C. elegans PVF-1 inhibits permissive UNC-40 signalling through CED-10 GTPase to position the male ray 1 sensillum.

Graded distributions of netrin and semaphorin guidance cues convey instructive polarity information to migrating cells and growth cones, but also have permissive (i.e. non-polarity determining) functions in mammalian development and repair. The permissive functions of these cues are largely uncharacterised at a molecular level. We found previously that UNC-6 (netrin) signals permissively through UNC-40 (DCC) and UNC-5 receptors to prevent anterior displacement of the ray 1 sensillum in the C. elegans male tail. UNC-6/UNC-40 signalling functions in parallel with SMP-1 (semaporin 1)/PLX-1 (plexin) signalling to prevent this defect. Here, we report that a deletion allele of pvf-1, which encodes a VEGF-related protein, causes no ray 1 defects, but enhances ray 1 defects of a plx-1 mutant, and unexpectedly also suppresses unc-6(ev400)-null mutant ray 1 defects. These mutant ray 1 inductive and suppressive effects are mimicked by the ability of unc-40(+) and ced-10(gain-of-function) multi-copy transgene arrays to induce ray 1 defects or suppress unc-6 mutant ray 1 defects, depending on their dosage, suggesting the pvf-1 mutation causes UNC-40 overactivity that interferes with signalling but is partially sensitive to UNC-6. Additional data suggest PVF-1 functions through four VEGF receptor-related proteins and inhibits only CED-10 (a GTPase), but not MIG-2-dependent UNC-40 activity, even though UNC-40 functions through both GTPases to position ray 1. pvf-1 and receptor mutant ray 1 defects are rescued by transgenes expressing mouse VEGF164 and human VEGF receptors, respectively. These data report the first case of VEGF-induced inhibition of the netrin signalling and a molecular conservation of VEGF function from worms to humans.

FACILITATE: A real-world, multicenter, prospective study investigating the utility of a rapid, fully automated real-time PCR assay versus local reference methods for detecting epidermal growth factor receptor variants in NSCLC.

Accurate testing for epidermal growth factor receptor (EGFR) variants is essential for informing treatment decisions in non-small cell lung cancer (NSCLC). Automated diagnostic workflows may allow more streamlined initiation of targeted treatments, where appropriate, while comprehensive variant analysis is ongoing. FACILITATE, a real-world, prospective, multicenter, European study, evaluated performance and analytical turnaround time of the Idylla™ EGFR Mutation Test compared with local reference methods. Sixteen sites obtained formalin-fixed paraffin-embedded biopsy samples with ≥ 10% neoplastic cells from patients with NSCLC. Consecutive 5 µm sections from patient samples were tested for clinically relevant NSCLC-associated EGFR variants using the Idylla™ EGFR Mutation Test and local reference methods; performance (concordance) and analytical turnaround time were compared. Between January 2019 and November 2020, 1,474 parallel analyses were conducted. Overall percentage agreement was 97.7% [n = 1,418; 95% confidence interval (CI): 96.8-98.3], positive agreement, 87.4% (n = 182; 95% CI: 81.8-91.4) and negative agreement, 99.2% (n = 1,236; 95% CI: 98.5-99.6). There were 38 (2.6%) discordant cases. Ninety percent of results were returned with an analytical turnaround time of within 1 week using the Idylla™ EGFR Mutation Test versus ∼22 days using reference methods. The Idylla™ EGFR Mutation Test performed well versus local methods and had shorter analytical turnaround time. The Idylla™ EGFR Mutation Test can thus support application of personalized medicine in NSCLC.

Neutrophil-to-Lymphocyte Ratio Is a Major Prognostic Factor in Non-small Cell Lung Carcinoma Patients Undergoing First Line Immunotherapy With Pembrolizumab.

BACKGROUND/AIM: Lung cancer is one of the most common malignant neoplastic diseases and by far the leading cause of cancer death worldwide. Recently, immune checkpoint inhibitors (ICIs) have received increasing attention for playing a crucial role in non-small cell lung cancer (NSCLC). Biomarkers, such as programmed cell death-ligand 1 (PD-L1) and tumor mutational burden (TMB), seemed to be helpful in selecting patients who are more likely to benefit from ICI treatment: however, their role has not yet been fully clarified. PATIENTS AND METHODS: In this retrospective study, we evaluated the relationship between pre-treatment peripheral blood neutrophil-to-lymphocyte ratio (NLR) and survival in 252 patients suffering from advanced NSCLC who had received pembrolizumab as their first-line immunotherapy. RESULTS: Compared to their NLR low counterparts who had a median overall survival (OS) of 34.8 months, patients with NLRs above 4.8 had a median OS of 7.6 months (HR=3.26, 95%Cl=2.3-4.6, p-value<0.0000001). In multivariate Cox regression analysis, alongside other variables, such as metastatic sites, age, and sex, NLR and PD-L1 predicted progression-free survival and OS; furthermore, a very high NLR - over 10 - seemed to forecast a very dismal prognosis in patients undergoing immunotherapy, with sudden deaths in the days immediately following therapy (median OS=3.8 months). CONCLUSION: NLR acts as a valuable and reliable prognostic factor in non-small cell lung carcinoma patients undergoing first line immunotherapy with pembrolizumab. Additional investigation is necessary to fully elucidate the underlying biological rationale, which can be found in myeloid derived suppressor cells, a heterogeneous population of cells with neutrophil-like immunophenotypic features.

An in silico pipeline approach uncovers a potentially intricate network involving spike SARS-CoV-2 RNA, RNA vaccines, host RNA-binding proteins (RBPs), and host miRNAs at the cellular level.

BACKGROUND: In the last 2 years, we have been fighting against SARS-CoV-2 viral infection, which continues to claim victims all over the world. The entire scientific community has been mobilized in an attempt to stop and eradicate the infection. A well-known feature of RNA viruses is their high mutational rate, particularly in specific gene regions. The SARS-CoV-2 S protein is also affected by these changes, allowing viruses to adapt and spread more easily. The vaccines developed using mRNA coding protein S undoubtedly contributed to the "fight" against the COVID-19 pandemic even though the presence of new variants in the spike protein could result in protein conformational changes, which could affect vaccine immunogenicity and thus vaccine effectiveness. RESULTS: The study presents the findings of an in silico analysis using various bioinformatics tools finding conserved sequences inside SARS-CoV-2 S protein (encoding mRNA) same as in the vaccine RNA sequences that could be targeted by specific host RNA-binding proteins (RBPs). According to the results an interesting scenario emerges involving host RBPs competition and subtraction. The presence of viral RNA in cytoplasm could be a new tool in the virus's armory, allowing it to improve its chances of survival by altering cell gene expression and thus interfering with host cell processes. In silico analysis was used also to evaluate the presence of similar human miRNA sequences within RBPs motifs that can modulate human RNA expression. Increased cytoplasmic availability of exogenous RNA fragments derived from RNA physiological degradation could potentially mimic the effect of host human miRNAs within the cell, causing modulation of the host cell network. CONCLUSIONS: Our in silico analysis could aid in shedding light on the potential effects of exogenous RNA (i.e. viruses and vaccines), thereby improving our understanding of the cellular interactions between virus and host biomolecules. Finally, using the computational approach, it is possible to obtain a safety assessment of RNA-based vaccines as well as indications for use in specific clinical conditions.

In Silico Analysis of Possible Interaction between Host Genomic Transcription Factors (TFs) and Zika Virus (ZikaSPH2015) Strain with Combinatorial Gene Regulation; Virus Versus Host-The Game Reloaded.

In silico analysis is a promising approach for understanding biological events in complex diseases. Herein we report on the innovative computational workflow allowed to highlight new direct interactions between human transcription factors (TFs) and an entire genome of virus ZikaSPH2015 strain in order to identify the occurrence of specific motifs on a genomic Zika Virus sequence that is able to bind and, therefore, sequester host's TFs. The analysis pipeline was performed using different bioinformatics tools available online (free of charge). According to obtained results of this in silico analysis, it is possible to hypothesize that these TFs binding motifs might be able to explain the complex and heterogeneous phenotype presentation in Zika-virus-affected fetuses/newborns, as well as the less severe condition in adults. Moreover, the proposed in silico protocol identified thirty-three different TFs identical to the distribution of TFBSs (Transcription Factor Binding Sites) on ZikaSPH2015 strain, potentially able to influence genes and pathways with biological functions confirming that this approach could find potential answers on disease pathogenesis.

SARS-CoV-2 in Urine May Predict a Severe Evolution of COVID-19.

We hypothesized that the spread of SARS-CoV-2 in urine during a severe COVID-19 infection may be the expression of the worsening disease evolution. Therefore, the aim of this study was to verify if the COVID-19 disease severity is related to the viral presence in urine samples. We evaluated the clinical evolution in acute COVID-19 patients admitted in the sub-intensive care and intensive care units between 28 of December 2020 and 15th of February 2021 and being positive for SARS-CoV-2 RNA in the respiratory tract, including repeated endotracheal aspirates (ETA), sputum, nasopharyngeal swabs (NPS) and urine. We found that those subjects with SARS-COV-2 in the urine at admittance (8 out of 60 eligible patients) had a more severe disease than those with negative SARS-CoV-2 in urine. Further, they showed an increase in fibrinogen and (C-reactive Protein) CRP serum levels, requiring mechanic ventilation. Of those with positive SARS-CoV-2 in the urine, 50% died. According to our preliminary results, it seems that the presence of SARS-CoV-2 in the urine characterizes patients with a more severe disease and is also related to a higher death rate.

A SARS-CoV-2 host infection model network based on genomic human Transcription Factors (TFs) depletion.

In December 2019 a new beta-coronavirus was isolated and characterized by sequencing samples from pneumonia patients in Wuhan, Hubei Province, China. Coronaviruses are positive-sense RNA viruses widely distributed among different animal species and humans in which they cause respiratory, enteric, liver and neurological symptomatology. Six species of coronavirus have been described (HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1) that cause cold-like symptoms in immunocompetent or immunocompromised subjects and two strains of sometimes fatal zoonotic origin that cause severe acute respiratory syndrome (SARS-CoV and MERS-CoV). The SARS-CoV-2 strain is the emerging seventh member of the coronavirus family, which is actually determining a global emergency. In silico analysis is a promising approach for understanding biological events in complex diseases and due to serious worldwide emergency and serious threat to global health, it is extremely important to use bioinformatics methods able to study an emerging pathogen like SARS-CoV-2. Herein, we report on in silico comparative analysis between complete genome of SARS-CoV, MERS-CoV, HCoV-OC43 and SARS-CoV-2 strains, to identify the occurrence of specific conserved motifs on viral genomic sequences which should be able to bind and therefore induce a subtraction of host's Transcription Factors (TFs) which lead to a depletion, an effect comparable to haploinsufficiency (a genetic dominant condition in which a single copy of wild-type allele at a locus, in heterozygous combination with a variant allele, is insufficient to produce the correct quantity of transcript and, therefore, of protein, for a correct standard phenotypic expression). In this competitive scenario, virus versus host, the proposed in silico protocol identified the TFs same as the distribution of TFBSs (Transcription Factor Binding Sites) on analyzed viral strains, potentially able to influence genes and pathways with biological functions confirming that this approach could brings useful insights regarding SARS-CoV-2. According to our results obtained by this in silico approach it is possible to hypothesize that TF-binding motifs could be of help in the explanation of the complex and heterogeneous clinical presentation in SARS-CoV-2 and subsequently predict possible interactions regarding metabolic pathways, and drug or target relationships.

The C. elegans pvf-1 gene encodes a PDGF/VEGF-like factor able to bind mammalian VEGF receptors and to induce angiogenesis.

Members of the platelet-derived growth factor/vascular endothelial growth factor (PDGF/VEGF) family have been implicated in a variety of functions in vertebrates, especially angiogenesis. Here we identify and characterize a PDGF/VEGF-like factor (named PVF-1) from the nematode C. elegans. We show that PVF-1 has biochemical properties similar to vertebrate PDGF/VEGF growth factors. More important, PVF-1 binds to the human receptors VEGFR-1 (Flt-1) and VEGFR-2 (KDR) and is able to induce angiogenesis in two model systems derived from vertebrates. Our results highlight the widespread evolutionary conservation of this important class of growth factors and raise the possibility that C. elegans can provide a simple experimental system in which to investigate how these factors function.

Fatty acid profile of beef from immunocastrated (BOPRIVA(®)) Nellore bulls.

Twenty Nellore bulls (ABW=357.7±9.65kg) were divided into 2 groups: intact and immunocastrated - Bopriva®. After the trial period, the cattle were slaughtered and carcass fat thickness was evaluated, ether extract and fatty acid composition of the longissimus thoracis analyses were performed, and the activity indices of relevant enzymes were calculated. The means were calculated and compared by Student's t-test and Pearson's correlation coefficients (p<0.05). The immunocastrated group showed higher back fat thickness, ether extract, monounsaturated fatty acids (MUFAs), and activity index of Δ(9) desaturase C18 and lower polyunsaturated fatty acids (PUFAs) and n-6 fatty acids when compared to the intact group. The correlations between ether extract and the saturated and monounsaturated fatty acids were positive, and negative with polyunsaturated, n-3, n-6 and PUFA/SFA. Therefore, immunocastration may improve the fatty acid profile in the longissimus thoracis by increasing MUFAs, mainly oleic acid that is the most representative fatty acid in the meat and is considered beneficial to health.

Identification of a deletion in the NDUFS4 gene using array-comparative genomic hybridization in a patient with suspected mitochondrial respiratory disease.

We evaluated a patient, born after a normal 38-week pregnancy, with psychomotor retardation, poor coordination of ocular movements, recurrent vomiting and severe lactic acidosis. The patient was admitted to hospital at 2 months of age because of a mitochondrial-like syndrome and died at the age of 4.5 months. Array-comparative genomic hybridization (a-CGH) analysis revealed a homozygous deletion in 5q11.2 involving NADH dehydrogenase (ubiquinone) Fe-S protein 4, 18 kDa (NADH-coenzyme Q reductase; NDUFS4). Both parents were heterozygous for the mutation. The array revealed a deletion of ~32kb that includes exon 2 of NDUFS4 subsequently confirmed by real time-PCR and multiplex PCR. NDUFS4 was previously correlated to Leigh syndrome since mutations in this gene block the assembly of complex I. This result demonstrates the relevance of a-CGH screening in patients affected by metabolic disorders of unknown etiology.

Microduplications in 22q11.2 and 8q22.1 associated with mild mental retardation and generalized overgrowth.

The 22q11.2 microduplication is a genomic disorder, characterized from a variable phenotype ranging from different defects to normality. The most common microduplication of 22q11.2 is 3 Mb in size, but there are also cases reported with atypical duplications between 0.8 Mb and 6Mb. Here, we describe a case of a child with macrocephaly, overgrowth with advanced bone age, attention deficits, evidence of mild mental retardation and dysmorphic features. An array-CGH analysis detected a 252 Kb duplication at the 22q11.2 region inherited from mother and 142 Kb duplication at 8q22.1 region inherited from father. Both parents show mild dysmorphic features. The duplicated genes in chromosomes 22q and 8q are TOP3B and PGCP, respectively. We describe for the first time a patient carrying the smaller atypical 22q11.2 duplication who also presents with mild mental retardation and generalized overgrowth. This patient has an additional duplication in 8q22.1 which may act as a genomic modifier of its clinical phenotype.

Detection of the t(11;14)(q13;q32) without CCND1/IGH fusion in a case of acute myeloid leukemia.

The t(11;14)(q13;q32) is a hallmark of mantle cell lymphoma. It has been found less frequently in other lymphoproliferative disorders, such as B-prolymphocytic leukemia, plasma cell leukemia, chronic lymphocytic leukemia, and multiple myeloma. Here, we describe a patient with acute myeloid leukemia (AML), categorized as M5b according to French-American-British classification, in which conventional cytogenetic analysis revealed a karyotype with t(11;14)(q13;q32). Fluorescence in situ hybridization analyses demonstrated no rearrangement of the immunoglobulin heavy-chain (IGH) (14q32) locus as well as of the cyclin D1 (CCND1) gene, suggesting that this is not the typical t(11;14) resulting from the CCND1/IGH fusion. The changes in the 11q13 region have been described in both myeloid and lymphoid neoplasm with different chromosomes serving as donors in translocation, but to the best of our knowledge, never with the chromosome 14.

Placenta growth factor is not required for exercise-induced angiogenesis.

Angiogenesis is a tightly regulated process, both during development and adult life. Animal models with mutations in the genes coding for placental growth factor (PlGF), a member of vascular endothelial growth factor (VEGF) family, or the tyrosine kinase domain of the PlGF receptor (Flt-1) have revealed differences between normal physiological angiogenesis and pathological angiogenesis associated with conditions such as tumor growth, arthritis and atherosclerosis. In the present paper, we investigated the potential role of PlGF in regulating physiological angiogenesis by analyzing vascular changes in heart and skeletal muscles of wild-type and Plgf-/- mice following prolonged and sustained physical training. Sedentary Plgf-/- mice showed a reduced capillary density in both heart and skeletal muscles as compared to wild-type mice (P < 0.05). However, after a 6-week training period, heart/body weight ratio, citrate synthase activity, vessel density and capillary/myocyte ratio were significantly increased in both wild-type and Plgf-/- mice (all P < 0.05). At the same time intercapillary distance was significantly reduced. Finally, acute exercise was not associated with any change in PlGF protein level in the skeletal muscle. Our results demonstrate that PlGF is not necessary for exercise-training-induced angiogenesis. We thus suggest that the role of PlGF is confined to the selective regulation of angiogenesis only under pathological conditions.