Expression of alternatively spliced human T-cell leukemia virus type 1 mRNAs is influenced by mitosis and by a novel cis-acting regulatory sequence

Human T-cell leukemia virus type 1 (HTLV-1) expression depends on the concerted action of Tax, which drives transcription of the viral genome, and Rex, which favors expression of incompletely spliced mRNAs and determines a 2-phase temporal pattern of viral expression. In the present study, we investigated the Rex dependence of the complete set of alternatively spliced HTLV-1 mRNAs. Analyses of cells transfected with Rex-wild-type and Rex-knockout HTLV-1 molecular clones using splice site-specific quantitative reverse transcription (qRT)-PCR revealed that mRNAs encoding the p30Tof, p13, and p12/8 proteins were Rex dependent, while the p21rex mRNA was Rex independent. These findings provide a rational explanation for the intermediate-late temporal pattern of expression of the p30tof, p13, and p12/8 mRNAs described in previous studies. All the Rex-dependent mRNAs contained a 75-nucleotide intronic region that increased the nuclear retention and degradation of a reporter mRNA in the absence of other viral sequences. Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) analysis revealed that this sequence formed a stable hairpin structure. Cell cycle synchronization experiments indicated that mitosis partially bypasses the requirement for Rex to export Rex-dependent HTLV-1 transcripts. These findings indicate a link between the cycling properties of the host cell and the temporal pattern of viral expression/latency that might influence the ability of the virus to spread and evade the immune system

COVID-19 Is a Multifaceted Challenging Pandemic Which Needs Urgent Public Health Interventions

Until less than two decades ago, all known human coronaviruses (CoV) caused diseases so mild that they did not stimulate further advanced CoV research. In 2002 and following years, the scenario changed dramatically with the advent of the new more pathogenic CoVs, including Severe Acute Respiratory Syndome (SARS-CoV-1), Middle Eastern respiratory syndrome (MERS)-CoV, and the new zoonotic SARS-CoV-2, likely originated from bat species and responsible for the present coronavirus disease (COVID-19), which to date has caused 15,581,007 confirmed cases and 635,173 deaths in 208 countries, including Italy. SARS-CoV-2 transmission is mainly airborne via droplets generated by symptomatic patients, and possibly asymptomatic individuals during incubation of the disease, although for the latter, there are no certain data yet. However, research on asymptomatic viral infection is currently ongoing worldwide to elucidate the real prevalence and mortality of the disease. From a clinical point of view, COVID-19 would be defined as “COVID Planet “ because it presents as a multifaceted disease, due to the large number of organs and tissues infected by the virus. Overall, based on the available published data, 80.9% of patients infected by SARS-CoV-2 develop a mild disease/infection, 13.8% severe pneumonia, 4.7% respiratory failure, septic shock, or multi-organ failure, and 3% of these cases are fatal, but mortality parameter is highly variable in dfferent countries. Clinically, SARS-CoV-2 causes severe primary interstitial viral pneumonia and a “cytokine storm syndrome”, characterized by a severe and fatal uncontrolled systemic inflammatory response triggered by the activation of interleukin 6 (IL-6) with development of endothelitis and generalized thrombosis that can lead to organ failure and death. Risk factors include advanced age and comorbidities including hypertension, diabetes, and cardiovascular disease. Virus entry occurs via binding the angiotensin-converting enzyme 2 (ACE2) receptor present in almost all tissues and organs through the Spike (S) protein. Currently, SARS-CoV-2 infection is prevented by the use of masks, social distancing, and improved hand hygiene measures. This review summarizes the current knowledge on the main biological and clinical features of the SARS-CoV-2 pandemic, also focusing on the principal measures taken in some Italian regions to face

Investigation of Metabolic Heterogeneity and Clonal Selection Driven by anti-VEGF therapy in Ovarian Cancer

The process of neoplastic transformation is associated with profound metabolic changes, including the widely studied dysregulated glucose metabolism. It is also increasingly recognized that tumors are metabolically heterogeneous, including both inter- and intra-tumor metabolic heterogeneity. However, whether this phenomenon depends on the existence of sub-populations endowed with different metabolic features or, rather, local modulation of the metabolism associated with microenvironment factors, such as hypoxia, has less been investigated. Along this line, we recently reported that anti-VEGF therapy (Bevacizumab) induces a stable metabolic change in epithelial ovarian cancer (EOC) xenografts that correlates with resistance to anti-angiogenic therapy. Aim of this Project is (I) to investigate whether metabolic heterogeneity exists at the clonal level in cancer cells; (II) to investigate the metabolic profile associated with this phenomenon and (III) establish whether anti-angiogenic therapy might shew tumor metabolism, leading to selection of metabolic variants poorly represented in the original tumor (IV) and whether it is possible to treat the resistant cell population with drugs targeting their key metabolic features. To achieve these aims, we initially isolated by limiting dilution several (n=10-35) clones from established ovarian cancer cell lines previously characterized for their glycolytic activity. Indeed, IGROV-1 and SKOV3 cells are prototypes of poorly glycolytic cells, whereas OC316 cells are highly glycolytic cells. OAW42, A2780 and A2774 ovarian cancer cell lines showed an intermediate glycolytic profile compared to IGROV1 and OC316 cells, according to measurements of glucose consumption and lactate production in vitro. We speculated that highly glycolytic cells could be relatively glucose addicted and hence tolerate less glucose starvation, compared with poorly glycolytic cells. To test this hypothesis, we cultivated clones either in the presence or in the absence of glucose (2 g/l) in the medium. Following 1-2 days in culture, clones were scored by optical microscopy. We used the acronym GDS to refer to a Glucose Deprivation Sensitive clone or GDR to indicate a clone relatively Resistant to Glucose Deprivation. Results showed that OC316-derived clones were exclusively of the GDS type, whereas IGROV-1 and SKOV3 cancer cell lines included both GDR (55%) and GDS (45%) clones. OAW42 cells were composed by 68% of GDR and 32% of GDS clones, A2780 and A2774 cells showed high enrichment in GDR clones. The GDR/GDS phenotype was substantially stable when clones were analyzed over separate weeks. Moreover, GDS clones did not express higher levels of transcripts link with glycolysis pathway or elevate lactate production compared with GDR clones. We performed a Seahorse analysis and we observed that Oxigen Consumption Rate was higher in GDR compared to GDS clones. Metabolomic analysis showed that the majority of pathways differentially active in GDS versus GDR clones were linked to mitochondrial functions. Unexpectedly, we could not discriminate GDS clones for their glycolytic activity, but we noticed an altered mitochondria pathway in GDR group compared to GDS clones. We also evaluated the modulation of different pathways in GDR and GDS clones under glucose starvation. Next, to investigate whether anti-angiogenic therapy would perturb the GDS/GDR ratio, we isolated GDS and GDR clones from ex vivo cultures of Control and Bevacizumab-treated IGROV-1 tumors. In the case of clones derived from Control tumors, the percentage of GDR and GDS clones was similar to that found in the parental IGROV-1 cell line. Cultures derived from Bevacizumab-treated tumors had an enrichment in GDR clones, suggesting that anti-VEGF therapy might perturb metabolic heterogeneity in tumors.Il processo di trasformazione neoplastica è associato ad un profondo cambiamento metabolico che comprende anche l’ampiamente studiata modulazione del metabolismo del glucosio. È inoltre sempre più apprezzato il fatto che i tumori sono metabolicamente eterogenei, compresa l'eterogeneità metabolica intra- e inter-tumorale. Tuttavia, non è ancora stato ampiamente studiato se questo fenomeno dipenda dall’esistenza di sottopopolazioni tumorali dotate di caratteristiche metaboliche diverse oppure da modulazioni locali del metabolismo associato a fattori del microambiente, come per esempio l’ipossia. In base a queste osservazioni, abbiamo recentemente dimostrato che la terapia che prevede l’utilizzo di un anticorpo monoclonale anti-VEGF, Bevacizumab, induce un cambiamento metabolico stabile in xenotrapianti di cancro ovarico epiteliale che correla con un’aumentata aggressività tumorale e la resistenza alla terapia anti-angiogenica. Lo scopo di questo progetto è quello di indagare se esiste eterogeneità a livello clonale in colture di cellule tumorali e di studiare l’aspetto metabolico associato a questo fenomeno. Abbiamo voluto stabilire se alcune terapie mirate possano modificare il metabolismo tumorale, grazie alla selezione di varianti metaboliche scarsamente rappresentate nel tumore originale e quando sia possibile, trattare queste cellule tumorali con un inibitore diverso dal Bevacizumab. Per raggiungere questi obiettivi, abbiamo inizialmente isolato, usando diluizioni seriali, diversi cloni (n = 10-35) da linee cellulari di cancro ovarico, precedentemente caratterizzate per la loro attività glicolitica. Infatti, le cellule IGROV-1 e SKOV3 sono prototipi di cellule scarsamente glicolitiche, mentre le cellule OC316 sono altamente glicolitiche. Le linee cellulari di cancro ovarico OAW42, A2780 e A2774 hanno mostrato un profilo glicolitico intermedio rispetto alle cellule IGROV-1 e OC316, in accordo con le misure della produzione di lattato e del consumo del glucosio in vitro. Abbiamo ipotizzato che cellule altamente glicolitiche potrebbero essere relativamente dipendenti dal glucosio e quindi meno tolleranti se nel terreno di coltura manca questo nutriente, rispetto alle cellule scarsamente glicolitiche. Per verificare questa ipotesi, abbiamo coltivato i cloni in presenza o in assenza di glucosio (2 g/l) nel terreno di coltura. Dopo 72h in cui i cloni sono stati sottoposti a deprivazione di glucosio, sono stati valutati attraverso la microscopia ottica. Abbiamo usato l'acronimo GDS per riferirci ad un clone sensibile alla deprivazione di glucosio e GDR per indicare un clone relativamente resistente alla deprivazione di glucosio. I risultati hanno mostrato che i cloni derivanti dalla coltura cellulare OC316 erano esclusivamente del tipo GDS, mentre nelle linee cellulari IGROV-1 e SKOV3 erano presenti entrambi i due sottogruppi di cloni GDR (55%) e cloni GDS (45%). La linea cellulare OAW42 era composta per il 68% da cloni GDR e per il 32% da cloni GDS, invece le linee A2780 e A2774 hanno mostrato un alto arricchimento nella % di cloni GDR. Il fenotipo GDR/GDS è sostanzialmente stabile anche dopo settimane di analisi. Inoltre, abbiamo osservato che i cloni GDS non esprimono livelli più elevati di trascritti collegati con la via glicolitica e neanche livelli più elevati di produzione di lattato rispetto ai cloni GDR. Abbiamo effettuato un'analisi con la metodica del Seahorse e abbiamo osservato che il tasso di consumo di ossigeno era aumentato nel gruppo dei GDR rispetto ai cloni GDS. L'analisi metabolomica ha mostrato che la maggior parte delle vie diversamente regolate era legata alla funzione mitocondriale nei cloni GDR. Inaspettatamente, non abbiamo potuto discriminare i cloni GDS per la loro attività glicolitica ma abbiamo notato una possibile alterazione dell’attività mitocondriale nel gruppo dei cloni GDR rispetto ai cloni GDS. Abbiamo anche valutato la modulazione di diverse vie metaboliche nei cloni GDR e GDS in condizioni di deprivazione di glucosio. Successivamente, abbiamo isolato i cloni GDR e GDS da colture cellulari ex vivo derivanti da tumori IGROV-1 trattati con Bevacizumab o di controllo. Abbiamo indagato se la terapia anti-angiogenica porta ad un’alterazione del rapporto GDR/GDS. Nel caso dei tumori di controllo, la percentuale dei cloni GDR e GDS isolati, era simile a quella trovata nelle cellule parentali IGROV-1. Le colture cellulari derivate da tumori trattati con Bevacizumab hanno mostrato un arricchimento nel numero di cloni GDR. Questo può suggerire che la terapia anti-VEGF perturba l’eterogeneità metabolica tumorale

Bioactive Materials for Soft Tissue Repair

Over the past decades, age-related pathologies have increased abreast the aging population worldwide. The increased age of the population indicates that new tools, such as biomaterials/scaffolds for damaged tissues, which display high efficiency, effectively and in a limited period of time, for the regeneration of the body’s tissue are needed. Indeed, scaffolds can be used as templates for three-dimensional tissue growth in order to promote the tissue healing stimulating the body’s own regenerative mechanisms. In tissue engineering, several types of biomaterials are employed, such as bioceramics including calcium phosphates, bioactive glasses, and glass–ceramics. These scaffolds seem to have a high potential as biomaterials in regenerative medicine. In addition, in conjunction with other materials, such as polymers, ceramic scaffolds may be used to manufacture composite scaffolds characterized by high biocompatibility, mechanical efficiency and load-bearing capabilities that render these biomaterials suitable for regenerative medicine applications. Usually, bioceramics have been used to repair hard tissues, such as bone and dental defects. More recently, in the field of soft tissue engineering, this form of scaffold has also shown promising applications. Indeed, soft tissues are continuously exposed to damages, such as burns or mechanical traumas, tumors and degenerative pathology, and, thereby, thousands of people need remedial interventions such as biomaterials-based therapies. It is known that scaffolds can affect the ability to bind, proliferate and differentiate cells similar to those of autologous tissues. Therefore, it is important to investigate the interaction between bioceramics and somatic/stem cells derived from soft tissues in order to promote tissue healing. Biomimetic scaffolds are frequently employed as drug-delivery system using several therapeutic molecules to increase their biological performance, leading to ultimate products with innovative functionalities. This review provides an overview of essential requirements for soft tissue engineering biomaterials. Data on recent progresses of porous bioceramics and composites for tissue repair are also presented

NLRP1 polymorphisms in patients with asbestos-associated mesothelioma

An increasing incidence of malignant mesothelioma (MM) cases in patients with low levels of asbestos exposure suggests the interference of alternative cofactors. SV40 infection was detected, as co-morbidity factor, only in 22% of asbestos-MM patients from a North-Eastern Italy area. An additional mechanism of injury related to asbestos exposure in MM development has been recently associated to inflammatory responses, principally driven by interleukin (IL)-1 beta (Ss) activated within the inflammasome complex. NLRP3 inflammosome has been described as the intracellular sensor for asbestos able to induce inflammasome activation and IL-1ss secretion while NLRP1 is expressed in lung epithelial cells and alveolar macrophages and contributes to the immune response and to survival/apoptosis balance. This study proposes to evaluate the impact of known NLRP3 and NLRP1 polymorphisms in the individual susceptibility to asbestos-induced mesothelioma in subjects from a hyperendemic area for MM. Methods 134 Italian patients with diagnosis of mesothelioma due (MMAE, n=69) or not (MMAF, n=65) to asbestos, 256 healthy Italian blood donors and 101 Italian healthy subjects exposed to asbestos (HCAE) were genotyped for NLRP1 (rs2670660 and rs12150220) and NLRP3 (rs35829419 and rs10754558) polymorphisms. Results While NLRP3 SNPs were not associated to mesothelioma, the NLRP1 rs12150220 allele T was significantly more frequent in MMAE (0.55) than in HCAE (0.41) (p=0.011; OR=1.79) suggesting a predisponent effect of this allele on the development of mesothelioma. This effect was amplified when the NLRP1 rs2670660 allele was combined with the NLRP1 rs12150220 allele (p=0.004; OR=0.52). Conclusion Although NLRP3 SNPs was not involved in mesothelioma predisposition, these data proposed NLRP1 as a novel factor possibly involved in the development of mesothelioma

SARS-CoV-2 Infection: New Molecular, Phylogenetic, and Pathogenetic Insights. Efficacy of Current Vaccines and the Potential Risk of Variants

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a newly discovered coronavirus responsible for the coronavirus disease 2019 (COVID-19) pandemic. COVID-19 has rapidly become a public health emergency of international concern. Although remarkable scientific achievements have been reached since the beginning of the pandemic, the knowledge behind this novel coronavirus, in terms of molecular and pathogenic characteristics and zoonotic potential, is still relatively limited. Today, there is a vaccine, or rather several vaccines, which, for the first time in the history of highly contagious infectious diseases that have plagued mankind, has been manufactured in just one year. Currently, four vaccines are licensed by regulatory agencies, and they use RNA or viral vector technologies. The positive effects of the vaccination campaign are being felt in many parts of the world, but the disappearance of this new infection is still far from being a reality, as it is also threatened by the presence of novel SARS-CoV-2 variants that could undermine the effectiveness of the vaccine, hampering the immunization control efforts. Indeed, the current findings indicate that SARS-CoV-2 is adapting to transmission in humans more efficiently, while further divergence from the initial archetype should be considered. In this review, we aimed to provide a collection of the current knowledge regarding the molecular, phylogenetic, and pathogenetic insights into SARS-CoV-2. The most recent findings obtained with respect to the impact of novel emerging SARS-CoV-2 variants as well as the development and implementation of vaccines are highlighted

Rewiring of Lipid Metabolism and Storage in Ovarian Cancer Cells after Anti-VEGF Therapy

Anti-angiogenic therapy triggers metabolic alterations in experimental and human tumors, the best characterized being exacerbated glycolysis and lactate production. By using both Liquid Chromatography-Mass Spectrometry (LC-MS) and Nuclear Magnetic Resonance (NMR) analysis, we found that treatment of ovarian cancer xenografts with the anti-Vascular Endothelial Growth Factor (VEGF) neutralizing antibody bevacizumab caused marked alterations of the tumor lipidomic profile, including increased levels of triacylglycerols and reduced saturation of lipid chains. Moreover, transcriptome analysis uncovered up-regulation of pathways involved in lipid metabolism. These alterations were accompanied by increased accumulation of lipid droplets in tumors. This phenomenon was reproduced under hypoxic conditions in vitro, where it mainly depended from uptake of exogenous lipids and was counteracted by treatment with the Liver X Receptor (LXR)-agonist GW3965, which inhibited cancer cell viability selectively under reduced serum conditions. This multi-level analysis indicates alterations of lipid metabolism following anti-VEGF therapy in ovarian cancer xenografts and suggests that LXR-agonists might empower anti-tumor e ects of bevacizumab

Dairy product intake and mortality in a cohort of 70-year-old Swedes : a contribution to the Nordic diet discussion

INTRODUCTION: Conflicting results in the literature exist on the role of dairy products in the context of a Nordic Healthy Diet (NHD). Two recent Swedish studies indicate both negative and positive associations with total mortality when comparing key dairy products. There is no consensus about how to include these foods into the NHD. PURPOSE: To study consumption of cheese and milk products (milk, sour milk and unsweetened yoghurt) by 70-year-old Swedes in relation to all-cause mortality. METHODS: Cox proportional hazard models, adjusted for potential confounders and stratified by follow-up duration, were used to assess the prediction of all-cause mortality by the above foods. The associations of fat from cheese and milk products with mortality were tested in separate models. RESULTS: Cheese intake inversely predicted total mortality, particularly at high protein intakes, and this association decreased in strength with increasing follow-up time. Milk products predicted increased mortality with stable HRs over follow-up. The association between milk products and mortality was strongly influenced by the group with the highest consumption. Fat from cheese mirrored the protective association of cheese intake with mortality, whereas fat from milk products predicted excess mortality, but only in an energy-adjusted model. CONCLUSION: Based on our results, it may be argued that the role of dairy products in the context of a Nordic healthy diet should be more clearly defined by disaggregating cheese and milk products and not necessarily focusing on dairy fat content. Future epidemiological research should consider dairy products as disaggregated food items due to their great diversity in health properties

Investigation on Silent Bacterial Infections in Specimens from Pregnant Women Affected by Spontaneous Miscarriage.

Miscarriage is one of the main complications occurring in pregnancy. The association between adverse pregnancy outcomes and silent bacterial infections has been poorly investigated. Ureaplasma parvum and urealiticum, Mycoplasma genitalium and hominis and Chlamydia trachomatis DNA sequences have been investigated by polymerase chain reaction (PCR) methods in chorionic villi tissues and peripheral blood mononuclear cells (PBMCs) from females with spontaneous abortion (SA, n = 100) and females who underwent voluntary interruption of pregnancy (VI, n=100). U. parvum DNA was detected in 14% and 15% of SA and VI, respectively, with a mean of bacterial DNA load of 1.3 × 10−1 copy/cell in SA and 2.8 × 10−3 copy/cell in VI; U. urealiticum DNA was detected in 3% and 2% of SA and VI specimens, respectively, with a mean DNA load of 3.3 × 10−3 copy/cell in SA and 1.6 × 10−3 copy/cell in VI; M. hominis DNA was detected in 5% of SA specimens with a DNA load of 1.3 × 10−4 copy/cell and in 6% of VI specimens with a DNA load of 1.4×10−4 copy/cell; C. trachomatis DNA was detected in 3% of SA specimens with a DNA load of 1.5 × 10−4 copy/cell and in 4% of VI specimens with a mean DNA load of 1.4 × 10−4 copy/cell. In PBMCs from the SA and VI groups, Ureaplasma spp, Mycoplasma spp and C. trachomatis DNAs were detected with a prevalence of 1%–3%. Bacteria were investigated, for the first time, by quantitative real‐time PCR (qPCR) in chorionic villi tissues and PBMCs from women affected by SA and VI. These data may help to understand the role and our knowledge of the silent infections in SA