TMBcalc: a computational pipeline for identifying pan-cancer Tumor Mutational Burden gene signatures.

Background: In the precision medicine era, identifying predictive factors to select patients most likely to benefit from treatment with immunological agents is a crucial and open challenge in oncology. Methods: This paper presents a pan-cancer analysis of Tumor Mutational Burden (TMB). We developed a novel computational pipeline, TMBcalc, to calculate the TMB. Our methodology can identify small and reliable gene signatures to estimate TMB from custom targeted-sequencing panels. For this purpose, our pipeline has been trained on top of 17 cancer types data obtained from TCGA. Results: Our results show that TMB, computed through the identified signature, strongly correlates with TMB obtained from whole-exome sequencing (WES). Conclusion: We have rigorously analyzed the effectiveness of our methodology on top of several independent datasets. In particular we conducted a comprehensive testing on: (i) 126 samples sourced from the TCGA database; few independent whole-exome sequencing (WES) datasets linked to colon, breast, and liver cancers, all acquired from the EGA and the ICGC Data Portal. This rigorous evaluation clearly highlights the robustness and practicality of our approach, positioning it as a promising avenue for driving substantial progress within the realm of clinical practice.

Drug resistant epilepsies: A multicentre case series of steroid therapy.

PURPOSE: Our study aimed to evaluate the effectiveness of corticosteroids on seizure control in drug-resistant epilepsies (DREs). Our primary goal was to assess the response to steroids for various underlying etiologies, interictal electroencephalographic (EEG) patterns and electroclinical seizure descriptions. Our second goal was to compare steroid responsiveness to different treatment protocols. METHODS: This is a retrospective multicentre cohort study conducted according to the STROBE guidelines (Strengthening the Reporting of Observational Studies in Epidemiology). The following data were collected for each patient: epilepsy etiology, interictal EEG pattern, seizure types and type of steroid treatment protocol administered. RESULTS: Thirty patients with DRE were included in the study. After 6 months of therapy, 62.7 % of patients experienced reduced seizure frequency by 50 %, and 6.6 % of patients experienced complete seizure cessation. Findings associated with favourable response to steroids included structural/lesional etiology of epilepsy, immune/infectious etiology and focal interictal abnormalities on EEG. Comparing four different steroid treatment protocols, the most effective for seizure control was treatment with methylprednisolone at the dose of 30 mg/kg/day administered for 3 days, leading to greater than 50 % seizure reduction at 6 months in 85.7 % of patients. Treatment with dexamethasone 6 mg/day for 5 days decreased seizure frequency in 71.4 % of patients. Hydrocortisone 10 mg/kg administered for 3 months showed a good response to treatment in 71 %. CONCLUSIONS: In our study, two-thirds of patients with DRE experienced a significant seizure reduction following treatment with steroids. We suggest considering steroids as a potential therapeutic option in children with epilepsy not responding to conventional antiseizure medicines (ASM).

GLUT-1DS resistant to ketogenic diet: from clinical feature to in silico analysis. An exemplificative case report with a literature review.

Glucose transporter type 1 deficiency syndrome (GLUT-1DS) is characterized by alterations in glucose translocation through the blood-brain barrier (BBB) due to mutation involving the GLUT-1 transporter. The fundamental therapy is ketogenic diet (KD) that provide an alternative energetic substrate - ketone bodies that across the BBB via MCT-1 - for the brain. Symptoms are various and include intractable seizure, acquired microcephalia, abnormal ocular movement, movement disorder, and neurodevelopment delay secondary to an energetic crisis for persistent neuroglycopenia. KD is extremely effective in controlling epileptic seizures and has a positive impact on movement disorders and cognitive impairment. Cases of KD resistance are rare, and only a few of them are reported in the literature, all regarding seizure. Our study describes a peculiar case of GLUT-1DS due to a new deletion involving the first codon of SLC2A1 gene determining a loss of function with a resistance to KD admitted to hospital due to intractable episodes of dystonia. This patient presented a worsening of symptomatology at higher ketonemia values but without hyperketosis and showed a complete resolution of symptomatology while maintaining low ketonemia values. Our study proposes an in-silico genomic and proteomic analysis aimed at explaining the atypical response to KD exhibited by our patient. In this way, we propose a new clinical and research approach based on precision medicine and molecular modelling to be applied to patients with GLUT-1DS resistant to first-line treatment with ketogenic diet by in silico study of genetic and altered protein product.

Role of transcriptomic and genomic analyses in improving the comprehension of cefiderocol activity in Acinetobacter baumannii.

The mechanisms of action and resistance of cefiderocol (FDC) in Acinetobacter baumannii are still not fully elucidated, but iron transport systems have been evoked in its entry into the cell to reach the penicillin-binding proteins (PBPs). To capture the dynamics of gene expression related to FDC action in various conditions, we report on the genomic and transcriptomic features of seven A. baumannii strains with different FDC susceptibility, focusing on the variants in genes associated with ß-lactam resistance and the expression of the siderophore biosynthesis and transport systems acinetobactin and baumannoferrin. We also investigated the expression of the TonB energy transduction system (ETS) and siderophore receptors piuA and pirA. The four clinical samples belonged to the same clonal complex (CC2), and the two strains with the highest FDC MICs showed peculiar variants in PBP2 and ampC. Similarly, the two clinical strains with the lowest MICs shared variants in an outer membrane protein as well as ampC. Gene expression analyses highlighted the up-regulation of the acinetobactin and baumannoferrin genes in response to iron depletion and a down-regulation in the presence of high iron concentrations. In response to FDC, gene expression seemed strain-dependent, probably due to the different metabolic features of each strain. Overall, FDC activates the ETS, confirming the active import of the drug; baumannoferrin, more than acinetobactin, appeared stimulated by FDC in an iron-depleted medium. In conclusion, iron transport systems play a clear role in the FDC uptake, and their expression likely contributes to MIC variation together with ß-lactam resistance determinants.IMPORTANCEAcinetobacter baumannii poses a threat to healthcare due to its ability to give difficult-to-treat infections as a consequence of our shortage of antibiotic molecules active on this multidrug-resistant bacterium. Cefiderocol (FDC) represents one of the few drugs active on A. baumannii, and to preserve its activity, this study explored the transcriptomic and genomic features of seven strains with varying susceptibility to FDC. Transcriptomic analyses revealed the different effects of FDC on iron transport systems, promoting mainly baumannoferrin expression-thus more likely related to FDC entry-and the energy transduction systems. These findings suggest that not all iron transport systems are equally involved in FDC entry into A. baumannii cells. Finally, mutations in PBPs and ß-lactamases may contribute to the resistance onset. Overall, the study sheds light on the importance of iron availability and metabolic differences in FDC resistance, offering insights into understanding the evolution of resistance in A. baumannii strains.

Ketogenic Diet in Neonates with Drug-Resistant Epilepsy: Efficacy and Side Effects-A Single Center's Initial Experience.

BACKGROUND: For patients with pharmacoresistant epilepsy, a therapeutic option is ketogenic diet. Currently, data on young infants are scarce, particularly during hospitalization in the neonatal intensive care unit (NICU). OBJECTIVE: The aim of the present study was to evaluate the short-term (3-month) efficacy and side effects of ketogenic diet in infants with "drugs-resistant" epilepsy treated during NICU stay. METHODS: This retrospective study included infants aged under 2 months started on ketogenic diet during NICU hospitalization to treat drug-resistant epilepsy from April 2018 to November 2022. RESULTS: Thirteen term-born infants were included, three (23.1%) of whom were excluded because they did not respond to the ketogenic diet. Finally, we included 10 infants. Six (60%) patients took three antiepileptics before starting the ketogenic diet, while four (40%) took more drugs. Diet had a good response in four (40%) patients. In four patients, the ketogenic diet was suspended because of the onset of serious side effects. The emetic levels of sodium, potassium, and chlorine, pH, and onset of diarrhea, constipation, and gastroesophageal reflux showed significant differences. Ketonuria was higher and blood pH lower in the group that took more than three drugs than in the group taking fewer than three drugs. CONCLUSION: The ketogenic diet is efficacious and safe in infants, but the early and aggressive management of adverse reactions is important to improve the safety and effectiveness of the ketogenic treatment.

Epileptic spasms in infants: can video-EEG reveal the disease's etiology? A retrospective study and literature review.

Objective: Epileptic spasms are a type of seizure defined as a sudden flexion or extension predominantly of axial and/or truncal limb muscles that occur with a noticeable periodicity. Routine electroencephalogram supports the diagnosis of epileptic spasms, which can occur due to different causes. The present study aimed to evaluate a possible association between the electro-clinical pattern and the underlying etiology of epileptic spasms in infants. Materials and methods: We retrospectively reviewed the clinical and video-EEG data on 104 patients (aged from 1 to 22 months), admitted to our tertiary hospital in Catania and the tertiary hospital in Buenos Aires, from January 2013 to December 2020, with a confirmed diagnosis of epileptic spasms. We divided the patient sample into structural, genetic, infectious, metabolic, immune, and unknown, based on etiology. Fleiss' kappa (К) was used to assess agreement among raters in the electroencephalographic interpretation of hypsarrhythmia. A multivariate and bivariate analysis was conducted to understand the role of the different video-EEG variables on the etiology of epileptic spasms. Furthermore, decision trees were constructed for the classification of variables. Results: The results showed a statistically significant correlation between epileptic spasms semiology and etiology: flexor spasms were associated with spasms due to genetic cause (87.5%; OR < 1); whereas mixed spasms were associated with spasms from a structural cause (40%; OR < 1). The results showed a relationship between ictal and interictal EEG and epileptic spasms etiology: 73% of patients with slow waves and sharp waves or slow waves on the ictal EEG, and asymmetric hypsarrhythmia or hemi hypsarrhythmia on the interictal EEG, had spasms with structural etiology, whereas 69% of patients with genetic etiology presented typical interictal hypsarrhythmia with high-amplitude polymorphic delta with multifocal spike or modified hypsarrhythmia on interictal EEG and slow waves on the ictal EEG. Conclusion: This study confirms that video-EEG is a key element for the diagnosis of epileptic spasms, also playing an important role in the clinical practice to determine the etiology.

SARS-CoV-2 and Swabs: Disease Severity and the Numbers of Cycles of Gene Amplification, Single Center Experience.

Pediatric COVID-19 determines a mild clinical picture, but few data have been published about the correlation between disease severity and PCR amplification cycles of SARS-CoV-2 from respiratory samples. This correlation is clinically important because it permits the stratification of patients in relation to their risk of developing a serious disease. Therefore, the primary endpoint of this study was to establish whether disease severity at the onset, when evaluated with a LqSOFA score, correlated with the gene amplification of SARS-CoV-2. LqSOFA score, also named the Liverpool quick Sequential Organ Failure Assessment, is a pediatric score that indicates the severity of illness with a range from 0 to 4 that incorporates age-adjusted heart rate, respiratory rate, capillary refill and consciousness level (AVPU). The secondary endpoint was to determine if this score could predict the days of duration for symptoms and positive swabs. Our study included 124 patients aged between 0 and 18 years. The LqSOFA score was negatively correlated with the number of PCR amplification cycles, but this was not significant (Pearson's index -0.14, p-value 0.13). Instead, the correlation between the LqSOFA score and the duration of symptoms was positively related and statistically significant (Pearson's index 0.20, p-value 0.02), such as the correlation between the LqSOFA score and the duration of a positive swab (Pearson's index 0.40, p-value < 0.01). So, the LqSOFA score upon admission may predict the duration of symptoms and positive swabs; the PCR amplification of SARS-CoV-2 appears not to play a key role at onset in the prediction of disease severity.

Long-Term Survivors in a Cohort of People Living with HIV Diagnosed between 1985 and 1994: Predictive Factors Associated with More Than 25 Years of Survival.

Although the mortality rate among individuals diagnosed during the pre-Highly Active Antiretroviral Treatment era has been substantial, a considerable number of them survived. Our study aimed to evaluate the prevalence of HIV long-term survivors in a cohort of People Living with HIV diagnosed between 1985 and 1994 and to speculate about potential predictive factors associated to long survival. This is a retrospective single-center study. Subjects surviving more than 300 months (25 years) from HIV diagnosis were defined as Long Term Survivors. Overall, 210 subjects were enrolled. More than 75.24% of the included people living with HIV were males, with a median age of 28 years (IQR 25-34). The prevalent risk factors for HIV infection were injection drug use (47.62%), followed by unprotected sex among heterosexual individuals (23.81%). Ninety-three individuals (44.29%) could be defined as LTS with a median (IQR) survival of 333 (312-377) months. A hazard ratio of 12.45 (95% CI 7.91-19.59) was found between individuals who were exposed to Highly Active AntiRetroviral Treatment (HAART) and individuals who were not, with the latter being at greater risk of death. The availability and accessibility of effective antiretroviral therapy for people living with HIV remain the cornerstone of survival.

Streptococcus salivarius 24SMBc Genome Analysis Reveals New Biosynthetic Gene Clusters Involved in Antimicrobial Effects on Streptococcus pneumoniae and Streptococcus pyogenes.

Streptococcus salivarius 24SMBc is an oral probiotic with antimicrobial activity against the otopathogens Streptococcus pyogenes and Streptococcus pneumoniae. Clinical studies have reinforced its role in reducing the recurrence of upper respiratory tract infections (URTIs) and rebalancing the nasal microbiota. In this study, for the first time, we characterized 24SMBc by whole genome sequencing and annotation; likewise, its antagonistic activity vs. Streptococcus pneumoniae and Streptococcus pyogenes was evaluated by in vitro co-aggregation and competitive adherence tests. The genome of 24SMBc comprises 2,131,204 bps with 1933 coding sequences (CDS), 44 tRNA, and six rRNA genes and it is categorized in 319 metabolic subsystems. Genome mining by BAGEL and antiSMASH tools predicted three novel biosynthetic gene clusters (BGCs): (i) a Blp class-IIc bacteriocin biosynthetic cluster, identifying two bacteriocins blpU and blpK; (ii) an ABC-type bacteriocin transporter; and (iii) a Type 3PKS (Polyketide synthase) involved in the mevalonate pathway for the isoprenoid biosynthetic process. Further analyses detected two additional genes for class-IIb bacteriocins and 24 putative adhesins and aggregation factors. Finally, in vitro assays of 24SMBc showed significant anti-adhesion and co-aggregation effects against Streptococcus pneumoniae strains, whereas it did not act as strongly against Streptococcus pyogenes. In conclusion, we identified a novel blpU-K bacteriocin-encoding BGC and two class-IIb bacteriocins involved in the activity against Streptococcus pneumoniae and Streptococcus pyogenes; likewise the type 3PKS pathway could have beneficial effects for the host including antimicrobial activity. Furthermore, the presence of adhesins and aggregation factors might be involved in the marked in vitro activity of co-aggregation with pathogens and competitive adherence, showing an additional antibacterial activity not solely related to metabolite production. These findings corroborate the antimicrobial activity of 24SMBc, especially against Streptococcus pneumoniae belonging to different serotypes, and further consolidate the use of this strain in URTIs in clinical settings.

Genomic Landscape Alterations in Primary Tumor and Matched Lymph Node Metastasis in Hormone-Naïve Prostate Cancer Patients.

BACKGROUND: Prostate cancer (PCa) is a disease with a wide range of clinical manifestations. Up to the present date, the genetic understanding of patients with favorable or unfavorable prognosis is gaining interest for giving the appropriate tailored treatment. We aimed to investigate genetic changes associated with lymph node metastasis in a cohort of hormone-naïve Pca patients. METHODS: We retrospectively analyzed data from 470 patients who underwent surgery for PCa between 2010 and 2020 at the Department of Urology, University of Catania. Inclusion criteria were patients with lymph node metastasis and patients with PCa with extra capsular extension (pT3) and negative lymph node metastasis. The final cohort consisted of 17 different patients (11 PCa with lymph node metastasis and 6 PCa without lymph node metastasis). Through the cBioPortal online tool, we analyzed gene alterations and their correlations with clinical factors. RESULTS: A total of 688 intronic, synonym and nonsynonym mutations were sequenced. The gene with the most sequenced mutations was ERBB4 (83 mutations, 12% of 688 total), while the ones with the lower percentage of mutations were AKT1, FGFR2 and MLH1 (1 mutation alone, 0.14%). CONCLUSION: In the present study we found mostly concordance concerning the ERBB4 mutation between both primary PCa samples and matched lymph node metastasis, underlining that the identification of alterations in the primary tumor is extremely important for cancer prognosis prediction.

Acinetobacter baumannii and Cefiderocol, between Cidality and Adaptability.

Among the bacterial species included in the ESKAPE group, Acinetobacter baumannii is of great interest due to its intrinsic and acquired resistance to many antibiotics and its ability to infect different body regions. Cefiderocol (FDC) is a novel cephalosporin that is active against Gram-negative bacteria, with promising efficacy for A. baumannii infections, but some studies have reported therapeutic failures even in the presence of susceptible strains. This study aims to investigate the interactions between FDC and 10 A. baumannii strains with different susceptibilities to this drug. We confirmed diverse susceptibility profiles, with resistance values close to the EUCAST-proposed breakpoints. The minimal bactericidal concentration (MBC)/MIC ratios demonstrated bactericidal activity of the drug, with ratio values of ≤4 for all of the strains except ATCC 19606; however, bacterial regrowth was evident after exposure to FDC, as were changes in the shapes of colonies and bacterial cells. A switch to a nonsusceptible phenotype in the presence of high FDC concentrations was found in 1 strain as an adaptation mechanism implemented to overcome the cidal activity of this antibiotic, which was confirmed by the presence of heteroresistant, unstable subpopulations in 8/10 samples. Genomic analyses revealed the presence of mutations in penicillin-binding protein 3 (PBP3) and TonB3 that were shared by all of the strains regardless of their resistance phenotype. Because our isolates harbored ß-lactamase genes, ß-lactamase inhibitors showed the ability to restore the antimicrobial activity of FDC despite the different nonsusceptibility levels of the tested strains. These in vitro results support the concept of using combination therapy to eliminate drug-adapted subpopulations and regain full FDC activity in this difficult-to-treat species. IMPORTANCE This work demonstrates the underrated presence of Acinetobacter baumannii heteroresistant subpopulations after exposure of A. baumannii strains to FDC and its instability. Both A. baumannii and FDC are of great interest for the scientific community, as well as for clinicians; the former represents a major threat to public health due to its resistance to antibiotics, with related costs of prolonged hospitalization, and the latter is a novel, promising cephalosporin currently under the magnifying glass.

Soil and Soilless Tomato Cultivation Promote Different Microbial Communities That Provide New Models for Future Crop Interventions.

The cultivation of soilless tomato in greenhouses has increased considerably, but little is known about the assembly of the root microbiome compared to plants grown in soil. To obtain such information, we constructed an assay in which we traced the bacterial and fungal communities by amplicon-based metagenomics during the cultivation chain from nursery to greenhouse. In the greenhouse, the plants were transplanted either into agricultural soil or into coconut fiber bags (soilless). At the phylum level, bacterial and fungal communities were primarily constituted in all microhabitats by Proteobacteria and Ascomycota, respectively. The results showed that the tomato rhizosphere microbiome was shaped by the substrate or soil in which the plants were grown. The microbiome was different particularly in terms of the bacterial communities. In agriculture, enrichment has been observed in putative biological control bacteria of the genera Pseudomonas and Bacillus and in potential phytopathogenic fungi. Overall, the study describes the different shaping of microbial communities in the two cultivation methods.

Virus finding tools: current solutions and limitations.

MOTIVATION: The study of the Human Virome remains challenging nowadays. Viral metagenomics, through high-throughput sequencing data, is the best choice for virus discovery. The metagenomics approach is culture-independent and sequence-independent, helping search for either known or novel viruses. Though it is estimated that more than 40% of the viruses found in metagenomics analysis are not recognizable, we decided to analyze several tools to identify and discover viruses in RNA-seq samples. RESULTS: We have analyzed eight Virus Tools for the identification of viruses in RNA-seq data. These tools were compared using a synthetic dataset of 30 viruses and a real one. Our analysis shows that no tool succeeds in recognizing all the viruses in the datasets. So we can conclude that each of these tools has pros and cons, and their choice depends on the application domain. AVAILABILITY: Synthetic data used through the review and raw results of their analysis can be found at https://zenodo.org/record/6426147. FASTQ files of real data can be found in GEO (https://www.ncbi.nlm.nih.gov/gds) or ENA (https://www.ebi.ac.uk/ena/browser/home). Raw results of their analysis can be downloaded from https://zenodo.org/record/6425917.

Computational Resources for the Interpretation of Variations in Cancer.

A broad ecosystem of resources, databases, and systems to analyze cancer variations is present in the literature. These are a strategic element in the interpretation of NGS experiments. However, the intrinsic wealth of data from RNA-seq, ChipSeq, and DNA-seq can be fully exploited only with the proper skill and knowledge. In this chapter, we survey relevant literature concerning databases, annotators, and variant prioritization tools.

Antimicrobial properties of Lactobacillus cell-free supernatants against multidrug-resistant urogenital pathogens.

The healthy vaginal microbiota is dominated by Lactobacillus spp., which provide an important critical line of defense against pathogens, as well as giving beneficial effects to the host. We characterized L. gasseri 1A-TV, L. fermentum 18A-TV, and L. crispatus 35A-TV, from the vaginal microbiota of healthy premenopausal women, for their potential probiotic activities. The antimicrobial effects of the 3 strains and their combination against clinical urogenital bacteria were evaluated together with the activities of their metabolites produced by cell-free supernatants (CFSs). Their beneficial properties in terms of ability to interfere with vaginal pathogens (co-aggregation, adhesion to HeLa cells, biofilm formation) and antimicrobial activity mediated by CFSs were assessed against multidrug urogenital pathogens (S. agalactiae, E. coli, KPC-producing K. pneumoniae, S. aureus, E. faecium VRE, E. faecalis, P. aeruginosa, P. mirabilis, P. vulgaris, C. albicans, C. glabrata). The Lactobacilli tested exhibited an extraordinary ability to interfere and co-aggregate with urogenital pathogens, except for Candida spp., as well as to adhere to HeLa cells and to produce biofilm in the Lactobacillus combination. Lactobacillus CFSs and their combination revealed a strong bactericidal effect on the multidrug resistant indicator strains tested, except for E. faecium and E. faecalis. The antimicrobial activity was maintained after heat treatment but decreased after enzymatic treatment. All Lactobacilli showed lactic dehydrogenase activity and production of D- and L-lactic acid isomers on Lactobacillus CFSs, while only 1A-TV and 35A-TV released hydrogen peroxide and carried helveticin J and acidocin A bacteriocins. These results suggest that they can be employed as a new vaginal probiotic formulation and bio-therapeutic preparation against urogenital infections. Further, in vivo studies are needed to evaluate human health benefits in clinical situations.