Analysis of the oral microbiome during hormonal cycle and its alterations in menopausal women: the "AMICA" project.

The maintenance of human health is dependent on a symbiotic relationship between humans and associated bacteria. The diversity and abundance of each habitat's signature microbes vary widely among body areas and among them the oral microbiome plays a key role. Significant changes in the oral cavity, predominantly at salivary and periodontal level, have been associated with changes in estrogen levels. However, whether the oral microbiome is affected by hormonal level alterations is understudied. Hence the main objective pursued by AMICA project was to characterize the oral microbiome (saliva) in healthy women through: profiling studies using "omics" technologies (NMR-based metabolomics, targeted lipidomics by LC-MS, metagenomics by NGS); SinglePlex ELISA assays; glycosidase activity analyses and bioinformatic analysis. For this purpose, thirty-nine medically healthy women aged 26-77 years (19 with menstrual cycle and 20 in menopause) were recruited. Participants completed questionnaires assessing detailed medical and medication history and demographic characteristics. Plasmatic and salivary levels of sexual hormones were assessed (FSH, estradiol, LH and progesteron) at day 3 and 14 for women with menstrual cycle and only once for women in menopause. Salivary microbiome composition was assessed through meta-taxonomic 16S sequencing and overall, the salivary microbiome of most women remained relatively stable throughout the menstrual cycle and in menopause. Targeted lipidomics and untargeted metabolomics profiling were assessed through the use of LC-MS and NMR spectroscopy technologies, respectively and significant changes in terms of metabolites were identified in saliva of post-menopausal women in comparison to cycle. Moreover, glycosyl hydrolase activities were screened and showed that the ß-D-hexosaminidase activity was the most present among those analyzed. Although this study has not identified significant alterations in the composition of the oral microbiome, multiomics analysis have revealed a strong correlation between 2-AG and α-mannosidase. In conclusion, the use of a multidisciplinary approach to investigate the oral microbiome of healthy women provided some indication about microbiome-derived predictive biomarkers that could be used in the future for developing new strategies to help to re-establish the correct hormonal balance in post-menopausal women.

Neurological complications in pediatric patients with SARS-CoV-2 infection: a systematic review of the literature.

OBJECTIVES: To describe clinical characteristics, laboratory tests, radiological data and outcome of pediatric cases with SARS-CoV-2 infection complicated by neurological involvement. STUDY DESIGN: A computerized search was conducted using PubMed. An article was considered eligible if it reported data on pediatric patient(s) with neurological involvement related to SARS-CoV-2 infection. We also described a case of an acute disseminated encephalomyelitis (ADEM) in a 5-year-old girl with SARS-CoV-2 infection: this case was also included in the systematic review. RESULTS: Forty-four articles reporting 59 cases of neurological manifestations in pediatric patients were included in our review. Most (32/59) cases occurred in the course of a multisystem inflammatory syndrome in children (MIS-C). Neurological disorders secondary to cerebrovascular involvement were reported in 10 cases: 4 children with an ischemic stroke, 3 with intracerebral hemorrhage, 1 with a cerebral sinus venous thrombosis, 1 with a subarachnoid hemorrhage, 1 with multiple diffuse microhemorrhages. Reversible splenial lesions were recognized in 9 cases, benign intracranial hypertension in 4 patients, meningoencephalitis in 4 cases, autoimmune encephalitis in 1 girl, cranial nerves impairment in 2 patients and transverse myelitis in 1 case. Five cases had Guillain-Barré syndrome (GBS) and two, including ours, had ADEM. Radiological investigations were performed in almost all cases (45/60): the most recurrent radiological finding was a signal change in the splenium of the corpus callosum. The presence of SARS-CoV-2 viral nucleic acid in the cerebrospinal fluid was proved only in 2 cases. The outcome was favorable in almost all, except in 5 cases. CONCLUSIONS: Our research highlights the large range of neurological manifestations and their presumed pathogenic pathways associated with SARS-CoV-2 infection in children. Nervous system involvement could be isolated, developing during COVID-19 or after its recovery, or arise in the context of a MIS-C. The most reported neurological manifestations are cerebrovascular accidents, reversible splenial lesions, GBS, benign intracranial hypertension, meningoencephalitis; ADEM is also a possible complication, as we observed in our patient. Further studies are required to investigate all the neurological complications of SARS-CoV-2 infection and their underlying pathogenic mechanism.

Influence of postharvest treatments on qualitative and chemical parameters of Tarocco blood orange fruits to be used for fresh chilled juice.

Tarocco "Sant'Alfio" is a late ripening blood orange cultivar. Blood oranges are more and more appreciated from consumers for their high nutraceutical value due to the presence of bioactive compounds including vitamin C, polyphenols, flavonoids and hydroxycinnamic acids. The aim of this work is to set up a reliable protocol for postharvest storage of the very-late Tarocco "Sant'Alfio" orange to prolong the availability of this product in the market to be used for fresh chilled orange juice production. Fruits were subjected to three storage treatments (20days at 1°C plus 50days at 4°C; 70days at 4°C; 70days at 20°C). The results indicate that cold treatments, in particular at 4°C constantly, can extend Tarocco "Sant'Alfio" shelf life enhancing total anthocyanin content. The defined protocols allow prolonging market availability of a high value product and could induce relevant benefits for the citrus industry and consumers.

A Case of Epididymo-orchitis after intravesical bacille Calmette-Guérin therapy for superficial bladder carcinoma in a patient with latent tuberculosis infection.

BACKGROUND: Intravesical instillation of bacille Calmette-Guérin (BCG) has been established as efficient therapy for superficial bladder carcinoma. Overall, intravesical BCG is well tolerated and results in complications of less than 5 %. However, adverse effects such as granulomatous prostatitis, pneumonitis, hepatitis, sepsis, and hypersensitivity reactions may occur. The reported rate for tuberculous orchitis after BCG intravesical therapy is 0.4 %. FINDINGS: We report a case of monolateral tuberculous orchitis occurring one month after the second course of intravescical instillation of bacille Calmette-Guérin in a patient with proven superficial bladder carcinoma and latent tuberculosis infection. CONCLUSIONS: In our opinion intravesical instillation of BCG should be considered on an individual patient basis, with full patient disclosure of the potentially significant risks. A screening with an intradermal Mantoux before starting the first cycle of BCG instillation should be recommended and isoniazid would be indicated as the treatment for latent tuberculosis infection.

[Mediterranean spotted fever in paediatric and adult patients: two clinical aspects of the same disease]. / La febbre bottonosa del Mediterraneo in età pediatricae in età adulta: due realtà cliniche differenti nella stessamalattia.

Mediterranean Spotted Fever is an acute febrile disease caused by Rickettsia conorii and transmitted to humans by the brown dog tick Rhipicephalus sanguineus. Nearly 400 cases are reported every year in Sicily, mainly from June to September. The aim of this study is to compare the clinical and laboratory features of two different groups of patients , one of adults and one of children. The analysis included all adult patients with MSF diagnosed at the Institute of Infectious Diseases, Paolo Giaccone University Polyclinic in Palermo, during the period January 2007- August 2010 and all the children diagnosed with MSF at the G. Di Cristina Children Hospital in Palermo during the period January 1997- December 2004. On admission, a complete physical and laboratory examination was carried out on every patient. An indirect immunofluorescence assay for Rickettsia conorii was performed in every case, a PCR assay was performed with blood samples from some adult patients. Analysis of the results confirms that MSF is a benign, and self-limiting disease in children, while severe complications can often arise in adults.

Presence of heterocyclic aromatic amines (HAS) in smoked "Provola" cheese from Calabria (Italy).

This study evaluated the residual levels of heterocyclic aromatic amines (HAs) in "Provola" cheese samples from Calabria, smoked using natural methods and using commercial buffered smoke. A comparative study of HAs concentrations was carried out on different portions of these samples: the rind, exterior part, core and on slice. Quantitative determination of HAs was carried out by HPLC using a fluorescence detector and analysis in HPLC-MS was conduced to confirm the presence of these amines. Residual levels of HAs were found in all naturally smoked "Provola" cheese samples. The results obtained showed that the smoking process performed using traditional methods contributes to HAs contamination while the use of commercial buffered smoke can be considered a safer technique for smoking of food.

Influence of a nonfragile FHIT transgene on murine tumor susceptibility.

FHIT, at a constitutively active chromosome fragile site, is often a target of chromosomal aberrations and deletion in a large fraction of human tumors. Inactivation of murine Fhit allelessignificantly increases susceptibility of mice to spontaneous and carcinogen-induced tumorigenesis. In this study, transgenic mice, carrying a human FHIT cDNA under control of the endogenous promoter, were produced to determine the effect of Fhit expression, from a nonfragile cDNA transgene outside the fragile region, on carcinogen-induced tumor susceptibility of wildtype and Fhit heterozygous mice. Mice received sufficient oral doses of N-nitrosomethybenzylamine (NMBA) to cause forestomach tumors in >80% of nontransgenic control mice. Although the level of expression of the FHIT transgene in the recombinant mouse strains was much lower than the level of endogenous Fhit expression, the tumor burden in NMBA-treated male transgenic mice was significantly reduced, while female transgenic mice were not protected. To determine if the difference in protection could be due to differences in epigenetic changes at the transgene loci in male versus female mice, we examined expression, hypermethylation and induced re-expression of FHIT transgenes in male and female mice or cells derived from them. The transgene was methylated in male and female mice and in cell lines established from male and female transgenic kidneys, the FHIT locus was both hypermethylated and deacetylated. It is likely that the FHIT transgene is more tightly silenced in female transgenic mice, leading to a lack of protection from tumor induction.

Skeletal dysplasia and male infertility locus on mouse chromosome 9.

In mice and humans, growth insufficiency and male infertility are common disorders that are genetically and phenotypically complex. We describe a spontaneously arising mouse mutant, chagun, that is affected by both dwarfism and male infertility. Dwarfism disproportionately affects long bones and is characterized by a defect in the proliferative zone of chondrocytes in the growth plate. Gonads of mutant males are small, with apparent germ cell loss and no evidence of mature sperm. The locus responsible for chagun is recessive and maps to distal chromosome 9, in a region homologous to human chromosome 3. This location is consistent with chagun defining a novel locus. Identification of the mutant gene will uncover the basis for another type of skeletal dysplasia and male infertility.

Molecular cloning of Ian4: a BCR/ABL-induced gene that encodes an outer membrane mitochondrial protein with GTP-binding activity.

Using the representation difference analysis technique, we have identified a novel gene, Ian4, which is preferentially expressed in hematopoietic precursor 32D cells transfected with wild-type versus mutant forms of the Bcr/Abl oncogene. Ian4 expression was undetectable in 32D cells transfected with v-src, oncogenic Ha-ras or v-Abl. Murine Ian4 maps to chromosome 6, 25 cM from the centromere. The Ian4 mRNA contains two open reading frames (ORFs) separated by 5 nt. The first ORF has the potential to encode for a polypeptide of 67 amino acids without apparent homology to known proteins. The second ORF encodes a protein of 301 amino acids with a GTP/ATP-binding site in the N-terminus and a hydrophobic domain in the extreme C-terminus. The IAN-4 protein resides in the mitochondrial outer membrane and the last 20 amino acids are necessary for this localization. The IAN-4 protein has GTP-binding activity and shares sequence homology with a novel family of putative GTP-binding proteins: the immuno-associated nucleotide (IAN) family.

Genomic organization, promoter analysis, and chromosomal mapping of the mouse gene encoding Cdk9.

Cdk9, previously known as PITALRE, belongs to the Cdc2 family of protein kinases. We report the isolation and characterization of the complete gene coding for the murine Cdk9 protein. The gene contains seven exons spanning over 6 kb of genomic DNA, and the exon/intron boundaries conformed to the GT/AG rule. The Cdk9 gene mapped on mouse chromosome 2, which is consistent with the known region of synteny with human chromosome 9q34.1. The length of the individual exons ranged from 82 to 850 bp, and introns ranged from 452 to 1,465 bp. The further 5' flanking region of the gene showed features of a housekeeping promoter, such as the lack of a canonical TATA box and the presence of a CCAAT box as well as several GC boxes, which are potential binding sites for numerous transcription factors. Additionally, we performed a basic analysis of the transcriptional activity of the promoter and found that the 364 bp of Cdk9 5' flanking region were able to elicit high transcriptional levels of a luciferase reporter gene in NIH3T3 cells. This study provides the molecular basis for understanding the transcriptional control of the Cdk9 gene, and could serve to facilitate the molecular genetic investigation of Cdk9 function during mouse embryonal development.

Muir-Torre-like syndrome in Fhit-deficient mice.

To investigate the role of the Fhit gene in carcinogen induction of neoplasia, we have inactivated one Fhit allele in mouse embryonic stem cells and produced (129/SvJ x C57BL/6J) F(1) mice with a Fhit allele inactivated (+/-). Fhit +/+ and +/- mice were treated intragastrically with nitrosomethylbenzylamine and observed for 10 wk posttreatment. A total of 25% of the +/+ mice developed adenoma or papilloma of the forestomach, whereas 100% of the +/- mice developed multiple tumors that were a mixture of adenomas, squamous papillomas, invasive carcinomas of the forestomach, as well as tumors of sebaceous glands. The visceral and sebaceous tumors, which lacked Fhit protein, were similar to those characteristic of Muir-Torre familial cancer syndrome.

Hepatitis B virus transgenic mouse model of chronic liver disease.

A model for hepatitis B virus-associated chronic liver disease has been made using cloned hepatitis B virus DNA as a transgene in a severe combined immunodeficient host. These mice consistently support virus gene expression and replication. After adoptive transfer of unprimed, syngeneic splenocytes, these mice cleared virus from liver and serum, and developed chronic liver disease. This model will permit identification of the host and virus contributions to chronic liver disease in the absence of tolerance.

Complete cDNA cloning, genomic organization, chromosomal assignment, functional characterization of the promoter, and expression of the murine Bamacan gene.

Bamacan is a chondroitin sulfate proteoglycan that abounds in basement membranes. To gain insights into the bamacan gene regulation and transcriptional control, we examined the genomic organization and identified the promoter region of the mouse bamacan gene. Secondary structure analysis of the protein reveals a sequential organization of three globular regions interconnected by two alpha-helix coiled-coils. The N- and the C-terminal ends carry a P-loop and a DA box motif that can act cooperatively to bind ATP. These features as well as the high sequence homology with members of the SMC (structural maintenance of chromosome) protein family led us to conclude that bamacan is a member of this protein family. The gene comprises 31 exons and is driven by a promoter that is highly enriched in GC sequences and lacks TATA and CAAT boxes. The promoter is highly functional in transient cell transfection assays, and step-wise 5' deletions identify a strong enhancer element between -659 and -481 base pairs that includes Jun/Fos proto-oncogene-binding elements. Using backcrossing experiments we mapped the Bam gene to distal chromosome 19, a locus syntenic to human chromosome 10q25. Bamacan is differentially expressed in mouse tissues with the highest levels in testes and brain. Notably, bamacan mRNA levels are low in normal cells and markedly reduced during quiescence but are highly increased when cells resume growth upon serum stimulation. In contrast, in all transformed cells tested, bamacan is constitutively overexpressed, and its levels do not change with cell cycle progression. These results suggest that bamacan is involved in the control of cell growth and transformation.

Characterization of a novel receptor that maps near the natural killer gene complex: demonstration of carbohydrate binding and expression in hematopoietic cells.

A novel type II integral membrane protein has been identified in the course of screening for genes overexpressed in a mouse model of chronic myelogenous leukemia blast crisis. This new protein, designated NKCL, consists of a 210-amino acid polypeptide with a short, NH2-terminal cytoplasmic tail of 17 amino acids preceding a transmembrane domain and a COOH-terminal extracellular region. The COOH-terminal 132 amino acids bear typical features of the C-type animal lectin carbohydrate-recognition domain. The Nkcl gene is unique in that it maps just proximal to the region of the genome that encodes group V members of the C-type animal lectin family near the natural killer gene complex on mouse chromosome 6, but its protein product also has features of several group II C-type animal lectins. Most notably, it has a complete Ca2+-binding site 2, which forms part of the sugar-binding site in other members of the family, and binds mannose in a Ca2+-dependent manner. Moreover, its expression is not restricted to natural killer cells, as reported for the majority of group V lectins. Nkcl is expressed in pluripotent myeloid precursors, precursor and mature macrophages, and neutrophils.

The murine Fhit locus: isolation, characterization, and expression in normal and tumor cells.

The murine Fhit locus maps near the centromere nu proximal Ptprg locus on mouse chromosome 14. The cDNA sequence and structure are similar to those of the human gene, with exons 5-9 encoding the protein. The predominant mRNA in the tissues and cell lines tested was an alternatively spliced form missing exon 3. Most murine cell lines tested, including lines established from normal mouse embryos and tumors, expressed very low or undetectable levels of Fhit mRNA. Most normal mouse tissues expressed wild-type Fhit mRNA, whereas approximately 40% of murine lung carcinomas expressed wild-type and aberrant Fhit RT-PCR products that lacked various exons. Several tumorigenic mouse cell lines exhibited homozygous deletions of Fhit exons. We conclude that the murine Fhit gene, like its human counterpart, is a target of alterations involved in murine carcinogenesis.

The Tight skin mouse: demonstration of mutant fibrillin-1 production and assembly into abnormal microfibrils.

Mice carrying the Tight skin (Tsk) mutation harbor a genomic duplication within the fibrillin-1 (Fbn 1) gene that results in a larger than normal in-frame Fbn 1 transcript. In this study, the consequences of the Tsk mutation for fibrillin-containing microfibrils have been examined. Dermal fibroblasts from Tsk/+ mice synthesized and secreted both normal fibrillin (approximately 330 kD) and the mutant oversized Tsk fibrillin-1 (approximately 450 kD) in comparable amounts, and Tsk fibrillin-1 was stably incorporated into cell layers. Immunohistochemical and ultrastructural analyses of normal and Tsk/+ mouse skin highlighted differences in the gross organization and distribution of microfibrillar arrays. Rotary shadowing of high Mr preparations from Tsk/+ skin demonstrated the presence of abundant beaded microfibrils. Some of these had normal morphology and periodicity, but others were distinguished by diffuse interbeads, longer periodicity, and tendency to aggregate. The presence of a structurally abnormal population of microfibrils in Tsk/+ skin was unequivocally demonstrated after calcium chelation and in denaturating conditions. Scanning transmission electron microscopy highlighted the presence of more mass in Tsk/+ skin microfibrils than in normal mice skin microfibrils. These data indicate that Tsk fibrillin-1 polymerizes and becomes incorporated into a discrete population of beaded microfibrils with altered molecular organization.