The bifunctional protein GlmU is a key factor in biofilm formation induced by alkylating stress in Mycobacterium smegmatis.

Living organisms have developed specific defence mechanisms to counteract hostile environmental conditions. Alkylation stress response mechanisms also occur in Mycobacterium tuberculosis (MTB) the pathogen responsible for tuberculosis. The effect of alkylating agents on the cellular growth of MTB was investigated using methyl methanesulfonate (MMS) as methyl donor demonstrating that limited doses of alkylating agents might affect MTB cell viability. A global investigation of Mycobacterium smegmatis response to alkylating stress was then pursued by differential proteomics to identify the most affected cellular pathways. Quantitative analysis of proteomic profiles demonstrated that most of the proteins upregulated in the presence of alkylating agents are involved in biofilm formation and/or cell wall biosynthesis. Tailored experiments confirmed that under stress conditions M. smegmatis elicits physical defence mechanisms by increasing biofilm formation. Among the upregulated proteins, we identified the GlmU bifunctional enzyme as a possible factor involved in biofilm production. Experiments with both conditional deletion and overexpressing glmU mutants demonstrated that down regulation of GlmU decreased M. smegmatis capabilities to produce biofilm whereas overexpression of the enzyme increased biofilm formation. These results were supported by inhibition of GlmU acetyltransferase activity with two different inhibitors, suggesting the involvement of this enzyme in the M. smegmatis defence mechanisms.

Levels of different cytokines in women and men with asymptomatic genital infection caused by Chlamydia.

INTRODUCTION: Immune response to genital Chlamydia trachomatis infection is involved in both immunity and pathology. The cytokine profile during infection has been implicated in the disease outcome, either resolution or severe sequelae. METHODOLOGY: In total, 3900 patients were analyzed for presence of genital infections caused by Chlamydia using molecular assays. Interleukins (IL) IL-10, IL-17, IL-6, IL-2 and chemokine IP-10 were estimated by ELISA in urine, cervical swabs and semen samples. Statistical analysis was performed using the T student test. RESULTS: A total of 47 out of 3900 samples (1.2%) were found to be positive for Chlamydia trachomatis based on the Real Time (RT) PCR results. Statistical analysis revealed that the differences between Chlamydia trachomatis positive and negative samples regarding levels of cytokines were not significant. CONCLUSIONS: Our results demonstrated that no significant difference in cytokine concentrations exists in Chlamydia trachomatis infected patients when compared to healthy controls. In further study, we aim to test on a greater number of positive samples a greater number of cytokines involved in the immune response to Chlamydia trachomatis infections.

In vitro activity of hybrid lavender essential oils against multidrug resistant strains of Pseudomonas aeruginosa.

INTRODUCTION: Lavender is an evergreen shrub native to Northern Africa and other mountainous Mediterranean regions. It grows throughout Southern Europe, the United States, and Australia. Lavender essential oil has been used since ancient times and is known for its anti-inflammatory, antidepressant, antiseptic, antifungal and antimicrobial properties. METHODOLOGY: in this study, the antimicrobial activity of two Lavender essential oils (Lavanda sumian and Lavanda grosso) against 16 multidrug-resistant P. aeruginosa strains from clinical ocular samples taken from migrant patients has been investigated. The in vitro cytotoxic activity on human Wong-Kilbourne derivative (WKD) conjunctiva cells from healthy patients and nitric oxide synthase (NOS) activity on murine macrophage (J774.1A) were also evaluated. RESULTS: L. sumian showed lower antimicrobial activity when compared to L. grosso. Both lavender oils tested had no cytotoxic effect at very low concentrations, mostly L. grosso. The essential oils extracted from L. sumian and L. grosso significantly reduced NOS in a cell model. CONCLUSION: Increase in drug resistance and lack of new antibiotics may encourage the development of natural antimicrobial treatments.

Tuberculosis in Sardinia: An investigation into the relationship between natives and immigrants.

OBJECTIVE/BACKGROUND: Tuberculosis (TB) has had a recrudescence in the last few decades in Italy as a result of many factors, among which migration from countries where TB is endemic is one of them. In Sardinia, a major island of Italy, there was no knowledge of the mechanisms of transmission of TB in the immigrant subpopulation and the impact it may have on the native subpopulation and on the community as a whole. Therefore, a molecular epidemiological study was carried out to get a clearer picture of the number and genetic features of Mycobacterium tuberculosis strains isolated from immigrants and from natives in Sardinia. METHODS: Two groups of clinical isolates of M. tuberculosis, one collected from immigrants and the other one from Sardinians, were analyzed in this study. The genotyping was executed through the variable number tandem repeat-mycobacterial interspersed repetitive units technique and a first-line antimycobacterial drug-susceptibility test was also carried out. RESULTS: Thirty-six clinical isolates from immigrants and 25 from Sardinians were analyzed. Variable number tandem repeat-mycobacterial interspersed repetitive units technique showed that all of them belonged to different strains and there was a quite high allelic diversity among them. Moreover, data collected allowed the finding of, with a good approximation, the phylogenetic relations among the strains isolated and the best-known phylogenetic groups. CONCLUSION: The study pointed out that since every strain is different, there was no TB transmission in any of the subpopulations and between immigrants and natives. This showed that the presence of immigrants was not a risk factor for contracting TB in the community.

Bacterial agents as a cause of infertility in humans.

Infertility is a problem affecting almost 15% of couples. There are many causes for this condition, among which urogenital bacterial infections seem to play an important role. Many studies have explained the mechanisms by which bacteria cause infertility both in men and women. Therefore we undertook this study to evaluate the presence of genito-urinary infections in infertile couples who sought counselling to investigate their condition. Microbiological analysis was performed on semen and vaginal/cervical samples of both partners of each couple. The percentage of individuals affected by a urogenital bacterial infection was between 14 and 20%. More significantly, most of the species isolated both in men and women have been described in the literature as potential causes of infertility.

Chemical composition, cytotoxicity, antimicrobial and antifungal activity of several essential oils.

Essential oils (EOs) are known and used for their biological, antibacterial, antifungal and antioxidant properties. Numerous studies have shown that EOs exhibit a large spectrum of biological activities in vitro. The incidence of drug-resistant pathogens and the toxicity of antibiotics have drawn attention to the antimicrobial activity of natural products, encouraging the development of alternative treatments. The aim of this study was to analyse the phytochemical and the cytotoxic characteristic of 36 EOs; we then evaluated the antimicrobial activity of the less-toxic EOs on Gram-positive, Gram-negative and fungi strains. The results showed low cytotoxicity in seven EOs and good activity against Gram-negative and Candida spp. strains. Based on our results, EOs could be proposed as a novel group of therapeutic agents. Further experiments are necessary to confirm their pharmacological effectiveness, and to determine potential toxic effects and the mechanism of their activity in in vivo models.

Essential oils in ocular pathology: an experimental study.

INTRODUCTION: The antimicrobial activity of essential oils (EOs) has been known for ages; in particular, the EOs of Melaleuca alternifolia, Thymus vulgaris, Mentha piperita, and Rosmarinus officinalis have been used for the treatment of fungal and bacterial infections. METHODOLOGY: This study focused on the in vitro cytotoxicity to normal human conjunctiva cells and antimicrobial activity of 20 EOs. RESULTS: The oils tested showed no cytotoxic effect at very low concentrations. Rosmarinus officinalis, Melaleuca alternifolia, and Thymus vulgaris L. red thyme geraniol sel oils had good antimicrobial activity against Gram-positive and Gram-negative strains. CONCLUSIONS: The results of this study are of great interest and may have a major impact on public health, providing useful tips to optimize the therapeutic use of some natural drugs.

Antitubercular activity of quinolizidinyl/pyrrolizidinylalkyliminophenazines.

Novel riminophenazine derivatives, characterized by the presence of the basic and cumbersome quinolizidinylalkyl and pyrrolizidinylethyl moieties, have been synthesized and tested (Rema test) against Mycobacterium tuberculosis H37Rv and H37Ra, and six clinical isolates of Mycobacterium avium and Mycobacterium tuberculosis. Most compounds exhibited potent activity against the tested strains, resulting more active than clofazimine, isoniazid and ethambutol. The best compounds (4, 5, 12 and 13) exhibited a MIC in the range 0.82-0.86µM against all strains of Mycobacterium tuberculosis and, with the exception of 4 a MIC around 3.3µM versus M. avium. The corresponding values for clofazimine (CFM) were 1.06 and 4.23µM, respectively. Cytotoxicity was evaluated against three cell lines and compound 4 displayed a selectivity index (SI) versus the human cell line MT-4 comparable with that of CFM (SI=5.23 vs 6.4). Toxicity against mammalian Vero 76 cell line was quite lower with SI=79.

Fear but not fright: re-evaluating traumatic experience attenuates anxiety-like behaviors after fear conditioning.

Fear allows organisms to cope with dangerous situations and remembering these situations has an adaptive role preserving individuals from injury and death. However, recalling traumatic memories can induce re-experiencing the trauma, thus resulting in a maladaptive fear. A failure to properly regulate fear responses has been associated with anxiety disorders, like Posttraumatic Stress Disorder (PTSD). Thus, re-establishing the capability to regulate fear has an important role for its adaptive and clinical relevance. Strategies aimed at erasing fear memories have been proposed, although there are limits about their efficiency in treating anxiety disorders. To re-establish fear regulation, here we propose a new approach, based on the re-evaluation of the aversive value of traumatic experience. Mice were submitted to a contextual-fear-conditioning paradigm in which a neutral context was paired with an intense electric footshock. Three weeks after acquisition, conditioned mice were treated with a less intense footshock (pain threshold). The effectiveness of this procedure in reducing fear expression was assessed in terms of behavioral outcomes related to PTSD (e.g., hyper-reactivity to a neutral tone, anxiety levels in a plus maze task, social avoidance, and learning deficits in a spatial water maze) and of amygdala activity by evaluating c-fos expression. Furthermore, a possible role of lateral orbitofrontal cortex (lOFC) in mediating the behavioral effects induced by the re-evaluation procedure was investigated. We observed that this treatment: (i) significantly mitigates the abnormal behavioral outcomes induced by trauma; (ii) persistently attenuates fear expression without erasing contextual memory; (iii) prevents fear reinstatement; (iv) reduces amygdala activity; and (v) requires an intact lOFC to be effective. These results suggest that an effective strategy to treat pathological anxiety should address cognitive re-evaluation of the traumatic experience mediated by lOFC.

Antifungal, anti-biofilm and adhesion activity of the essential oil of Myrtus communis L. against Candida species.

Candida species belong to the normal microbiota of the oral cavity, gastrointestinal tract and vagina. The increasing incidence of drug-resistant pathogens and the toxicity of the antifungal compounds have drawn the attention towards the antimicrobial activity of natural products, an inexpensive alternative. The aim of this work was to evaluate the adhesion activity, the biofilm formation and the action of the Myrtus communis L. essential oil (EO) on the biofilm formation towards three species isolated from clinical samples: Candida albicans, Candida parapsilosis and Candida tropicalis. Furthermore, we evaluated the antimycotic activity of the EO towards the three species, and the results were compared with the minimum inhibitory concentration of six antimycotics. The activity of the EO against C. albicans and C. parapsilosis was better than that obtained against C. tropicalis; moreover, the strains used in the assay were adhesive and biofilm producer, and the effect of myrtle EO on the biofilm formation yielded encouraging results.

Tis21 is required for adult neurogenesis in the subventricular zone and for olfactory behavior regulating cyclins, BMP4, Hes1/5 and Ids.

Bone morphogenic proteins (BMPs) and the Notch pathway regulate quiescence and self-renewal of stem cells of the subventricular zone (SVZ), an adult neurogenic niche. Here we analyze the role at the intersection of these pathways of Tis21 (Btg2/PC3), a gene regulating proliferation and differentiation of adult SVZ stem and progenitor cells. In Tis21-null SVZ and cultured neurospheres, we observed a strong decrease in the expression of BMP4 and its effectors Smad1/8, while the Notch anti-neural mediators Hes1/5 and the basic helix-loop-helix (bHLH) inhibitors Id1-3 increased. Consistently, expression of the proneural bHLH gene NeuroD1 decreased. Moreover, cyclins D1/2, A2, and E were strongly up-regulated. Thus, in the SVZ Tis21 activates the BMP pathway and inhibits the Notch pathway and the cell cycle. Correspondingly, the Tis21-null SVZ stem cells greatly increased; nonetheless, the proliferating neuroblasts diminished, whereas the post-mitotic neuroblasts paradoxically accumulated in SVZ, failing to migrate along the rostral migratory stream to the olfactory bulb. The ability, however, of neuroblasts to migrate from SVZ explants was not affected, suggesting that Tis21-null neuroblasts do not migrate to the olfactory bulb because of a defect in terminal differentiation. Notably, BMP4 addition or Id3 silencing rescued the defective differentiation observed in Tis21-null neurospheres, indicating that they mediate the Tis21 pro-differentiative action. The reduced number of granule neurons in the Tis21-null olfactory bulb led to a defect in olfactory detection threshold, without effect on olfactory memory, also suggesting that within olfactory circuits new granule neurons play a primary role in odor sensitivity rather than in memory.

Clinical considerations on Buruli ulcer employing two molecular tests for the detection of Mycobacterium ulcerans in 100 skin biopsies.

OBJECTIVES: Buruli ulcer (BU) is an infected cutaneous lesion, the etiological agent of which is Mycobacterium ulcerans. Diagnosis is confirmed by the identification of acid-fast bacilli and culture. In clinically suspicious forms with negative bacteriological or Ziehl-Neelsen (ZN) findings, molecular tests are used. This study compared the concordance of nested polymerase chain reaction (PCR) (targeting IS2404) and PCR (targeting IS2606) in different clinical situations. METHODS: A total of 57 samples were sourced from 39 BU patients. Control samples (n = 43) were obtained from non-BU ulcers in 38 patients. Samples were divided into two pieces and submitted to, respectively, histological examination and ZN staining, and PCR. Subsamples submitted to PCR were divided and submitted to nested PCR IS2404 and PCR IS2606, respectively. RESULTS: Of the 57 BU biopsies, positive results were obtained by nested PCR in 18 (31.6%) and by IS2606 PCR in 37 (64.9%) cases. Sequencing of the positive samples confirmed the specificity of amplicons in all nested PCR samples and in 26 of 37 (70.2%) samples positive to IS2606. Hence, nested PCR was more specific (100% vs. 93%) and less sensitive (32% vs. 46%) than IS2606 PCR. In the BU samples, nested PCR was negative in 15 instances, and IS2606 PCR was negative in 11 instances in which ZN histology had been positive (false negatives). Both PCRs were positive in six ZN-negative smears. CONCLUSIONS: We considered 57 samples from 39 BU patients in various clinical stages and at different times after the beginning of therapy. These provided positive results in 18 cases with IS2404 nested PCR and in 37 cases with PCR IS2606; only 26 of the latter remained positive subsequent to sequencing. Hence, even if IS2404 PCR is considered more specific, in subjects who appear to fail to respond to therapy, it is advisable to also carry out IS2606 PCR. A possible interpretation of the discordance between the two techniques due to unavoidable technical errors as well as to different sensitivity of the two tests at M. ulcerans DNA low concentration (i.e. in recent infection and in well-treated cases) is discussed.

Identification of non-tuberculous mycobacteria from clinical samples.

Non-tuberculous mycobacterial infections cause morbidity worldwide. NTM are considered opportunistic pathogens, and several species have been associated with human disease which has typically pulmonary, skin and soft tissue, lymphatic or disseminated presentation. This study evaluated the distribution of non-tuberculous mycobacteria in Sardinia. Mycobacterium avium, Mycobacterium gordonae and Mycobacterium xenopi were frequently found. Our results agreed with literature data both for the frequent isolation of M. avium, M. xenopi and M. gordonae, and the symptoms and radiological evidence of the patients analysed.

Molecular identification of Mycobacterium avium subspecies paratuberculosis in oral biopsies of Crohn's disease patients.

Oral lesions may be found in patients with Crohn's disease (CD), in a percentage up to 20%. The aim of this study was to investigate a possible relationship between Mycobacterium avium subsp. paratuberculosis (MAP) and oral lesions in CD patients. 23 oral biopsies were examined performing IS900 Nested PCR; 9 of them were positive: 8 from CD patients and 1 from a control. Our purpose is to go on with this study, amplifying the number of subjects examined and testing subjects with oral lesions related to diseases other than CD to verify the specific association between MAP and oral lesions in CD patients.

Impact of N-tau on adult hippocampal neurogenesis, anxiety, and memory.

Different pathological tau species are involved in memory loss in Alzheimer's disease, the most common cause of dementia among older people. However, little is known about how tau pathology directly affects adult hippocampal neurogenesis, a unique form of structural plasticity implicated in hippocampus-dependent spatial learning and mood-related behavior. To this aim, we generated a transgenic mouse model conditionally expressing a pathological tau fragment (26-230 aa of the longest human tau isoform, or N-tau) in nestin-positive stem/progenitor cells. We found that N-tau reduced the proliferation of progenitor cells in the adult dentate gyrus, reduced cell survival and increased cell death by a caspase-3-independent mechanism, and recruited microglia. Although the number of terminally differentiated neurons was reduced, these showed an increased dendritic arborization and spine density. This resulted in an increase of anxiety-related behavior and an impairment of episodic-like memory, whereas less complex forms of spatial learning remained unaltered. Understanding how pathological tau species directly affect neurogenesis is important for developing potential therapeutic strategies to direct neurogenic instructive cues for hippocampal function repair.

Tuberculin skin test and QuantiFERON in children.

Until some time ago, the tuberculin skin test was the only available screening test for the diagnosis of tubercular infection. Now the new interferon-? release assay QuantiFERON-TB Gold shows promise of greater accuracy in the detection of Mycobacterium tuberculosis-infected subjects. The aim of our study was to evaluate the use of QuantiFERONTB Gold in children and to verify its agreement with the tuberculin skin test. A total of 27 children had a positive tuberculin skin test, 76 subjects were negative and the remaining 2 had a dubious Mantoux test. A positive QuantiFERONTB Gold result was obtained in 21 children while in 84 it was negative. No statistically significant difference was detected between the two assays, which showed a concordance of 90.57%. Our results demonstrated a good concordance between the tuberculin skin test and the interferon-? release assay, though the QuantiFERON-TB may have several advantages over the Mantoux test.

Btg1 is Required to Maintain the Pool of Stem and Progenitor Cells of the Dentate Gyrus and Subventricular Zone.

Btg1 belongs to a family of cell cycle inhibitory genes. We observed that Btg1 is highly expressed in adult neurogenic niches, i.e., the dentate gyrus and subventricular zone (SVZ). Thus, we generated Btg1 knockout mice to analyze the role of Btg1 in the process of generation of adult new neurons. Ablation of Btg1 causes a transient increase of the proliferating dentate gyrus stem and progenitor cells at post-natal day 7; however, at 2 months of age the number of these proliferating cells, as well as of mature neurons, greatly decreases compared to wild-type controls. Remarkably, adult dentate gyrus stem and progenitor cells of Btg1-null mice exit the cell cycle after completing the S phase, express p53 and p21 at high levels and undergo apoptosis within 5 days. In the SVZ of adult (two-month-old) Btg1-null mice we observed an equivalent decrease, associated to apoptosis, of stem cells, neuroblasts, and neurons; furthermore, neurospheres derived from SVZ stem cells showed an age-dependent decrease of the self-renewal and expansion capacity. We conclude that ablation of Btg1 reduces the pool of dividing adult stem and progenitor cells in the dentate gyrus and SVZ by decreasing their proliferative capacity and inducing apoptosis, probably reflecting impairment of the control of the cell cycle transition from G1 to S phase. As a result, the ability of Btg1-null mice to discriminate among overlapping contextual memories was affected. Btg1 appears, therefore, to be required for maintaining adult stem and progenitor cells quiescence and self-renewal.

Epidemic of tuberculosis in a high school in Northern Sardinia.

The aim of this study was to investigate the Mycobacterium tuberculosis transmission among high school student and teacher populations in a high school in Northern Sardinia. Tuberculin skin-test screening, chest-X-rays, QuantiFERON-TB Gold, microbiological examination, spoligotyping and variable numbers of tandem repeats (VNTR) analysis of M. tuberculosis isolates were performed. This study indicates the effectiveness of the epidemiological investigation.