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Abstract Background Methicillin-resistant Staphylococcus aureus (MRSA) is highly prevalent worldwide and can cause severe diseases. MRSA is associated with other antibiotic resistance. COVID-19 pandemic increased antimicrobial resistance in adult patients. Only a few data report the antimicrobial susceptibility of S. aureus in the Italian pediatric population, before and during the COVID-19 pandemic. Methods We included all the S. aureus positive samples with an available antibiogram isolated from pediatric patients (

Abstract Background Investigations on haematological alterations in paediatric COVID-19 have been focused mostly on lymphocytes and clotting profiles. Neutropenia has been occasionally reported and its course and impact on the disease have not been elucidated. The aim of this study was to describe the epidemiology, course, and impact of neutropenia in children with COVID-19 hospitalised in a tertiary care referral paediatric ward. Methods A single-centre retrospective study was conducted. Hospitalised children between 1 month and 18 years with confirmed COVID-19 and neutropenia were included and compared to non neutropenic patients. Complete blood picture with differential blood count, serum biochemistry, clotting profiles were performed; clinical data, length of hospitalisation, and prescription of drugs were collected. Results Twelve out of 95 patients (12.63%) with documented SARS-CoV-2 infection were neutropenic and met the inclusion criteria. The mean age was 161 days (range 38—490 days). The mean duration of symptoms in neutropenic children was 3.82 days, while the mean length of hospitalisation was 7.67 days. These findings were not significantly different in the two study groups. All patients had mild clinical manifestations and were discharged without sequelae. Conclusions We provided the first comprehensive study on neutropenia in mild paediatric COVID-19 infection. Our findings show that the main features of this haematological disorder in COVID-19 are analogous to the well-known transient benign neutropenia associated with other common viral infections. In our setting, neutropenia does not emerge as a potential negative prognostic factor in paediatric COVID-19.

Abstract Background Budding yeast, Saccharomyces cerevisiae, has been extensively favored as a model organism in aging and age-related studies, thanks to versatile microfluidic chips for cell dynamics assay and replicative lifespan (RLS) determination at single-cell resolution. However, previous microfluidic structures aiming to immobilize haploid yeast may impose excessive spatial constraint and mechanical stress on cells, especially for larger diploid cells that sprout in a bipolar pattern. Results We developed a high-throughput microfluidic chip for diploid yeast long-term culturing (DYLC), optical inspection and cell-aging analysis. The DYLC chip features 1100 “leaky bowl”-shaped traps formatted in an array to dock single cells under laminar-perfused medium and effectively remove daughter cells by hydraulic shear forces. The delicate microstructures of cell traps enable hydrodynamic rotation of newborn buds, so as to ensure bud reorientation towards downstream and concerted daughter dissection thereafter. The traps provide sufficient space for cell-volume enlargement during aging, and thus properly alleviate structural compression and external stress on budding yeast. Trapping efficiency and long-term maintenance of single cells were optimized according to computational fluid dynamics simulations and experimental characterization in terms of critical parameters of the trap and array geometries. Owing to the self-filling of daughter cells dissected from traps upstream, an initial trapping efficiency of about 70% can rapidly reach a high value of over 92% after 4-hour cell culturing. During yeast proliferation and aging, cellular processes of growth, budding and daughter dissection were continuously tracked for over 60 h by time-lapse imaging. Yeast RLS and budding time interval (BTI) were directly calculated by the sequential two-digit codes indicating the budding status in images. With the employed diploid yeast strain, we obtained an RLS of 24.29 ± 3.65 generations, and verified the extension of BTI in the first couple of generations after birth and the last several generations approaching death, as well as cell de-synchronization along diploid yeast aging. Conclusions The DYLC chip offers a promising platform for reliable capture and culturing of diploid yeast cells and for life-long tracking of cell dynamics and replicative aging processes so that grasping comprehensive insights of aging mechanism in complex eukaryotic cells. Graphical Abstract

Machine learning (ML) is widely used today, especially through deep neural networks (DNNs); however, increasing computational load and resource requirements have led to cloud-based solutions. To address this problem, a new generation of networks has emerged called spiking neural networks (SNNs), which mimic the behavior of the human brain to improve efficiency and reduce energy consumption. These networks often process large amounts of sensitive information, such as confidential data, and thus privacy issues arise. Homomorphic encryption (HE) offers a solution, allowing calculations to be performed on encrypted data without decrypting them. This research compares traditional DNNs and SNNs using the Brakerski/Fan-Vercauteren (BFV) encryption scheme. The LeNet-5 and AlexNet models, widely-used convolutional architectures, are used for both DNN and SNN models based on their respective architectures, and the networks are trained and compared using the FashionMNIST dataset. The results show that SNNs using HE achieve up to 40% higher accuracy than DNNs for low values of the plaintext modulus t, although their execution time is longer due to their time-coding nature with multiple time steps.

Abstract Introduction The incidence of acute respiratory tract infections (ARTIs) in children is difficult to estimate because they are typically treated in outpatient settings and the majority of epidemiological data originate from hospital settings and refer to the most severe illnesses. Therefore, the incidence of ARTIs in a real-world setting remains largely unexplored. Therefore, this study aims to estimate the incidence of ARTIs, upper respiratory tract infections (URTIs), and lower respiratory tract infections (LRTIs) in children aged 0–5 years in an outpatient setting. Methods This prospective cohort study was conducted in Lombardy, Italy, from October 1st, 2019, to March 31st, 2021, before and during the COVID-19 pandemic that began in March 2020. Caucasian healthy children aged 0–5 years were recruited from 69 Family Pediatricians (FP) and followed-up in an outpatient setting. Data were collected whenever a child was referred to FP and ARTI was diagnosed (Covid-19 related ARTI were excluded). The primary outcome was an estimate of the incidence of ARTIs. The incidence of ARTIs in different age groups and the effect of the COVID-19 pandemic on the incidence of ARTIs were secondary outcomes. Results We enrolled 484 children, 249 male (51.8%), mean age of 2.39 ± 1.68 years. The mean estimated incidence of ARTIs was 12.1/100 children × 30 days (95% CIs: 9.5–12.9), with the highest value observed in infants aged 1–12 months (24.9/100 children × 30 days; 95% CIs: 17.6–28.9). The mean estimated incidence of URTIs was higher than that of LRTIs (8.3 – CIs: 7.6–8.9 vs 3.8/100 children × 30 days – CIs: 6.4–4.3, respectively). The comparison of ARTIs, which occurred in the pre-pandemic winter, to those measured during the COVID-19 pandemic, revealed an impressive 82.1% drop in the incidence rate (CIs: 77.8–85.7). Conclusions This study showed that infants aged 1–12 months are more likely to develop ARTIs than older children and that COVID-19 pandemic has dramatically altered the epidemiology of ARTIs in children aged 0–5 years.

Neural Architecture Search (NAS) algorithms aim at finding efficient Deep Neural Network (DNN) architectures for a given application under given system constraints. DNNs are computationally-complex as well as vulnerable to adversarial attacks. In order to address multiple design objectives, we propose RoHNAS, a novel NAS framework that jointly optimizes for adversarial-robustness and hardware-efficiency of DNNs executed on specialized hardware accelerators. Besides the traditional convolutional DNNs, RoHNAS additionally accounts for complex types of DNNs such as Capsule Networks. For reducing the exploration time, RoHNAS analyzes and selects appropriate values of adversarial perturbation for each dataset to employ in the NAS flow. Extensive evaluations on multi - Graphics Processing Unit (GPU) - High Performance Computing (HPC) nodes provide a set of Pareto-optimal solutions, leveraging the tradeoff between the above-discussed design objectives. For example, a Pareto-optimal DNN for the CIFAR-10 dataset exhibits 86.07% accuracy, while having an energy of 38.63 mJ, a memory footprint of 11.85 MiB, and a latency of 4.47 ms.

Adversarial training is exploited to develop a robust Deep Neural Network (DNN) model against the malicious altered data. These attacks may have catastrophic effects on DNN models but are indistinguishable for a human being. For example, an external attack can modify an image adding noises invisible for a human eye, but a DNN model misclassifies the image. A key objective for developing robust DNN models is to use a learning algorithm that is fast but can also give model that is robust against different types of adversarial attacks. Especially for adversarial training, enormously long training times are needed for obtaining high accuracy under many different types of adversarial samples generated using different adversarial attack techniques. This paper aims at accelerating the adversarial training to enable fast development of robust DNN models against adversarial attacks. The general method for improving the training performance is the hyperparameters fine-tuning, where the learning rate is one of the most crucial hyperparameters. By modifying its shape (the value over time) and value during the training, we can obtain a model robust to adversarial attacks faster than standard training. First, we conduct experiments on two different datasets (CIFAR10, CIFAR100), exploring various techniques. Then, this analysis is leveraged to develop a novel fast training methodology, AccelAT, which automatically adjusts the learning rate for different epochs based on the accuracy gradient. The experiments show comparable results with the related works, and in several experiments, the adversarial training of DNNs using our AccelAT framework is conducted up to $2\times $ faster than the existing techniques. Thus, our findings boost the speed of adversarial training in an era in which security and performance are fundamental optimization objectives in DNN-based applications. To facilitate reproducible research this is the AccelAT open-source framework: https://github.com/Nikfam/AccelAT.

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in children is characterized by a wide variety of expressions ranging from asymptomatic to, rarely, critical illness. The basis of this variability is not yet fully understood. The aim of this study was to identify clinical and genetic risk factors predisposing to disease susceptibility and progression in children.MethodsWe enrolled 181 consecutive children aged less than 18 years hospitalized with or for SARS-CoV-2 infection during a period of 24 months. Demographic, clinical, laboratory, and microbiological data were collected. The development of coronavirus disease 2019 (COVID-19)-related complications and their specific therapies were assessed. In a subset of 79 children, a genetic analysis was carried out to evaluate the role of common COVID-19 genetic risk factors (chromosome 3 cluster; ABO-blood group system; FUT2, IFNAR2, OAS1/2/3, and DPP9 loci).ResultsThe mean age of hospitalized children was 5.7 years, 30.9% of them being under 1 year of age. The majority of children (63%) were hospitalized for reasons different than COVID-19 and incidentally tested positive for SARS-CoV-2, while 37% were admitted for SARS-CoV-2 infection. Chronic underlying diseases were reported in 29.8% of children. The majority of children were asymptomatic or mildly symptomatic; only 12.7% developed a moderate to critical disease. A concomitant pathogen, mainly respiratory viruses, was isolated in 53.3%. Complications were reported in 7% of children admitted for other reasons and in 28.3% of those hospitalized for COVID-19. The respiratory system was most frequently involved, and the C-reactive protein was the laboratory test most related to the development of critical clinical complications. The main risk factors for complication development were prematurity [relative risk (RR) 3.8, 95% confidence interval (CI) 2.4–6.1], comorbidities (RR 4.5, 95% CI 3.3–5.6), and the presence of coinfections (RR 2.5, 95% CI 1.1–5.75). The OAS1/2/3 risk variant was the main genetic risk factor for pneumonia development [Odds ratio (OR) 3.28, 95% CI 1-10.7; p value 0.049].ConclusionOur study confirmed that COVID-19 is generally less severe in children, although complications can develop, especially in those with comorbidities (chronic diseases or prematurity) and coinfections. Variation at the OAS1/2/3 genes cluster is the main genetic risk factor predisposing to COVID-19 pneumonia in children.

Omalizumab, which downregulates the immunoglobulin E (IgE) receptor site on plasmacytoid dendritic cells and thereby increases interferon-α (INF-α) production, may shorten the duration of viral infections by enhancing the antiviral immunity.A systematic review was conducted to investigate whether previous anti-IgE treatment with omalizumab could protect against SARS-CoV-2 disease (“COVID-19”) (infection, disease duration, and severity), and whether IFN-α upregulation could be involved. The research included articles published from March 2020 to January 2022. An accurate search was performed on bibliographic biomedical database (MEDLINE – Pubmed, SCOPUS, EMBASE, BIOMED CENTRAL, Google scholar, COCHRANE LIBRARY, ClinicalTrial.gov) including cohorts, case reports and reviews. Different methods were used, based on the study design, to assess the quality of eligible studies.Several authors link omalizumab to a possible protection against viruses, but they often refer to studies carried out before the pandemic and with viruses other than SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) (eg, rhinoviruses -RV). Few cases of COVID-19 patients treated with omalizumab have been recorded, and, in most of them, no increased susceptibility to severe disease was observed. According to these data, the current indication is to continue omalizumab therapy during the pandemic.Moreover, although omalizumab may enhance the antiviral immune response even for SARS-CoV-2, further studies are needed to confirm this hypothesis. It would be helpful to establish a registry of omalizumab-treated (or in treatment) patients who have developed COVID-19. Finally, randomized controlled trials could be able to demonstrate the effect of omalizumab in protecting against severe SARS-CoV-2, through IFN-α upregulation or other immunological pathways.

This study aims to observe the clinical characteristics and outcomes of recovered patients from Coronavirus Disease (COVID-19) related to the vaccination status. We examined results of 205 COVID-19-recovered patients from 15 December 2021 to 1 March 2022 in two hospitals of Local Health Authority of Alessandria (Italy) during the fourth pandemic wave. 77% of patients were hospitalized for acute respiratory failure (ARF) with radiological pneumonia pattern (recovered for COVID), 23% for other causes with occasional positivity finding (recovered with COVID). 32% of patients were not vaccinated for Sars-COV-2, 37% had three doses, 25% two doses, 5% only one dose. All patients without vaccination were hospitalized for ARF and they had a 7 times higher risk of hospitalization than the vaccinated. 60% of all patients had >3 comorbidities, of these 50% was vaccinated with three doses. In the fourth pandemic wave compared to the other not all patients were hospitalized for ARF and pneumonia and the presence of comorbidities >3 is a risk factor for hospitalization regardless of vaccination status. This justifies the administration of the fourth dose to frail patients.

Breast metastases are uncommon findings compared to primary breast cancer and in particular bilateral secondary breast lesions from neuroendocrine tumor (NET)s are extremely rare with just less over 13 cases described in literature. We reported herewith the case of a 54-year-old woman who presented to our Breast Unit after noticing multiple, mobile, bilateral breast lumps. Imaging studies confirmed the presence of multiple, circumscribed, bilateral breast masses with slightly spiculated margins, classified as suspicious for malignancy (BI-RADS 4). A tru-cut biopsy was carried out on the largest lesion of each side and histopathologic and immunohistochemistry examination was consistent with metastases from pancreatic neuroendocrine tumor (PNET). Total-body CT revealed the presence of a mass located in the pancreatic body - tail with associated abdominal lymphadenopathies and multiple secondary nodules in bilateral breast and in the liver. Stage IV disease was diagnosed, patient did not undergo surgery and started LAR – octreotide therapy. Although rare, breast metastases from NETs represent an important diagnostic challenge for practitioners because of the difficulty to differentiate from a primary breast carcinoma or even from benign breast lesions. Clinicians should be aware of the possibility of bilateral breast metastases in differential diagnosis of breast lesions in order to ensure the correct diagnosis and the most appropriate management of these patients.

The need for co-ordinate, high-level, and stable expression of multiple genes is essential for the engineering of biosynthetic circuits and metabolic pathways. This work outlines the functionality and design of IRES- and 2 A-peptide-based constructs by comparing different strategies for co-expression in polycistronic vectors. In particular, 2 A sequences are small peptides, mostly derived from viral polyproteins, that mediate a ribosome-skipping event such that several, different, separate proteins can be generated from a single open reading frame. When applied to metabolic engineering and synthetic gene circuits, 2 A peptides permit to achieve co-regulated and reliable expression of various genes in eukaryotic cells.

Ceftriaxone, a third-generation cephalosporin, is commonly used in pediatric patients and is generally well tolerated. Its more frequent adverse effects are biliary pseudolithiasis, urolithiasis, and hemolytic anemia. On the other hand, ceftriaxone-induced acute cholestatic hepatitis is a very rare condition, especially in children. Here, we describe a case of this condition in a young male child to highlight the importance of suspecting this drug-induced liver injury to achieve a prompt diagnosis.

Abstract The present study aimed to characterize clinical and molecular data of a large cohort of subjects with childhood-onset hereditary spastic paraplegias (HSPs). A multicenter historical cohort was performed at five centers in Brazil, in which probands and affected relatives' data from consecutive families with childhood-onset HSP (onset

Abstract CD157/BST-1 (a member of the ADP-ribosyl cyclase family) is expressed at variable levels in 97% of patients with acute myeloid leukemia (AML), and is currently under investigation as a target for antibody-based immunotherapy. We used peripheral blood and bone marrow samples from patients with AML to analyse the impact of CD157-directed antibodies in AML survival and in response to cytarabine (AraC) ex vivo. The study was extended to the U937, THP1 and OCI-AML3 AML cell lines of which we engineered CD157-low versions by shRNA knockdown. CD157-targeting antibodies enhanced survival, decreased apoptosis and reduced AraC toxicity in AML blasts and cell lines. CD157 signaling activated the PI3K/AKT/mTOR and MAPK/ERK pathways and increased expression of Mcl-1 and Bcl-XL anti-apoptotic proteins, while decreasing expression of Bax pro-apoptotic protein, thus preventing Caspase-3 activation. The primary CD157-mediated anti-apoptotic mechanism was Bak sequestration by Mcl-1. Indeed, the Mcl-1-specific inhibitor S63845 restored apoptosis by disrupting the interaction of Mcl-1 with Bim and Bak and significantly increased AraC toxicity in CD157-high but not in CD157-low AML cells. This study provides a new role for CD157 in AML cell survival, and indicates a potential role of CD157 as a predictive marker of response to therapies exploiting Mcl-1 pharmacological inhibition.

Abstract Recurrent respiratory infections (RRIs) are a common clinical condition in children, in fact about 25% of children under 1 year and 6% of children during the first 6 years of life have RRIs. In most cases, infections occur with mild clinical manifestations and the frequency of episodes tends to decrease over time with a complete resolution by 12 years of age. However, RRIs significantly reduce child and family quality of life and lead to significant medical and social costs. Despite the importance of this condition, there is currently no agreed definition of the term RRIs in the literature, especially concerning the frequency and type of infectious episodes to be considered. The aim of this consensus document is to propose an updated definition and provide recommendations with the intent of guiding the physician in the complex process of diagnosis, management and prevention of RRIs.

Este artículo fue pensado y escrito desde una ciudad mediterránea y universitaria que gestó, en 1918, la Reforma Universitaria que aún nos marca y nos permite hackear continuamente las verdades instaladas; estoy efectivamente escribiendo desde Córdoba, la Docta; más precisamente desde la Universidad Nacional de Córdoba. Sin embargo, nada de esto hubiera sido posible sin la invalorable mirada crítica y propositiva de un amigo colombiano, que a estas alturas podría ya ser colaborador directo de este artículo. Mi amigo es doctor, pero sobre todo es Alfredo. Imagino que a estas alturas ya todos saben que me refiero al Dr. Alfredo Gutiérrez Borrero. Con Alfredo hemos compartido textos, charlas y revisiones críticas de distintos borradores previos a este ensayo. Por eso lo menciono especialmente, convencida de la necesidad de la construcción colaborativa de miradas y revisiones críticas de conceptos y reflexiones que siempre enmarcan nuestros modos ser y estar en los lugares que ocupamos circunstancialmente.

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Abstract Background Acute hematogenous osteomyelitis (AHOM) is an insidious infection of the bone that more frequently affects young males. The etiology, mainly bacterial, is often related to the patient’s age, but it is frequently missed, owing to the low sensitivity of microbiological cultures. Thus, the evaluation of inflammatory biomarkers and imaging usually guide the diagnosis and follow-up of the infection. The antibiotic treatment of uncomplicated AHOM, on the other hand, heavily relies upon the clinician experience, given the current lack of national guidelines for the management of this infection. Methods A systematic review of the studies on the empirical treatment of uncomplicated AHOM in children published in English or Italian between January 1, 2009, and March 31, 2020, indexed on Pubmed or Embase search engines, was carried out. All guidelines and studies reporting on non-bacterial or complicated or post-traumatic osteomyelitis affecting newborns or children older than 18 years or with comorbidities were excluded from the review. All other works were included in this study. Results Out of 4576 articles, 53 were included in the study. Data on different topics was gathered and outlined: bone penetration of antibiotics; choice of intravenous antibiotic therapy according to the isolated or suspected pathogen; choice of oral antibiotic therapy; length of treatment and switch to oral therapy; surgical treatment. Conclusions The therapeutic management of osteomyelitis is still object of controversy. This study reports the first Italian consensus on the management of uncomplicated AHOM in children of pediatric osteomyelitis, based on expert opinions and a vast literature review.

Abstract Background Panton-Valentine leukocidin (PVL) is one of the major virulence factor of Staphylococcus aureus (SA) that might be associated with invasive life-threating infections. A prompt diagnosis and adequate treatment are essential in achieving the best outcome and avoiding serious sequelae. We describe a case of severe invasive PVL-SA infection in an infant. A literature review starting from 2010 was also performed in order to discuss clinical presentations, radiological findings, treatment and outcome. Case presentation This is a case of a 6-month-old boy who rapidly developed high fever and poor general condition. He was diagnosed as having multiple muscular abscesses, multiple foci of osteomyelitis and bloodstream infections caused by Panton-Valentine leukocidin Methicillin-resistant Staphylococcus aureus. He received intravenous antibiotics and surgical drainage of the abscess with progressive recovery. Conclusion Our report highlights the importance of improving awareness of this severe infection, as a prompt diagnosis and adequate manage is essential in order to save life and to prevent serious complications.

Abstract Background A few studies have suggested that the Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) was present in Northern Italy several weeks before its official detection on February 21, 2020. On the other hand, no clinical data have been provided so far to support such hypothesis. We investigated clinical-epidemiological evidence of SARS-CoV-2 infection among children and adults referring to emergency department (ED) in the main hospital of the center of Milan (Italy) before February 21, 2020. Methods A retrospective analysis of medical records of ED visits at the Fondazione Ca′ Granda Policlinico, Milan between January 11 and February 15 in 2017, 2018, 2019 and in 2020 was performed. The number of subjects referring with fever, cough or dyspnea was compared between the studied period of 2020 and the previous 3 years, by calculating a standardized referral ratio (SRR, number of observed cases in 2020 divided by the number of expected cases according to 2017–2019) and the corresponding 95% confidence interval (CI). Results In the pediatric ED, 7709 (average 2570/year) and 2736 patients were visited during the period 2017–2019 and in the 2020, respectively. Among adults, 13,465 (average 4488/year) and 4787 were visited during the period 2017–2019 and in the 2020, respectively. The SRR was 1.16 (95% CI 1.10–1.23) in children and 1.25 (95% CI 1.16–1.35) in adults. The ratio for the two (children and adults) SRRs was 0.93 (0.84–1.02), suggesting a trend towards a higher frequency in adults compared to children. Conclusions This study suggests that SARS-CoV-2 might have spread in Milan before February 21, 2020 with a minor trend among children.

Fungal laccase (Lac) has become a very useful biocatalyst in different industries, bio-refineries and, most importantly, bioremediation. Many reports have also linked hydrocarbon tolerance and degradation by various microorganisms with Lac secretion. In this study, Trametes trogii Lac (Ttlcc1) was engineered into Saccharomyces cerevisiae strain CEN.PK2–1 C under the constitutive GPD promoter (pGPD) for multi-fold synthesis with efficient hydrocarbon tolerance and degradation. Protein expression in heterologous hosts is strictly strain-specific, it can also be influenced by the synthetic design and culture conditions. We compared synthetic designs with different shuttle vectors for the yeast strains and investigated the best culture conditions by varying the pH, temperature, carbon, nitrogen sources, and CuSO4 amount. Two S. cerevisiae strains were built in this study: byMM935 and byMM938. They carry the transcription unit pGPD-Ttlcc1-CYC1t either inside the pRSII406 integrative plasmid (byMM935) or the pRSII426 multicopy plasmid (byMM938). The performance of these two synthetic strains were studied by comparing them to the wild-type strain (byMM584). Both byMM935 and byMM938 showed significant response to different carbon sources (glucose, galactose, lactose, maltose, and sucrose), nitrogen sources (NH4Cl, NH4NO3, KNO3, malt extract, peptone, and yeast extract), and solid state fermentation of different plant biomasses (bagasse, banana peels, corn cob, mandarin peels, and peanut shells). They performed best in optimized growth conditions with specific carbon and nitrogen sources, and a preferred pH in the range 3.5–4.5, temperature between 30 and 40 0C, and 1 mM CuSO4. In optimized yeast-growth medium, strain byMM935 showed the highest laccase activities of 1.621 ± 0.063 U/mL at 64 h, whereas byMM938 gave its highest activity (1.417 ± 0.055 U/mL) at 48 h. In this work, we established, by using Bushnell Hass synthetic medium, that the new Ttlcc1-yeast strains tolerated extreme pH and complex hydrocarbon mixture (CHM) toxicity. They degraded 60–90% of the key components in CHM within 48 h, including poly-cyclic aromatic hydrocarbons, alkyl indenes, alkyl tetralines, alkyl benzenes, alkyl biphenyls, and BTEX (Benzene, Toluene, Ethylbenzene, and Xylenes). This is the first report on the hydrocarbon degradation potential of a Ttlcc1-yeast. Compared to the native organism, such synthetic strains are better suited for meeting growing demands and have potentials for application in large-scale in situ bioremediation of hydrocarbon-polluted sites.

CRISPR–Cas systems provide powerful biological tools for genetic manipulation and gene expression regulation. Class 2 systems, comprising type II, type V, and type VI, have the significant advantage to require a single effector Cas protein (Cas9, Cas12, and Cas13 respectively) to cleave nucleic acids upon binding the crRNA. Both Cas9 and Cas12 recognize DNA and induce a double-strand break in it. In contrast, Cas13 bind and cleave RNA exclusively. However, some Cas9 homologs have shown RNase activity as well. Here, we harnessed Nme1Cas9, LwaCas13a, and RfxCas13d to carry out gene downregulation in Saccharomyces cerevisiae by triggering mRNA degradation. To avoid potential DNA damage, we mutated Nme1Cas9 into d16ANme1Cas9 that lost the nuclease activity of the RuvC domain but retained the active HNH domain, able to act on the target DNA strand and, therefore, on the corresponding transcript. Our results showed that d16ANme1Cas9 is a functional RNase in vivo, although with moderate activity since it provoked a fluorescence reduction from 21% to 32%. Interestingly, d16ANme1Cas9 works in a PAM-independent way nor demands helper PAMmer molecules. LwaCas13a and RfxCas13d appeared substantially unfunctional in S. cerevisiae, though they were shown to perform well in mammalian cells. To the best of our knowledge, this is the first report about the working in vivo of a variant of Nme1Cas9 as an RNase and the issues connected with the usage of Cas13 proteins in S. cerevisiae.

COVID-19 has proven to be particularly serious and life-threatening for patients presenting with pre-existing pathologies. Patients affected by rheumatic musculoskeletal disease (RMD) are likely to have impaired immune responses against SARS-CoV-2 infection due to their compromised immune system and the prolonged use of disease-modifying anti-rheumatic drugs (DMARDs), which include conventional synthetic (cs) DMARDs or biologic and targeted synthetic (b/ts) DMARDs. To provide an integrated analysis of the immune response following SARS-CoV-2 infection in RMD patients treated with different classes of DMARDs we carried out an immunological analysis of the antibody responses toward SARS-CoV-2 nucleocapsid and RBD proteins and an extensive immunophenotypic analysis of the major immune cell populations. We showed that RMD individuals under most DMARD treatments mount a sustained antibody response to the virus, with neutralizing activity. In addition, they displayed a sizable percentage of effector T and B lymphocytes. Among b-DMARDs, we found that anti-TNFα treatments are more favorable drugs to elicit humoral and cellular immune responses as compared to CTLA4-Ig and anti-IL6R inhibitors. This study provides a whole picture of the humoral and cellular immune responses in RMD patients by reassuring the use of DMARD treatments during COVID-19. The study points to TNF-α inhibitors as those DMARDs permitting elicitation of functional antibodies to SARS-CoV-2 and adaptive effector populations available to counteract possible re-infections.

Abstract Protease proprotein convertase subtilisin/kexin type 9 (PCSK9) is a regulator of LDL cholesterol clearance and has been associated with cardiovascular risk. PCSK9 inhibitors increase in vivo circulating endothelial progenitor cells (EPCs), a subtype of immature cells involved in ongoing endothelial repair. We hypothesized that the effect of PCSK9 on vascular homeostasis may be mediated by EPCs in patients with or without type 2 diabetes mellitus (T2DM). Eighty-two patients (45 with, 37 without T2DM) at high cardiovascular risk were enrolled in this observational study. Statin treatment was associated with higher circulating levels of PCSK9 in patients with and without T2DM (p

Abstract The 90K protein, also known as Mac-2 BP or LGALS3BP, can activate the immune response in part by increasing major histocompatibility (MHC) class I levels. In studies on a non-immune cell model, the rat FRTL-5 cell line, we observed that transforming growth factor (TGF)-β1, like γ-interferon (IFN), increased 90K levels, despite its immunosuppressive functions and the ability to decrease MHC class I. To explain this paradoxical result, we investigated the mechanisms involved in the TGF-β1 regulation of 90K expression with the aim to demonstrate that TGF-β1 utilizes different molecular pathways to regulate the two genes. We found that TGF-β1 was able to increase the binding of Upstream Stimulatory Factors, USF1 and USF2, to an E-box element, CANNTG, at −1926 to −1921 bp, upstream of the interferon response element (IRE) in the 90K promoter. Thyrotropin (TSH) suppressed constitutive and γ-IFN-induced 90K expression by decreasing USF binding to the E-box. TGF-β1 was able to overcome TSH suppression at the transcriptional level by increasing USF binding to the E-box. We suggest that the ability of TGF-β1 to increase 90K did not result in an increase in MHC class I because of a separate suppressive action of TGF-β1 directly on the MHC class I gene. We propose that the increased levels of 90K may play a role, rather than in immune response, in the context of the TGF-β1-induced changing of the cellular microenvironment that predisposes to cell motility and cancer progression. Consistently, analyzing the publicly available cancer patient data sets cBioPortal, we found that 90K expression directly correlated with TGF-β1 and USFs and that high levels of 90K were significantly associated with increased mortality in patients affected by different types of cancer.

Abstract Background Children often develop an asymptomatic form of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), but it is debated if children are at higher risk than adults to be asymptomatic carriers of SARS-CoV-2, especially during the school reopening. The main aim of this study was to investigate the frequency of SARS-CoV-2 asymptomatic carriers in children and adults during the reopening of the schools in Milan, Italy. Methods We conducted a cross-sectional study at the pediatric and adult Emergency Department (ED) of the Ca' Granda Ospedale Maggiore Policlinico (Milan) between October 1 and 31, 2020, i.e. 3 weeks after the reopening of schools. Patients admitted to the ED short stay observation and without any sign or symptom consistent with a SARS-CoV-2 were eligible. These patients underwent a nasopharyngeal swab specimen for the detection of SARS-CoV-2. The odds ratio and its 95% confidence interval (CI) was calculated to assess the risk of asymptomatically carrying the SARS-CoV-2 infection in children and adults. Results A total of 69 (27 females, median age 8.7 years) children and 251 (107 females, median age 71 years) adults were enrolled. Pediatric and adult subjects tested positive for SARS-CoV-2 with a similar frequency (1/69 [1.4%] vs 4/251 [1.6%]). Children had an odds ratio to be a carrier of 0.91 (CI 0.02– 9.38) compared to adults. Conclusions The frequency of asymptomatic SARS-CoV-2 carriers was similar among children and adults. Considering the emerging diffusion of new SARS-CoV-2 variants, the asymptomatic spread of SARS-CoV-2 infection among children and adults should be monitored.

Abstract Clinical data provided by the patient are not always reliable or could be difficult to collect. In this case, a difficult history collection resulted in a diagnostic delay. Major complications were avoided performing an urgent surgical intervention.

Abstract Background De novo pathogenic variants in the DDX3X gene are reported to account for 1–3% of unexplained intellectual disability (ID) in females, leading to the rare disease known as DDX3X syndrome (MRXSSB, OMIM #300958). Besides ID, these patients manifest a variable clinical presentation, which includes neurological and behavioral defects, and abnormal brain MRIs. Case presentation We report a 10-year-old girl affected by delayed psychomotor development, delayed myelination, and polymicrogyria (PMG). We identified a novel de novo missense mutation in the DDX3X gene (c.625C > G) by whole exome sequencing (WES). The DDX3X gene encodes a DEAD-box ATP-dependent RNA-helicase broadly implicated in gene expression through regulation of mRNA metabolism. The identified mutation is located just upstream the helicase domain and is suggested to impair the protein activity, thus resulting in the altered translation of DDX3X-dependent mRNAs. The proband, presenting with the typical PMG phenotype related to the syndrome, does not show other clinical signs frequently reported in presence of missense DDX3X mutations that are associated with a most severe clinical presentation. In addition, she has brachycephaly, never described in female DDX3X patients, and macroglossia, that has never been associated with the syndrome. Conclusions This case expands the knowledge of DDX3X pathogenic variants and the associated DDX3X syndrome phenotypic spectrum.

Late preterm infants constitute the largest subset of premature infants and are more likely to experience feeding issues leading to delayed oral feeding independence and low breastfeeding rates. Considering the increased parental concern about their infants’ nutrition and growth, we performed a literature review to provide an update on the feeding challenges faced by late preterm infants and the impact of these issues on maternal mental health and the mother–infant relationship. Based on our findings, late preterm infants have a high prevalence of feeding difficulties which need to be addressed by targeted support interventions to promote breastfeeding success and the establishment of a harmonious dyadic interaction between the mother and her infant, all of which contribute to the prevention of altered feeding behavior later in life. There is still a need for additional research to develop a standardized and shared strategy that can be proven to be effective. Should this be accomplished, it will be possible to offer appropriate support for mothers, encourage the oral skills and maturation of late preterm infants, and improve the relationship quality within the dyad.

Nowadays, the adoption of In Vitro Fertilization (IVF) techniques is undergoing an impressive increase. In light of this, one of the most promising strategies is the novel use of non-physiological materials and naturally derived compounds for advanced sperm preparation methods. Here, sperm cells were exposed during capacitation to MoS2/Catechin nanoflakes and catechin (CT), a flavonoid with antioxidant properties, at concentrations of 10, 1, 0.1 ppm. The results showed no significant differences in terms of sperm membrane modifications or biochemical pathways among the groups, allowing the hypothesis that MoS2/CT nanoflakes do not induce any negative effect on the parameters evaluated related to sperm capacitation. Moreover, the addition of CT alone at a specific concentration (0.1 ppm) increased the spermatozoa fertilizing ability in an IVF assay by increasing the number of fertilized oocytes with respect to the control group. Our findings open interesting new perspectives regarding the use of catechins and new materials obtained using natural or bio compounds, which could be used to implement the current strategies for sperm capacitation.

Abstract Background Hookworm infections (Necator americanus, Ancylostoma duodenale) are common in rural areas of tropical and subtropical countries. Human acquisition results from direct percutaneous invasion of infective larvae from contaminated soil. Overall, almost 472 million people in developing rural countries are infected. According to simulation models, hookworm disease has a global financial impact of over US$100 billion a year. Hookworm infection in newborn or infancy is rare, and most of the cases reported in literature are from endemic countries. Here, we describe the case of an infant with an Ancylostoma duodenale infection and review the literature currently available on this topic. Case presentation An Italian 2-month-old infant presented with vomit and weight loss. Her blood exams showed anemia and eosinophilia and stool analysis resulted positive for hookworms’ eggs, identified as Ancylostoma duodenale with real time-PCR. Parasite research on parents’ stools resulted negative, and since the mother travelled to Vietnam and Thailand during pregnancy, we assumed a transplacental transmission of the infection. The patient was treated successfully with oral Mebendazole and discharged in good conditions. Discussion Hookworm helminthiasis is a major cause of morbidity in children in the tropics and subtropics, but rare in developed countries. Despite most of the patients is usually asymptomatic, children are highly exposed to negative sequelae such as malnutrition, retarded growth and impaired cognitive development. In infants and newborns, the mechanism of infection remains unclear. Although infrequent, vertical transmission of larvae can occur through breastfeeding and transplacentally. Hookworm infection should be taken into account in children with abdominal symptoms and unexplained persistent eosinophilia. The treatment of infants infected by hookworm has potential benefit, but further studies are needed to define the best clinical management of these cases.

Abstract Background 22q11.2 deletion syndrome is one of the most common genomic disorders, characterized by the variable presence of facial dysmorphisms, congenital cardiac defects, velopharyngeal insufficiency/cleft palate, thymic hypoplasia/aplasia, immunodeficiency, parathyroid hypoplasia, developmental delay, learning disabilities, psychiatric disorders, renal, ocular, and skeletal malformations, hearing loss and laryngeal abnormalities. Chromosomal microarray (CMA) hybridization is one of the most performed diagnostic tests but as a genome wide analysis, it can point out relevant incidental copy number variations. Case presentation We report the case of a 2-year-old boy that came to our attention for mild psychomotor delay, poor growth, and minor facial anomalies. Considering a diagnosis of 22q11.2 deletion syndrome, we performed CMA that not only confirmed our diagnosis, but also pointed out an additional de novo 5q21.3q22.2 microdeletion, encompassing APC gene. As a result of the genetic testing we enrolled the patient in a tailored surveillance protocol that enabled the early detection of a hepatoblastoma. The child underwent surgical and chemotherapic treatments with complete cancer eradication. Conclusions The concurrent finding of an expected result and an additional deletion of APC gene represents an example of a relevant issue about the health and ethical management of secondary findings revealed by genome-wide tests. Furthermore, this report highlights the need to develop dedicated surveillance guidelines for children with APC-related polyposis and raise the question whether to suspect and screen for APC-related conditions in cases of sporadic hepatoblastomas.

Abstract Background Many fungi grow as saprobic organisms and obtain nutrients from a wide range of dead organic materials. Among saprobes, fungal species that grow on wood or in polluted environments have evolved prolific mechanisms for the production of degrading compounds, such as ligninolytic enzymes. These enzymes include arrays of intense redox-potential oxidoreductase, such as laccase, catalase, and peroxidases. The ability to produce ligninolytic enzymes makes a variety of fungal species suitable for application in many industries, including the production of biofuels and antibiotics, bioremediation, and biomedical application as biosensors. However, fungal ligninolytic enzymes are produced naturally in small quantities that may not meet the industrial or market demands. Over the last decade, combined synthetic biology and computational designs have yielded significant results in enhancing the synthesis of natural compounds in fungi. Main body of the abstract In this review, we gave insights into different protein engineering methods, including rational, semi-rational, and directed evolution approaches that have been employed to enhance the production of some important ligninolytic enzymes in fungi. We described the role of metabolic pathway engineering to optimize the synthesis of chemical compounds of interest in various fields. We highlighted synthetic biology novel techniques for biosynthetic gene cluster (BGC) activation in fungo and heterologous reconstruction of BGC in microbial cells. We also discussed in detail some recombinant ligninolytic enzymes that have been successfully enhanced and expressed in different heterologous hosts. Finally, we described recent advance in CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR associated) protein systems as the most promising biotechnology for large-scale production of ligninolytic enzymes. Short conclusion Aggregation, expression, and regulation of ligninolytic enzymes in fungi require very complex procedures with many interfering factors. Synthetic and computational biology strategies, as explained in this review, are powerful tools that can be combined to solve these puzzles. These integrated strategies can lead to the production of enzymes with special abilities, such as wide substrate specifications, thermo-stability, tolerance to long time storage, and stability in different substrate conditions, such as pH and nutrients.

The impact of Coronavirus disease 2019 (COVID-19) on the pediatric population is increasingly recognized. A widespread vaccination in childhood would provide benefits for children and might help ending the pandemic by enhancing community protection. Following recent approval by the European Medicines Agency (EMA) of Comirnaty (Pfizer-BioNTech) for children aged 5–11 years, we aimed to investigate caregivers' intention to vaccinate their children 98%). Three questionnaires were excluded due to compiling errors and 601 were included in the analysis. A total of 311 (51.7%) caregivers stated they would have their child vaccinated, 138 (23%) would refuse to vaccinate their child and 152 (25.3%) were unsure. The intention to vaccinate the child was higher in caregivers vaccinated against COVID-19, in those with a bachelor's degree or higher level of education, and in those with friends/acquaintances who became ill or died due to COVID-19. This study shows that increasing efforts are necessary to provide evidence-based tailored information to caregivers and to promote vaccination in this pediatric age group.