Prevalence of Enterococci and Vancomycin Resistance in the Throat of Non-Hospitalized Individuals Randomly Selected in Central Italy.

Enterococci are commonly found in the environment and humans as a part of the normal microbiota. Among these, Enterococcus faecalis and Enterococcus faecium can convert into opportunistic pathogens, making them a major cause of nosocomial infections. The rapid diffusion of vancomycin-resistant strains and their impact on nosocomial settings is of considerable concern. Approximately one-third of the E. faecium infections in Italy are caused by vancomycin-resistant strains. This study explored the hypothesis that the oral cavity could represent a silent reservoir of virulent enterococci. A sample of 862 oral flora specimens collected from healthy human volunteers in Central Italy was investigated by real-time PCR to detect E. faecalis and E. faecium, as well as the genetic elements that most frequently determine vancomycin resistance. The prevalence of E. faecalis was 19%, a value that was not associated with alcohol consumption, tobacco smoking, or age of the subjects. Less frequently detected, with an overall prevalence of 0.7%, E. faecium was more common among people older than 49 years of age. The genes conferring vancomycin resistance were detected in only one sample. The results indicate that the oral cavity can be considered a reservoir of clinically relevant enterococci; however, our data suggest that healthy individuals rarely carry vancomycin-resistant strains.

Ultra-Rare Variants Identify Biological Pathways and Candidate Genes in the Pathobiology of Non-Syndromic Cleft Palate Only.

In recent decades, many efforts have been made to elucidate the genetic causes of non-syndromic cleft palate (nsCPO), a complex congenital disease caused by the interaction of several genetic and environmental factors. Since genome-wide association studies have evidenced a minor contribution of common polymorphisms in nsCPO inheritance, we used whole exome sequencing data to explore the role of ultra-rare variants in this study. In a cohort of 35 nsCPO cases and 38 controls, we performed a gene set enrichment analysis (GSEA) and a hypergeometric test for assessing significant overlap between genes implicated in nsCPO pathobiology and genes enriched in ultra-rare variants in our cohort. GSEA highlighted an enrichment of ultra-rare variants in genes principally belonging to cytoskeletal protein binding pathway (Probability Density Function corrected p-value = 1.57 × 10-4); protein-containing complex binding pathway (p-value = 1.06 × 10-2); cell adhesion molecule binding pathway (p-value = 1.24 × 10-2); ECM-receptor interaction pathway (p-value = 1.69 × 10-2); and in the Integrin signaling pathway (p-value = 1.28 × 10-2). Two genes implicated in nsCPO pathobiology, namely COL2A1 and GLI3, ranked among the genes (n = 34) with nominal enrichment in the ultra-rare variant collapsing analysis (Fisher's exact test p-value < 0.05). These genes were also part of an independent list of genes highly relevant to nsCPO biology (n = 25). Significant overlap between the two sets of genes (hypergeometric test p-value = 5.86 × 10-3) indicated that enriched genes are likely to be implicated in physiological palate development and/or the pathological processes of oral clefting. In conclusion, ultra-rare variants collectively impinge on biological pathways crucial to nsCPO pathobiology and point to candidate genes that may contribute to the individual risk of disease. Sequencing can be an effective approach to identify candidate genes and pathways for nsCPO.

Polymorphic variants of IGF2BP3 and SENCR have an impact on predisposition and/or progression of Ewing sarcoma.

Ewing sarcoma (EWS), the second most common malignant bone tumor in children and adolescents, occurs abruptly without clear evidence of tumor history or progression. Previous association studies have identified some inherited variants associated with the risk of developing EWS but a common picture of the germline susceptibility to this tumor remains largely unclear. Here, we examine the association between thirty single nucleotide polymorphisms (SNPs) of the IGF2BP3, a gene that codes for an oncofetal RNA-binding protein demonstrated to be important for EWS patient's risk stratification, and five SNPs of SENCR, a long non-coding RNA shown to regulate IGF2BP3. An association between polymorphisms and EWS susceptibility was observed for three IGF2BP3 SNPs - rs112316332, rs13242065, rs12700421 - and for four SENCR SNPs - rs10893909, rs11221437, rs12420823, rs4526784 -. In addition, IGF2BP3 rs34033684 and SENCR rs10893909 variants increased the risk for female respect to male subgroup when carried together, while IGF2BP3 rs13242065 or rs76983703 variants reduced the probability of a disease later onset (> 14 years). Moreover, the absence of IGF2BP3 rs10488282 variant and the presence of rs199653 or rs35875486 variant were significantly associated with a worse survival in EWS patients with localized disease at diagnosis. Overall, our data provide the first evidence linking genetic variants of IGF2BP3 and its modulator SENCR to the risk of EWS development and to disease progression, thus supporting the concept that heritable factors can influence susceptibility to EWS and may help to predict patient prognosis.

Prevalence of Staphylococcus aureus and mec-A Cassette in the Throat of Non-Hospitalized Individuals Randomly Selected in Central Italy.

Methicillin-resistant Staphylococcus aureus (MRSA) is a cause of life-threatening infections that are difficult to treat because of resistance to several antibiotics. Most documented MRSA infections are acquired nosocomially or among community with frequent contact with health facilities. However, an increasing attention to community acquired MRSA strains appears justified. A population of Central Italy was investigated for the presence of S. aureus and for the methicillin-resistance determinant mec-A gene. Exclusion was due to systemic diseases, pathologies or therapies inducing systemic immunosuppression, facial trauma or poor oral hygiene. Throat swabs obtained from 861 randomly selected participants were tested for the presence of DNA sequences of S. aureus and the mec-A gene by real-time PCR. The DNA of S. aureus was detected in 199 specimens (23.1%), while the mec-A gene was detected in 27 samples (3.1%). The prevalence of patients carrying methicillin-resistant strains was higher in younger and older strata. The prevalence of mec-A among S. aureus positive samples was 7.5%. Our data confirm that S. aureus and methicillin-resistant strains are common in the throat of the general population of Central Italy. Although the PCR methods used in this study are different from traditional culture-based approaches, the observed prevalence was consistent to those observed in Italians and other populations. Considering that carriers have a higher risk to develop post surgically life-threatening infections, it is worth evaluating a preventive approach based on rapid PCR screening of incoming patients to reduce the risk of developing health-care-associated infections.

Prevalence of Human Papillomavirus in the Oropharynx of Healthy Individuals in an Italian Population.

Oral cavity and oropharynx cancer associated with human papil loma virus infection, particularly in young people who are continuously exposed to this virus, is a serious public health problem worldwide, especially for high-risk strains that are most associated with premalignant lesions and tumors. These neoplasms remain asymptomatic for a long time and, when they occur, they are already at an advanced stage. If diagnosed and treated early, oral cancer induced by human papilloma virus allows for high survival, as it often has a more favorable prognosis than oral cancers not directly related to viral infection. In this study, the presence of different high-risk and low-risk HPV strains was investigated to assess the epidemiological status in a population of healthy individuals. Two types of samples, one from the tonsils and one from the base of the tongue, were collected from 2015 healthy individuals of different sex and age. A total of 1885 DNA samples belonging to 1285 patients were tested for the presence of 11 high-risk HPV types plus 4 low-risk HPV types using real-time PCR. Of the patients' DNA samples screened for 15 HPV types, only four samples were positive, all of which were taken from male smokers. These results indicate that newly acquired oral oncogenic HPV infections in the healthy population are rare and, in many cases, controversial. Therefore, more studies are needed to ensure fewer variations in outcomes and a greater clarification of HPV infection and its prevalence in the oropharynx of the healthy population, and to guide efforts to prevent the development of this infection which, if undiagnosed, can lead to the onset of malignant tumors in the oral cavity.

Short-term variation in the subgingival microbiota in two groups of patients treated with clear aligners and vestibular fixed appliances: A longitudinal study.

OBJECTIVE: To evaluate the subgingival microbiological changes during the first six months of therapy with clear aligners (CAs) and fixed appliances (FAs). The null hypothesis was that there would be no microbiological differences between the two. SETTING/SAMPLE: Two groups of patients to be treated, respectively, with CAs (14 patients; 9 females and 5 males; mean age 21 years ± 0.25) and FAs (13 patients; 8 females and 5 males; mean 14 years ± 0.75) were consecutively recruited. MATERIALS AND METHODS: Subgingival microbiological samples were obtained at the right upper central incisor and right first molar at four different time points: before appliance fitting (T0), and at 1 month (T1), 3 months (T3) and 6 months (T6) thereafter. Total bacterial load (TBL) and counts of the bacteria Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Fusobacterium nucleatum, Campylobacter rectus, Treponema denticola and Tannerella forsythia were determined using real-time PCR. RESULTS: Total bacterial load did not vary in the CA group, while a significant increase was detected after 3 and 6 months of treatment in the FA group. Unlike red complex species, C rectus and F nucleatum were often detected: levels remained stable in the CA group but increased progressively in the FA group. CONCLUSION: The type of orthodontic appliance influences the subgingival microbiota. TBL increased in the FA group but not in the CA group, although the levels of the individual periodontal pathogenic bacteria species did not significantly increase during the observation period.

Non-syndromic Cleft Palate: An Overview on Human Genetic and Environmental Risk Factors.

The epithelial and mesenchymal cells involved in early embryonic facial development are guided by complex regulatory mechanisms. Any factor perturbing the growth, approach and fusion of the frontonasal and maxillary processes could result in orofacial clefts that represent the most common craniofacial malformations in humans. The rarest and, probably for this reason, the least studied form of cleft involves only the secondary palate, which is posterior to the incisive foramen. The etiology of cleft palate only is multifactorial and involves both genetic and environmental risk factors. The intention of this review is to give the reader an overview of the efforts made by researchers to shed light on the underlying causes of this birth defect. Most of the scientific papers suggesting potential environmental and genetic causes of non-syndromic cleft palate are summarized in this review, including genome-wide association and gene-environment interaction studies.

ROCK1 is associated with non-syndromic cleft palate.

BACKGROUND: Craniofacial morphogenesis is the result of an intricate multistep network of tightly controlled spatial and temporal signalling that involves several molecules and transcription factors organized into highly coordinated pathways. Any alteration in even one step of this delicate process can lead to congenital malformations such as cleft palate. One of the first steps in embryonal orofacial development is the migration of cells from the neural crests to the branchial arches. Next, the cells have to proliferate, differentiate, move and connect to each other in order to correctly form the palate. Cell contraction, promoted by the interaction of non-muscle myosin II and actin A, is a crucial step in morphogenesis and is regulated by ROCK1 protein. METHODS: A family-based association study was carried out in order to verify whether or not genetic variants of ROCK1 were associated with non-syndromic cleft palate (nsCP). Two cohorts from Italy and Iran, a total of 189 nsCP cases and their parents were enrolled. RESULTS: The rs35996865-G allele was under-transmitted in cases of nsCP [P = .006, odds ratio (OR) = 0.63 (95% CI 0.45-0.88)]. CONCLUSION: This investigation reveals for the first time data supporting a role for ROCK1 in nsCP aetiology.

Copy number variation analysis of twin pairs discordant for cleft lip with or without cleft palate.

Non-syndromic cleft lip with or without cleft palate (nsCL/P) is a frequent orofacial malformation. The comparison of concordance rate observed in monozygotic and dizygotic twins supports high level of heritability and a strong genetic component. However, phenotype concordance for orofacial cleft in monozygotic twins is about 50%. The aim of the present investigation was to detect postzygotic events that may account for discordance in monozygotic twins. High-density SNP microarrays hybridization was used to genotype two pairs of monozygotic twins discordant for nsCL/P. Discordant SNP genotypes and copy number variants were analyzed to identify genetic differences responsible of phenotype discrepancy. A number of differences were observed, none involving known nsCL/P candidate genes or genomic regions. Considering the limitation of the study, related to the small sample size and to the large-scale investigation method, the results suggest that the detection of discordant events in other monozygotic twin pairs would be remarkable and warrant further investigations.

Association between oral cleft and transcobalamin 2 polymorphism in a sample study from Nassiriya, Iraq.

Orofacial clefts are common congenital defects whose prevalence differs between geographical regions and ethnic groups. The inheritance is complex, involving the contribution of both genetic and environmental factors. The involvement of genes belonging to the folate pathway is still matter of debate, with strong evidences of association and conflicting results. After demonstrating the contribution, for a sample from the Italian population, of common mutations mapping on three genes of the folate pathway, our group tried to unravel their contribution in independent sample studies with different ethnicity. In the present investigation a set of 34 triads with oral cleft from Nassiriya, Iraq, has been genotyped for rs1801133 of MTHFR, rs1801198 of TCN2, and rs4920037 of CBS polymorphisms. Association analysis evidenced a decreased risk of cleft for children carrying the 667G allele at TCN2 gene (P = 0.02). This evidence further supported the relationship between polymorphisms of folate related genes and oral clefts, and outlined the relevance of studying populations having different ethnicity.

Berberine and Tinospora cordifolia exert a potential anticancer effect on colon cancer cells by acting on specific pathways.

Berberine (BBR) is a natural active principle with potential antitumor activity. The compound targets multiple cell signaling pathways, including proliferation, differentiation, and epithelial-mesenchymal transition. The aim of this study was to elucidate the mechanisms behind the anticancer activity of BBR by comparing the effects of purified BBR with those of the extract of Tinospora cordifolia, a medicinal plant that produces this metabolite. The expression levels of a panel of 44 selected genes in human colon adenocarcinoma (HCA-7) cell line were quantified by real-time polymerase chain reaction (PCR). BBR treatment resulted in a time- and dose-dependent down regulation of 33 genes differently involved in cell cycle, differentiation, and epithelial-mesenchymal transition. The trend was confirmed across the two types of treatment, the two time points, and the different absolute dosage of BBR. These findings suggest that the presence of BBR in T. cordifolia extract significantly contributes to its antiproliferative activity.

Non-syndromic cleft palate: Association analysis on three gene polymorphisms of the folate pathway in Asian and Italian populations.

Periconceptional folic acid supplementation can reduce the risk of inborn malformations, including orofacial clefts. Polymorphisms of MTHFR, TCN2, and CBS folate-related genes seem to modulate the risk of cleft lip with or without cleft palate (CL/P) in some populations. CL/P and cleft palate only (CPO) are different malformations that share several features and possibly etiological causes. In the present investigation, we conducted a family-based, candidate gene association study of non-syndromic CPO. Three single nucleotide polymorphisms, namely, rs1801133 of MTHFR, rs1801198 of TCN2, and rs4920037 of CBS, were investigated in a sample that included 129 Italian and 65 Asian families. No evidence of association between the three genotyped polymorphisms and CPO was found in the Italian and Asian cases, indeed the transmission disequilibrium test did not detect any asymmetry of transmission of alleles. This investigation, although with some limitation, further supports that CL/P and CPO diverge in their genetic background.

Gabapentin affects the expression of inflammatory mediators on healthy gingival cells.

Gabapentin is one of the most used drugs to treat postoperative pain with antihyperalgesic properties and has a unique mechanism of action, which differentiates it from other commonly used drugs. Various studies have shown that the perioperative use of gabapentin reduces postoperative pain. In our study, fragments of gingival tissue of healthy volunteers were collected during operation. Gene expression of 29 genes was investigated in gingival fibroblasts cell culture treated with gabapentin, compared with untreated cells. Of the different chemokines and interleukins studied, only 10 were statistically significant (CCL1, CCR1, CCR4, CCR5, CCR6, ILI1A, ILI1B, IL5, IL6R, TNFSF10). The overexpression of these cytokines, obtained in many studies, leads us to think that gabapentin can interact and cause post-inflammatory gingival hyperplasia, but, probably, in our study the gabapentin has not the same effect, because we used gingival fibroblasts of healthy people.

Molecular tools for preventing and improving diagnosis of peri-implant diseases.

Peri-implantitis is an inflammatory disease of tissues surrounding osseointegrated dental implants. Inflammation affecting soft and hard peri-implant tissues can cause alveolar bone resorption and subsequent implant loss. Clinical surveillance and early diagnosis are of paramount importance to reduce clinical failures and improve implant survival. Current diagnosis of implants is based on clinical and radiological signs. Molecular tests are an emerging diagnostic methodology, which potentially can help to detect and prevent early peri-implantitis and monitor the efficacy of therapy as well. A plethora of potential biomarkers are potentially available to support the clinical diagnosis of peri-implantitis. However, conflicting diagnostic conclusions have been reached, probably related to weak statistical results due to limited sample size or disease heterogeneity. The present paper reviews candidate diagnostic biomarkers for peri-implantitis, including infective agents, genetic susceptibility factors, and key proteins related to inflammation and tissue remodeling.

A Noninvasive Test for MicroRNA Expression in Oral Squamous Cell Carcinoma.

MicroRNAs have recently been proposed as non-invasive biomarkers in Oral Squamous Cell Carcinoma (OSCC). The aim of this study was to analyze the expression of a panel of miRNAs in epithelial cells collected by oral brushing from OSCCs from regenerative areas after OSCC surgical resection and from their respective normal distant mucosa. Oral brushing specimens were collected from 24 healthy donors, 14 OSCC patients with specimens from tumour and normal distant mucosa, and from 13 patients who had OSCC resection, with samples from regenerative areas after OSCC resection and normal distant mucosa. Expression levels of eight targets (miR-21, miR-375, miR-345, miR-181b, miR-146a, miR-649, miR-518b, and miR-191) were evaluated by real-time Polymerase Chain Reaction (PCR). A highly significant between-group difference was found for miR-21 (F = 6.58, p < 0.001), miR-146a (F = 6.974, p < 0.001), and miR-191 (F = 17.07, p < 0.001). The major difference was observed between samples from healthy donors and from OSCC brushing, whereas no significant differences were observed between areas infiltrated by OSCC and their respective normal distant mucosa. Furthermore, altered expression of miR-146a and miR-191 was also observed in regenerative areas after OSCC resection. CONCLUSIONS: Oral brushing could be proposed as a noninvasive method to study microRNA expression in oral mucosa in OSCC patients.

Possible effect of SNAIL family transcriptional repressor 1 polymorphisms in non-syndromic cleft lip with or without cleft palate.

OBJECTIVE: Orofacial development is a complex process subjected to failure impairing. Indeed, the cleft of the lip and/or of the palate is among the most frequent inborn malformations. The JARID2 gene has been suggested to be involved in non-syndromic cleft lip with or without cleft palate (nsCL/P) etiology. JARID2 interacts with the polycomb repressive complex 2 (PRC2) in regulating the expression patterns of developmental genes by modifying the chromatin state. MATERIALS AND METHODS: Genes coding for the PRC2 components, as well as other genes active in cell differentiation and embryonic development, were selected for a family-based association study to verify their involvement in nsCL/P. A total of 632 families from Italy and Asia participated to the study. RESULTS: Evidence of allelic association was found with polymorphisms of SNAI1; in particular, the rs16995010-G allele was undertransmitted to the nsCL/P cases [P = 0.004, odds ratio = 0.69 (95% C.I. 0.54-0.89)]. However, the adjusted significance value corrected for all the performed tests was P = 0.051. CONCLUSIONS: The findings emerging by the present study suggest for the first time an involvement of SNAI1 in the nsCL/P onset. CLINICAL RELEVANCE: Interestingly, SNAI1 is known to promote epithelial to mesenchymal transition by repressing E-cadherin expression, but it needs an intact PRC2 to act this function. Alterations of this process could contribute to the complex etiology of nsCL/P.

CD99 polymorphisms significantly influence the probability to develop Ewing sarcoma in earlier age and patient disease progression.

Ewing sarcoma (EWS), the second most common primary bone tumor in pediatric age, is known for its paucity of recurrent somatic abnormalities. Apart from the chimeric oncoprotein that derives from the fusion of EWS and FLI genes, recent genome-wide association studies have identified susceptibility variants near the EGR2 gene that regulate DNA binding of EWS-FLI. However, to induce transformation, EWS-FLI requires the presence of additional molecular events, including the expression of CD99, a cell surface molecule with critical relevance for the pathogenesis of EWS. High expression of CD99 is a common and distinctive feature of EWS cells, and it has largely been used for the differential diagnosis of the disease. The present study first links CD99 germline genetic variants to the susceptibility of EWS development and its progression. In particular, a panel of 25 single nucleotide polymorphisms has been genotyped in a case-control study. The CD99 rs311059 T variant was found to be significantly associated [P value = 0.0029; ORhet = 3.9 (95% CI 1.5-9.8) and ORhom = 5.3 (95% CI 1.2-23.7)] with EWS onset in patients less than 14 years old, while the CD99 rs312257-T was observed to be associated [P value = 0.0265; ORhet = 3.5 (95% CI 1.3-9.9)] with a reduced risk of relapse. Besides confirming the importance of CD99, our findings indicate that polymorphic variations in this gene may affect either development or progression of EWS, leading to further understanding of this cancer and development of better diagnostics/prognostics for children and adolescents with this devastating disease.

Non-syndromic cleft lip with or without cleft palate in Asian populations: Association analysis on three gene polymorphisms of the folate pathway.

OBJECTIVE: Orofacial clefts (OFCs) are one of the most common birth defects in humans. They are the subject of a number of investigations aimed at elucidating the bases of their complex mode of inheritance involving both genetic and environmental factors. Genes belonging to the folate pathway have been among the most studied. The aim of the investigation was to replicate previous studies reporting evidence of association between polymorphisms of folate related genes and the occurrence of non-syndromic cleft lip with or without cleft palate (NSCL/P), using three independent samples of different ancestry: from Tibet, Bangladesh and Iran, respectively. DESIGN: Specifically, the polymorphisms rs1801133 of MTHFR, rs1801198 of TCN2, and rs4920037 of CBS, were tested. RESULTS: A decreased risk of NSCL/P was observed in patients presenting the C677T variant at MTHFR gene (relative risk for heterozygotes=0.53; 95% confidence interval [C.I.]=0.32-0.87). The investigated polymorphisms mapping at TCN2 and CBS genes did not provide any evidence of association. CONCLUSION: Overall, these results indicate that NSCL/P risk factors differ among populations and confirm the importance of testing putative susceptibility variants in different genetic backgrounds.

Replication analysis of 15 susceptibility loci for nonsyndromic cleft lip with or without cleft palate in an italian population.

BACKGROUND: Nonsyndromic cleft lip with or without cleft palate (nsCL/P) is one of the most common congenital malformations in humans. Its average global incidence is 1.7 per 1000 live births, with wide variation according to geographical location and ethnicity. Its etiology involves both genetic and environmental factors. The aim of the present study was to confirm genetic association of a selection of 15 candidate nsCL/P loci using an independent sample of the Italian population. METHODS: At least one single-nucleotide polymorphism (SNP) for each locus was genotyped in 380 nuclear trios. RESULTS: Transmission disequilibrium analysis revealed significant associations for three variants at two loci (8q24 and 1p22). Two SNPs at 8q24 showed the strongest level of association, the rs987525 (PTDT = 6.81 × 10(-6) ; homozygous relative risk = 3.60 [95% confidence interval, 2.12-6.13]), and the rs17241253 (PTDT = 1.03 × 10(-5) ; homozygous relative risk = 3.75 [95% confidence interval, 2.10-6.67]). Four additional loci (at 1q32, 3q12, 8q21, and 10q25) achieved nominally significant p-values. Two SNPs at 1p36 achieved p-values of < 0.1. The present data suggest that 6 of the 15 analyzed nsCL/P risk loci contribute significantly to nsCL/P risk in the Italian population. These include the 1p22 locus, which previous research has implicated predominantly in Asian populations. CONCLUSION: Different loci, including 8q24 and 1p22 have been found associated with nsCL/P in multiple populations. Further efforts are needed to identify causative variants and transfer knowledge to clinical application, such as personal genetic risk assessment.

Human Multidrug Resistance 1 gene polymorphisms and Idiopathic Pulmonary Fibrosis.

BACKGROUND: For the first time we tested an association between the human multidrug resistance gene 1 (MDR1) polymorphisms (SNPs) and idiopathic pulmonary fibrosis (IPF). Several MDR1 polymorphisms are associated with pathologies in which they modify the drug susceptibility and pharmacokinetics. MATERIALS AND METHODS: We genotyped three MDR1 polymorphisms of 48 IPF patients and 100 control subjects with Italian origins. RESULTS: No evidence of association was detected. CONCLUSION: There are 50 known MDR1 SNPs, and their role is explored in terms of the effectiveness of drug therapy. We consider our small-scale preliminary study as a starting point for further research.