable number of such variants in affected individuals were inherited from non-affected parents. Here we provide suggestive evidence that common variation in the FOXI3 allele in trans with the pathogenic variant could modify the phenotypic severity and accounts for the incomplete penetrance.

ECHOPAEDIA: Echography in Paediatric Patients in the Age of Coronavirus Disease 2019: Utility of Lung Ultrasound and Chest X-Ray in Diagnosis of Community-Acquired Pneumonia and Severe Acute Respiratory Syndrome Coronavirus 2 Pneumonia.

Background: In recent years, lung ultrasound (LUS) has spread to emergency departments and clinical practise gaining great support, especially in time of pandemic, but only a few studies have been done on children. The aim of the present study is to compare the diagnostic accuracy of LUS (using Soldati LUS score) and that of chest X-ray (CXR) in CAP and COVID-19 pneumonia in paediatric patients. Secondary objective of the study is to examine the association between LUS score and disease severity. Finally, we describe the local epidemiology of paediatric CAP during the study period in the era of COVID-19 by comparing it with the previous 2 years. Methods: This is an observational retrospective single-centre study carried out on patients aged 18 or younger and over the month of age admitted to the Paediatric Unit of our Foundation for suspected community-acquired pneumonia or SARS-CoV-2 pneumonia during the third pandemic wave of COVID-19. Quantitative variables were elaborated with Shapiro-Wilks test or median and interquartile range (IQR). Student's t-test was used for independent data. Association between quantitative data was evaluated with Pearson correlation. ROC curve analysis was used to calculate best cut-off of LUS score in paediatric patients. Area under the ROC curve (AUC), sensibility, and specificity are also reported with 95% confidence interval (CI). Results: The diagnostic accuracy of the LUS score in pneumonia, the area underlying the ROC curve (AUC) was 0.67 (95% CI: 0.27-1) thus showing a discrete discriminatory power, with a sensitivity of 89.66% and specificity 50% setting a LUS score greater than or equal to 1 as the best cut-off. Nine patients required oxygen support and a significant statistical correlation (p = 0.0033) emerged between LUS score and oxygen therapy. The mean LUS score in patients requiring oxygen therapy was 12. RCP was positively correlated to the patient's LUS score (p = 0.0024). Conclusions: Our study has shown that LUS is a valid alternative to CXR. Our results show how LUS score can be applied effectively for the diagnosis and stratification of paediatric pneumonia.

Pathophysiology and Therapy of High-Altitude Sickness: Practical Approach in Emergency and Critical Care.

High altitude can be a hostile environment and a paradigm of how environmental factors can determine illness when human biological adaptability is exceeded. This paper aims to provide a comprehensive review of high-altitude sickness, including its epidemiology, pathophysiology, and treatments. The first section of our work defines high altitude and considers the mechanisms of adaptation to it and the associated risk factors for low adaptability. The second section discusses the main high-altitude diseases, highlighting how environmental factors can lead to the loss of homeostasis, compromising important vital functions. Early recognition of clinical symptoms is important for the establishment of the correct therapy. The third section focuses on high-altitude pulmonary edema, which is one of the main high-altitude diseases. With a deeper understanding of the pathogenesis of high-altitude diseases, as well as a reasoned approach to environmental or physical factors, we examine the main high-altitude diseases. Such an approach is critical for the effective treatment of patients in a hostile environment, or treatment in the emergency room after exposure to extreme physical or environmental factors.

Carriers of ADAMTS13 Rare Variants Are at High Risk of Life-Threatening COVID-19.

Thrombosis of small and large vessels is reported as a key player in COVID-19 severity. However, host genetic determinants of this susceptibility are still unclear. Congenital Thrombotic Thrombocytopenic Purpura is a severe autosomal recessive disorder characterized by uncleaved ultra-large vWF and thrombotic microangiopathy, frequently triggered by infections. Carriers are reported to be asymptomatic. Exome analysis of about 3000 SARS-CoV-2 infected subjects of different severities, belonging to the GEN-COVID cohort, revealed the specific role of vWF cleaving enzyme ADAMTS13 (A disintegrin-like and metalloprotease with thrombospondin type 1 motif, 13). We report here that ultra-rare variants in a heterozygous state lead to a rare form of COVID-19 characterized by hyper-inflammation signs, which segregates in families as an autosomal dominant disorder conditioned by SARS-CoV-2 infection, sex, and age. This has clinical relevance due to the availability of drugs such as Caplacizumab, which inhibits vWF-platelet interaction, and Crizanlizumab, which, by inhibiting P-selectin binding to its ligands, prevents leukocyte recruitment and platelet aggregation at the site of vascular damage.

Genetic counseling during COVID-19 pandemic: Tuscany experience.

BACKGROUND: COVID-19 outbreak prompted health centres to reorganize their clinical and surgical activity. In this paper, we show how medical genetics department's activity, in our tertiary pediatric hospital, has changed due to pandemic. METHODS: We stratified all our scheduled visits, from March 9th through April 30th, and assessed case-by-case which genetic consultations should be maintained as face-to-face visit, or postponed/switched to telemedicine. RESULTS: Out of 288 scheduled appointments, 60 were prenatal consultations and 228 were postnatal visits. We performed most of prenatal consultations as face-to-face visits, as women would have been present in the hospital to perform other procedures in addition to our consult. As for postnatal care, we suspended all outpatient first visits and opted for telemedicine for selected follow-up consultations: interestingly, 75% of our patients' parents revealed that they would have cancelled the appointment themselves for the fear to contract an infection. CONCLUSIONS: Spread of COVID-19 in Italy forced us to change our working habits. Given the necessity to optimize healthcare resources and minimize the risk of in-hospital infections, we experienced the benefits of telegenetics. Current pandemic made us familiar with telemedicine, laying the foundations for its application to deal with the increasing number of requests in clinical genetics.

Identification, molecular characterization and segregation analysis of a variant DMPK pre-mutation allele in a three-generation Italian family.

DM1 is an autosomal dominant multisystemic disease caused by an unstable CTG repeat expansion in the 3'-untranslated region (UTR) of the DMPK gene. The complex variant DMPK expanded the alleles containing CAG, CCG, CTC and/or GGC interruptions repetition sequences have been reported in 3-8% of DM1 patients. To date, very few information is available about the frequency and clinical consequences of pre-mutated DMPK variant allele. In this study, we describe a three-generation Italian family showing the segregation of an interrupted DMPK allele within the premutation range. TP-PCR with primers complementary to CCG repetitions and direct sequencing allow us to identify a hetero-triplet (CTG)6(CCGCTG)15(CTG)5 repeat structure. The haplotype analysis demonstrated that this variant allele is associated with the European founder DM1 haplotype. The pyrosequencing analysis of the CpG islands contained in the flanking regions of the CTG array, did not show the presence of a cis effect of the CCG interruptions on the methylation profile of the DM1 locus. The analysis of both meiotic transmissions, one maternal and one paternal, revealed the intrafamilial stability of the DM1 premutation among relatives. Our findings further support the hypothesis of a stabilizing effect of CCG interruptions on the mutational dynamics of the DM1 locus, also in intermediate DMPK alleles.

Polymeric Microporous Nanofilms as Smart Platforms for in Vitro Assessment of Nanoparticle Translocation and Caco-2 Cell Culture.

The study of nanomaterial translocation across epithelial barriers is often hindered by the low permeability of transwell membranes to nanoparticles. To address this issue ultra-thin poly(L-lactic acid) nanofilms with zero tortuosity micropores were developed for use in nanoparticle passage tests. In this study we demonstrate that microporous polymeric nanofilms allow a significantly higher passage of silver nanoparticles in comparison with commercial membranes normally used in transwell inserts. A reliable procedure for collecting free-standing nanofilms which enables their manipulation and use in lab-on-chip systems is described. We also demonstrate the cytocompatibility of porous nanofilms and their ability to sustain the adhesion and proliferation of Caco-2 cells. Ultra-thin microporous membranes show promise as low-cost nanomaterial screening tools and may be used as matrices for the development of bioengineered systems for mimicking the intestinal epithelium.

A bioreactor with an electro-responsive elastomeric membrane for mimicking intestinal peristalsis.

This study describes an actuated bioreactor which mimics the pulsatile contractile motion of the intestinal barrier using electro-responsive elastomers as smart materials that undergo deformation upon electrical stimulation. The device consists of an annular dielectric elastomer actuator working as a radial artificial muscle able to rhythmically contract and relax a central cell culture well. The bioreactor maintained up to 4 h of actuation at a frequency of 0.15 Hz and a strain of 8%-10%, to those of the cyclic contraction and relaxation of the small intestine. In vitro tests demonstrated that the device was biocompatible and cell-adhesive for Caco-2 cells, which formed a confluent monolayer following 21 days of culture in the central well. In addition, cellular adhesion and cohesion were maintained after 4 h of continuous cyclic strain. These preliminary results encourage further investigations on the use of dielectric elastomer actuation as a versatile technology that might overcome the limitations of commercially available pneumatic driving systems to obtain bioreactors that can cyclically deform cell cultures in a biomimetic fashion.