Comparative genomics provides an operational classification system and reveals early emergence and biased spatio-temporal distribution of SARS-CoV-2

Effective systems for the analysis of molecular data are of fundamental importance for real-time monitoring of the spread of infectious diseases and the study of pathogen evolution. While the Nextstrain and GISAID portals offer widely used systems for the classification of SARS-CoV-2 genomes, both present relevant limitations. Here we propose a highly reproducible method for the systematic classification of SARS-CoV-2 viral types. To demonstrate the validity of our approach, we conduct an extensive comparative genomic analysis of more than 20,000 SARS-CoV-2 genomes. Our classification system delineates 12 clusters and 4 super-clusters in SARS-CoV-2, with a highly biased spatio-temporal distribution worldwide, and provides important observations concerning the evolutionary processes associated with the emergence of novel viral types. Based on the estimates of SARS-CoV-2 evolutionary rate and genetic distances of genomes of the early pandemic phase, we infer that SARS-CoV-2 could have been circulating in humans since August-November 2019. The observed pattern of genomic variability is remarkably similar between all clusters and super-clusters, being UTRs and the s2m element, a highly conserved secondary structure element, the most variable genomic regions. While several polymorphic sites that are specific to one or more clusters were predicted to be under positive or negative selection, overall, our analyses also suggest that the emergence of novel genome types is unlikely to be driven by widespread convergent evolution and independent fixation of advantageous substitutions. While, in the absence of rigorous experimental validation, several questions concerning the evolutionary processes and the phenotypic characteristics (increased/decreased virulence) remain open, we believe that the approach outlined in this study can be of relevance for the tracking and functional characterization of different types of SARS-CoV-2 genomes

Comparative genomics suggests limited variability and similar evolutionary patterns between major clades of SARS-CoV-2

Phylogenomic analysis of SARS-CoV-2 as available from publicly available repositories suggests the presence of 3 prevalent groups of viral episomes (super-clades), which are mostly associated with outbreaks in distinct geographic locations (China, USA and Europe). While levels of genomic variability between SARS-CoV-2 isolates are limited, to our knowledge, it is not clear whether the observed patterns of variability in viral super-clades reflect ongoing adaptation of SARS-CoV-2, or merely genetic drift and founder effects. Here, we analyze more than 1100 complete, high quality SARS-CoV-2 genome sequences, and provide evidence for the absence of distinct evolutionary patterns/signatures in the genomes of the currently known major clades of SARS-CoV-2. Our analyses suggest that the presence of distinct viral episomes at different geographic locations are consistent with founder effects, coupled with the rapid spread of this novel virus. We observe that while cross species adaptation of the virus is associated with hypervariability of specific protein coding regions (including the RDB domain of the spike protein), the more variable genomic regions between extant SARS-CoV-2 episomes correspond with the 3\u2019 and 5\u2019 UTRs, suggesting that at present viral protein coding genes should not be subjected to different adaptive evolutionary pressures in different viral strains. Although this study can not be conclusive, we believe that the evidence presented here is strongly consistent with the notion that the biased geographic distribution of SARSCoV-2 isolates should not be associated with adaptive evolution of this novel pathogen

Managing multiple pressures for cetaceans’ conservation with an Ecosystem-Based Marine Spatial Planning approach

Despite the recognized important ecological role that cetaceans play in the marine environment, their protection is still scarcely enforced in the Mediterranean Sea even though this area is strongly threatened by local human pressures and climate change. The piecemeal of knowledge related to cetaceans' ecology and distribution in the basin undermines the capacity of addressing cetaceans' protection and identifying effective conservation strategies. In this study, an Ecosystem-Based Marine Spatial Planning (EB-MSP) approach is applied to assess human pressures on cetaceans and guide the designation of a conservation area in the Gulf of Taranto, Northern Ionian Sea (Central-eastern Mediterranean Sea). The Gulf of Taranto hosts different cetacean species that accomplish important phases of their life in the area. Despite this fact, the gulf does not fall within any area-based management tools (ABMTs) for cetacean conservation. We pin down the Gulf of Taranto being eligible for the designation of diverse ABMTs for conservation, both legally and non-legally binding. Through a risk-based approach, this study explores the cause-effect relationships that link any human activities and pressures exerted in the study area to potential effects on cetaceans, by identifying major drivers of potential impacts. These were found to be underwater noise, marine litter, ship collision, and competition and disturbance on preys. We draw some recommendations based on different sources of available knowledge produced so far in the area (i.e., empirical evidence, scientific and grey literature, and expert judgement) to boost cetaceans’ conservation. Finally, we stress the need of sectoral coordination for the management of human activities by applying an EB-MSP approach and valuing the establishment of an ABMT in the Gulf of Taranto

Predictive Role of p53 Protein as a Single Marker or Associated to Ki67 Antigen in Oral Carcinogenesis

p53 over-expression has been proposed as a reliable marker associated to oral carcinogenesis, although only about 50% of oral carcinomas (OSCC) are associated with p53 over-expression and even p53-negative lesions can progress to OSCC. The aim of the study was to determine whether the combination of p53 over-expression and p53 low-expression associated with Ki67 over-expression (high Ki67/p53 ratio) could lead to a more sensitive parameter. Immunohistochemical expression of Ki67 and p53 was measured in 54 specimens from OSCC; 27 specimens from moderate/severe epithelial dysplasia; 32 specimens from oral leukoplakias without epithelial dysplasia, and 13 specimens with normal epithelium. p53 over-expression was found in 31 (53%) samples from OSCC, in 10 (37%) samples from severe dysplasias, and in 5 (15%) samples from non-dysplastic lesions, while the combination of high p53 values with high Ki67/p53 ratio was observed in 93% of OSCC, in 81% of dysplastic lesions, and in 50% of non-dysplastic lesions. This parameter may have a clinical implication to detect early lesions with an impairment of p53 pathway, and probably at risk of progress to OSCC

CD200 as a Potential New Player in Inflammation during Rotator Cuff Tendon Injury/Repair: An In Vitro Model

Rotator cuff tendon (RCT) disease results from multifactorial mechanisms, in which inflammation plays a key role. Pro-inflammatory cytokines and tendon stem cell/progenitor cells (TSPCs) have been shown to participate in the inflammatory response. However, the underlying molecular mechanism is still not clear. In this study, flow cytometry analyses of different subpopulations of RCT-derived TSPCs demonstrate that after three days of administration, TNFα alone or in combination with IFNγ significantly decreases the percentage of CD146+CD49d+ and CD146+CD49f+ but not CD146+CD109+ TSPCs populations. In parallel, the same pro-inflammatory cytokines upregulate the expression of CD200 in the CD146+ TSPCs population. Additionally, the TNFα/IFNγ combination modulates the protein expression of STAT1, STAT3, and MMP9, but not fibromodulin. At the gene level, IRF1, CAAT (CAAT/EBPbeta), and DOK2 but not NF-κb, TGRF2 (TGFBR2), and RAS-GAP are modulated. In conclusion, although our study has several important limitations, the results highlight a new potential role of CD200 in regulating inflammation during tendon injuries. In addition, the genes analyzed here might be new potential players in the inflammatory response of TSPCs

Mitogenomics reveals two cryptic species in Ciona intestinalis

Individual mitochondrial genes or genomic features are commonly used as phylogenetic markers at many taxonomic levels. We used a mitogenomics approach to demonstrate the existence of two cryptic species in the ascidian Ciona intestinalis, a model chordate whose status as a single species has recently been questioned. Comprehensive comparative analysis of the mitochondrial genome of the two cryptic species revealed significant differences in gene order, size and number of noncoding regions, compositional features and divergence of protein-coding genes

Phylogenomics and systematics of botryllid ascidians, and implications for the evolution of allorecognition

Allorecognition, the ability of an organism to distinguish kin from non-kin, or self from non-self, has been studied extensively in a group of invertebrate chordates, the colonial ascidians called botryllids (Subphylum Tunicata, Class Ascidiacea, Family Styelidae). When two conspecific botryllid colonies come in contact, there are two potential outcomes to an allorecognition reaction: fusion or rejection. The rejection outcome of allorecognition varies by species, and has been classified by type (referred to as R-Type). R-Type is defined according to how far the fusion process progresses before the rejection begins, since the rejection reaction appears as an interference of the fusion process. Here, we map the evolution of R-Types onto an extended and robust phylogeny of the botryllids. In this study, we have reconstructed the largest phylogenomic tree of botryllids, including 97 samples and more than 40 different species, and mapped on it nine of the 13 species for which the R-Type is known. Based on the R-Type known in a single outgroup species (Symplegma reptans), we infer that at least R-Type B and E-like could be ancestral to the Botrylloides/Botryllus group. However, the application of ancestral character state reconstructions does not provide conclusive results since several clades show more than one equiparsimonious R-Type state. Notably, all R-Type A species are clustered together and certainly evolved later than other R-Types. Our phylogenomic tree has been built on 177 nuclear loci and nearly all clades are well supported. Moreover, our phylogenetic analyses also take into account the results of species delimitation analyses based on the mitochondrial COI gene and of careful morphological analyses of the samples. The implementation of this integrated taxonomic approach, combining morphological as well as nuclear and mitochondrial data, has allowed the description of six new species, and the identification of a number of putative unnamed taxa. Thus, our results also demonstrate the existence of an unexplored hidden diversity within botryllids

Shared epigenetic alterations between oral cancer and periodontitis: A preliminary study

Introduction: We recently developed a non-invasive sampling procedure for oral squamous cell carcinoma (OSCC) detection based on DNA methylation analysis of a panel of 13 genes. Oral cancer, as well as acute and chronic inflammatory diseases, may influence the methylation level of several genes in the oral cavity. In the present study, we evaluated the presence of periodontal disease (PD) and the methylation status using our 13-gene panel. Methods: Oral brushing specimens were collected from three different patient groups: 23 gingival OSCC patients, 15 patients affected by PD, and 15 healthy volunteers lacking evidence of PD. DNA methylation analysis was performed and each sample was determined to be positive or negative based on a predefined cut-off value. Results: Positive results were found for 23/23 OSCC patients, 3/15 PD patients, and 0/15 samples from healthy volunteers. The GP1BB and MIR193 genes in the PD group exhibited mean methylation levels similar to OSCC patients. ZAP70 showed different methylation levels among three groups. Conclusion: Preliminary data identified shared epigenetic alterations between PD and OSCC patients in two inflammatory genes (GP1BB and MIR193). This study may help to identify potential links between the two diseases and serve as a starting point for the future research focused on pathogenesis

Extracellular vesicles from rat-bone-marrow mesenchymal stromal/stem cells improve tendon repair in rat Achilles tendon injury model in dose-dependent manner: A pilot study

Mesenchymal stromal/stem cells (MSCs) are increasingly employed for tissue regeneration, largely mediated through paracrine actions. Currently, extracellular vesicles (EVs) released by MSCs are major mediators of these paracrine effects. We evaluated whether rat-bone-marrow-MSC-derived EVs (rBMSCs-EVs) can ameliorate tendon injury in an in vivo rat model. Pro-collagen1A2 and MMP14 protein are expressed in rBMSC-EVs, and are important factors for extracellular-matrix tendon-remodeling. In addition, we found pro-collagen1A2 in rBMSC-EV surface-membranes by dot blot. In vitro on cells isolated from Achilles tendons, utilized as rBMSC -EVs recipient cells, EVs at both low and high doses induce migration of tenocytes; at higher concentration, they induce proliferation and increase expression of Collagen type I in tenocytes. Pretreatment with trypsin abrogate the effect of EVs on cell proliferation and migration, and the expression of collagen I. When either low- or high-dose rBMSCs-EVs were injected into a rat-Achilles tendon injury-model (immediately after damage), at 30 days, rBMSC-EVs were found to have accelerated the remodeling stage of tendon repair in a dose-dependent manner. At histology and histomorphology evaluation, high doses of rBMSCs-EVs produced better restoration of tendon architecture, with optimal tendon-fiber alignment and lower vascularity. Higher EV-concentrations demonstrated greater expression of collagen type I and lower expression of collagen type III. BMSC-EVs hold promise as a novel cell-free modality for the management of tendon injuries

Expression profiling of microRNAs and isomiRs in conventional central chondrosarcoma

Conventional central chondrosarcoma (CCC) is a malignant bone tumor that is characterized by the production of chondroid tissue. Since radiation therapy and chemotherapy have limited effects on CCC, treatment of most patients depends on surgical resection. This study aimed to identify the expression profiles of microRNAs (miRNAs) and isomiRs in CCC tissues to highlight their possible participation to the regulation of pathways critical for the formation and growth of this type of tumor. Our study analyzed miRNAs and isomiRs from Grade I (GI), Grade II (GII), and Grade III (GIII) histologically validated CCC tissue samples. While the different histological grades shared a similar expression profile for the top abundant miRNAs, we found several microRNAs and isomiRs showing a strong different modulation in GII + GIII vs GI grade samples and their involvement in tumor biology could be consistently hypothesized. We then in silico validated these differently expressed miRNAs in a larger chondrosarcoma public dataset and confirmed the expression trend for 17 out of 34 miRNAs. Our results clearly suggests that the contribution of miRNA deregulation, and their targeted pathways, to the progression of CCC could be relevant and strongly indicates that when studying miRNA deregulation in tumors, not only the canonical miRNAs, but the whole set of corresponding isomiRs should be taken in account. Improving understanding of the precise roles of miRNAs and isomiRs over the course of central chondrosarcoma progression could help identifying possible targets for precision medicine therapeutic intervention