Dim artificial light at night alters gene expression rhythms and growth in a key seagrass species (Posidonia oceanica).

Artificial light at night (ALAN) is a globally spreading anthropogenic stressor, affecting more than 20% of coastal habitats. The alteration of the natural light/darkness cycle is expected to impact the physiology of organisms by acting on the complex circuits termed as circadian rhythms. Our understanding of the impact of ALAN on marine organisms is lagging behind that of terrestrial ones, and effects on marine primary producers are almost unexplored. Here, we investigated the molecular and physiological response of the Mediterranean seagrass, Posidonia oceanica (L.) Delile, as model to evaluate the effect of ALAN on seagrass populations established in shallow waters, by taking advantage of a decreasing gradient of dim nocturnal light intensity (from < 0.01 to 4 lx) along the NW Mediterranean coastline. We first monitored the fluctuations of putative circadian-clock genes over a period of 24 h along the ALAN gradient. We then investigated whether key physiological processes, known to be synchronized with day length by the circadian rhythm, were also affected by ALAN. ALAN influenced the light signalling at dusk/night in P. oceanica, including that of shorter blue wavelengths, through the ELF3-LUX1-ZTL regulatory network, and suggested that the daily perturbation of internal clock orthologs in seagrass might have caused the recruitment of PoSEND33 and PoPSBS genes to mitigate the repercussions of a nocturnal stress on photosynthesis during the day. A long-lasting impairment of gene fluctuations in sites characterised by ALAN could explain the reduced growth of the seagrass leaves when these were transferred into controlled conditions and without lighting during the night. Our results highlight the potential contribution of ALAN to the global loss of seagrass meadows, posing questions about key interactions with a variety of other human-related stressors in urban areas, in order to develop more efficient strategies to globally preserve these coastal foundation species.

Short-term stability of rocky intertidal biofilm to nitrogen and phosphorus pulses.

Coastal environments experience both natural and anthropogenic inputs of nitrogen (N) and phosphorus (P). Agricultural fertilisers, organic run-offs, and edaphic characteristics of coastal environments may generate mosaics of nutrient concentrations that ultimately influence the coastal primary productivity. Here, we experimentally assessed the effects of repeated pulses of N and P on multiple components of ecological stability (sensitivity, resilience, temporal stability and recovery) of phototrophic rocky intertidal biofilm. We performed a repeated-pulses factorial experiment crossing increasing N and P concentrations chosen to reflect a range of nutrient enrichment conditions, from oligotrophic to eutrophic. N and P, regardless of concentration or whether they occurred in isolation or combination, enhanced biofilm's sensitivity (increased biomass or physiological performance compared to controls) without altering resilience. Our experiment illustrates how the stability of an essential coastal primary producer responds to increasing N and P supply levels. Furthermore, notwithstanding the importance of decomposing the multiple dimensions of stability, the transitory increase of the sole sensitivity indicated that rocky shore biofilm is robust against a wide range of nutrient enrichment.

Lack of circulating toxoplasma gondii DNA in seropositive patients with bipolar or schizophrenia spectrum disorders.

Toxoplasmosis has been previously associated with an increased risk of having Schizophrenia or Bipolar disorder in several epidemiological studies. The aim of this observational, cross-sectional study was to examine the seroprevalence of Toxoplasma infection in a cohort of Italian psychiatric inpatients and to verify the presence of circulating Toxoplasma gondii DNA in the seropositive subjects. Sixty-three patients affected by bipolar or schizoaffective disorders according to DSM-5 criteria were enrolled. The presence of Toxoplasma infection was firstly examined using an indirect serological method (ELFA), and three different direct PCR-based methods were performed to detect circulating DNA in the seropositive patients. The seroprevalence of infection was 28.6%, with a significant association between higher age and the infection status. PCR, nested-PCR and Real-Time PCR revealed no positive samples for Toxoplasma gondii. This result is in contrast with recent data from case-control studies that detected parasite genome in patients with different neuropsychiatric diagnosis without clinical evidence of acute toxoplasmosis. Our findings are to be interpreted with caution, because of the small sample size, the heterogeneity of enrolled patients and the observational nature of the study. Further studies are needed to better define the clinical features correlated to the seropositive status in neuropsychiatric patients.

Experimental evidence of spatial signatures of approaching regime shifts in macroalgal canopies.

Developing early warning signals to predict regime shifts in ecosystems is a central issue in current ecological research. While there are many studies addressing temporal early warning indicators, research into spatial indicators is far behind, with field experiments even more rare. Here, we tested the performance of spatial early warning signals in an intertidal macroalgal system, where removal of algal canopies pushed the system toward a tipping point (corresponding to approximately 75% of canopy loss), marking the transition between a canopy- to a turf-dominated state. We performed a two-year experiment where spatial early warning indicators were assessed in transects where the canopy was differentially removed (from 0 to 100%). Unlike Moran correlation coefficient at lag-1, spatial variance, skewness, and spatial spectra at low frequency increased along the gradient of canopy degradation and dropped, or did not show any further increase beyond the transition point from a canopy- to a turf-dominated state (100% canopy removal). Our study provides direct evidence of the suitability of spatial early warning signals to anticipate regime shifts in natural ecosystems, emphasizing the importance of field experiments as a powerful tool to establish causal relationships between environmental stressors and early warning indicators.

Impact of immigration on tuberculosis in a low-incidence area of Italy: a molecular epidemiological approach.

The effects that immigration might have on the epidemiology of tuberculosis (TB) in a low-incidence area of Italy was investigated by determining, in autochthonous and immigrant TB patients, the molecular characteristics of the Mycobacterium tuberculosis complex (MTBC) isolates, which may provide information on their phylogeographical origin. A total of 1080 MTBC strains, collected during a 4- year period in Tuscany from 614 Italian-born and 466 foreign-born patients, were genotyped by spoligotyping and assigned to the different phylogeographical lineages that constitute the MTBC. The autochthonous Euro-American phylogeographical lineage, which includes the spoligotype families T, Haarlem, Latin American­Mediterranean (LAM), S and X, was highly prevalent among Italian-born patients, with a total of 477 cases (77.7%), and foreign-born TB patients, with a total of 270 cases (57.9%); 24 Italian-born (3.9%) and 141 foreign- born (30.3%) TB cases were due to MTBC genotypic families associated with distant geographical areas, i.e. East African­Indian (EAI), Beijing, Central Asian (CAS), and Mycobacterium africanum. Strains of Mycobacterium bovis and strains of undefined genotype, which are all considered together, as it is not possible to assign a specific geographical origin, accounted for 113 (18.4%) Italian cases and 55 (11.8%) foreign-born cases. A total of 79 Italian TB cases (12.9%) have been attributed to transmission from immigrants to the local population. No significant contribution to drug resistance appeared to be associated with imported MTBC strains. It is concluded that, at present, the overall impact of imported TB on public health in the low-incidence study area is relatively modest and of the same order as in other western countries.

Association of Mycobacterium tuberculosis complex isolates of BOVIS and Central Asian (CAS) genotypic lineages with extrapulmonary disease.

The association between isolate genotype, defined as in the international spoligotype database SpolDB4, and extrapulmonary tuberculosis was determined among 1009 patients in a population-based, 4-year survey performed in Tuscany, Italy. Extrapulmonary disease occurred in 24.2% of patients. A statistically significant association with extrapulmonary disease was found for the BOVIS (adjusted OR 3.2; 95% CI 1.2-8.1) and for the Central Asian (CAS) lineages (adjusted OR 2.3; 95% CI 1.0-5.1). These findings support the view that Mycobacterium tuberculosis strains within individual genotypic lineages might have evolved unique pathogenic characteristics that are capable of influencing the clinical outcome of the infection.

Mutations responsible for Mycobacterium tuberculosis isoniazid resistance in Italy.

SETTING: The incidence of tuberculosis (TB) and drug resistance in Italy is low compared to other countries. Mutations in several genomic regions of Mycobacterium tuberculosis are involved in the occurrence of isoniazid (INH) resistance. OBJECTIVE: To investigate the mutations responsible for INH resistance among Italian isolates of M. tuberculosis, to assess the feasibility of predicting drug resistance using a genetic approach. DESIGN: The mutations responsible for INH resistance were looked for in selected regions of genes katG, kasA and ndh and in the promoter regions of inhA and ahpC by nucleotide sequencing, and the results were compared with data reported in other studies. RESULTS: Prevalent INH resistance mutations were found at codon 315 of the katG gene and at position -15 of the inhA regulatory region (respectively 37.8% and 20.0% of isolates). The prevalence of mutations at position -24 of inhA, in ahpC, and in kasA ranged from 2.2% to 4.4%. No mutations were found in 35.6% of the isolates. CONCLUSION: The identification of INH resistance by genetic analysis of the selected regions may be inappropriate in areas with a low prevalence of TB, such as Italy, as the genetic mechanisms of resistance remain unidentified for approximately one third of the isolates.

Isolation of a novel sequevar of Mycobacterium flavescens from the synovial fluid of an AIDS patient.

This report describes the characterisation of a mycobacterium involved in a case of septic arthritis in an AIDS patient that was treated successfully with specific anti-mycobacterial drugs. The biochemical and cultural features, and the mycolic acid pattern as assessed by high-performance liquid chromatography, were fully compatible with the isolate being Mycobacterium flavescens. However, the isolate's 16S rDNA sequence differed by five nucleotides from the two known sequevars of M. flavescens, thus indicating that this isolate belonged to a new 16S rDNA sequevar.

Requirement of gene fadD33 for the growth of Mycobacterium tuberculosis in a hepatocyte cell line.

Gene fadD33 of Mycobacterium tuberculosis, one of the 36 homologues of gene fadD of Escherichia coli identified in the M. tuberculosis genome, predictively encodes an acyl-CoA synthase, an enzyme involved in fatty acids metabolism. The gene is underexpressed in the attenuated strain M. tuberculosis H37Ra relative to virulent H37Rv and plays a role in M. tuberculosis virulence in BALB/c mice by supporting mycobacterial replication in the liver. In the present paper, we investigated the role of fadD33 expression in bacterial growth within the hepatocyte cell line HepG2, as well as in human monocyte-derived THP-1 cells and peripheral blood mononuclear cells. M. tuberculosis H37Rv proved able to grow within HepG2 cells, while the intracellular replication of M. tuberculosis H37Ra was markedly impaired; complementation of strain H37Ra with gene fadD33 restored its replication to the levels of H37Rv. Moreover, disruption of gene fadD33 by allelic exchange mutagenesis reduced the intracellular growth of M. tuberculosis H37Rv, and complementation of the fadD33-disrupted mutant with gene fadD33 restored bacterial replication. Conversely, fadD33 expression proved unable to influence M. tuberculosis growth in human phagocytes, as fadD33-disrupted M. tuberculosis H37Rv mutant, as well as fadD33-complemented M. tuberculosis H37Ra, grew within THP-1 cells and peripheral monocytes basically at the same rates as parent H37Rv and H37Ra strains. The results of these experiments indicate that gene fadD33 expression confers growth advantage to M. tuberculosis in immortalized hepatocytes, but not in macrophages, thus emphasizing the importance of fadD33 in liver-specific replication of M. tuberculosis.

Mycobacterium elephantis: not an exceptional finding in clinical specimens.

Following the recent report of new 16S rDNA sequences of Mycobacterium elephantis, three clinical strains suspected to belong to such species were investigated using biochemical and cultural tests, high performance liquid chromatography of cell wall mycolic acids and genetic sequencing. Antimicrobial susceptibility was also determined. The findings confirmed recent data concerning human isolates of this new mycobacterium and identified a new 16S rDNA sequevar for this species.

Burden of unidentifiable mycobacteria in a reference laboratory.

Modern identification techniques at the genomic level have greatly improved the taxonomic knowledge of mycobacteria. In adjunct to nucleic acid sequences, mycobacterial identification has been endorsed by investigation of the lipidic patterns of unique mycolic acids in such organisms. In the present investigation, the routine use of high-performance liquid chromatography (HPLC) of mycolic acids, followed by the sequencing of the 16S rRNA, allowed us to select 72 mycobacterial strains, out of 1,035 screened, that do not belong to any of the officially recognized mycobacterial species. Most strains (i.e., 47) were isolated from humans, 13 were from the environment, 3 were from animals, and 9 were from unknown sources. The majority of human isolates were grown from the respiratory tract and were therefore most likely not clinically significant. Some, however, were isolated from sterile sites (blood, pleural biopsy, central venous catheter, or pus). Many isolates, including several clusters of two or more strains, mostly slow growers and scotochromogenic, presented unique genetic and lipidic features. We hope the data reported here, including the results of major conventional identification tests, the HPLC profiles of strains isolated several times, and the whole sequences of the 16S rRNA hypervariable regions of all 72 mycobacteria, may encourage reporting of new cases. The taxonomy of the genus Mycobacterium is, in our opinion, still far from being fully elucidated, and the reporting of unusual strains provides the best background for the recognition of new species. Our report also shows the usefulness of the integration of novel technology to routine diagnosis, especially in cases involving slow-growing microorganisms such as mycobacteria.

Genes of Mycobacterium tuberculosis H37Rv downregulated in the attenuated strain H37Ra are restricted to M. tuberculosis complex species.

By comparing gene expression of virulent Mycobacterium tuberculosis H37Rv and attenuated strain H37Ra, we previously detected six genes that appear to be markedly downregulated in the attenuated strain compared with the virulent one. Three of these genes, i.e. Rv1345, Rv2770c, and Rv0288, code for proteins that can be predictively associated to immunological or pathogenetic aspects of M. tuberculosis infection; the other genes, i.e. Rv2336, Rv1320c, and Rv2819c, code for proteins with unknown functions (Rindi et al., 1999). In this paper we searched for the above mentioned genes in Pvu II-digested genomic DNA of a number of mycobacterial species by southern blot analysis employing PCR-generated probes in high-stringency conditions. Hybridization signals were only found in species belonging to the M. tuberculosis complex, i.e., M. tuberculosis, M. bovis, including the BCG strain, and M. microti, but not in other mycobacterial species, including M. avium, M. intracellulare, M. malmoense, M. xenopi, M. kansasii, M. simiae, M. marinum, M. scrofulaceum, M. gordonae, M. fortuitum, and M. smegmantis. These results indicate that genes Rv1345, Rv2770c, Rv0288, Rv2336, Rv1320c, and Rv2819c are associated with the most virulent mycobacteria and further support their potential role in M. tuberculosis virulence.

IS6110-based restriction fragment length polymorphism (RFLP) analysis of Mycobacterium tuberculosis H37Rv and H37Ra.

IS6110-based restriction fragment length polymorphism (RFLP) analysis of Mycobacterium tuberculosis H37Rv and its avirulent mutant H37Ra was performed by a number of restriction enzymes, including Nru I, EcoN I, Pst I, and Pvu II. No differences were found in the IS 6110-fingerprints of the study strains by Nru I. One differential IS6110-positive restriction fragment was detected by EcoN I in strain H37Ra, while analysis by Pst I revealed that two fragments of the strain H37Rv were replaced by four novel IS6110-positive fragments in the strain H37Ra. By using Pvu II, a restriction enzyme that cleaves IS 6110 once, and by probing for an IS6110 specific target sequence located to the right of the Pvu II site, we found that the strains H37Rv and H37Ra share 13 IS6110-positive restriction fragments and that one IS6110-positive restriction fragment of H37Rv is replaced by four novel fragments in H37Ra; by probing for an IS6110-specific target sequence to the left of the Pvu II site, 13 shared restriction fragments and 2 differential bands in strain H37Ra were detected. These findings demonstrate that novel insertions of the IS6110 element exist in the avirulent strain H37Ra and raise the question of the role, if any, of IS6110-insertional mutagenesis in the establishment of the avirulent M. tuberculosis H37Ra phenotype.

Search for genes potentially involved in Mycobacterium tuberculosis virulence by mRNA differential display.

An mRNA differential display (DD) assay was developed to compare gene expression between Mycobacterium tuberculosis H37Rv and its avirulent mutant H37Ra. The DD protocol made use of an oligo(dT) to prime reverse-transcriptase (RT)-dependent transcription of poly-A tailed mRNAs and a PCR amplification of the RT products by using ten 12-base arbitrary primers in all their pair combinations. This analysis yielded 745 and 708 bands, including 52 and 15 differentially generated bands, in the strains H37Rv and H37Ra, respectively. Six cDNAs that appeared to be expressed in H37Rv, but not in H37Ra, were reamplified and cloned and at least 10 inserts were sequenced for each cloned cDNA. After resolving discrepant results, 6 inserts were found highly homologous to M. tuberculosis H37Rv genes. Three of these, i.e., genes Rv2770c, Rv1345, and Rv0288, coding respectively for a member of the PPE protein family, a probable polyketide synthase, and a member of the protein family containing ESAT-6, have been predictively associated to immunological or pathogenetic aspects of M. tuberculosis infection; the other genes, i.e., Rv2336, Rv1320c, and Rv2819c, code for proteins with unknown functions. These results show that mRNA DD methodology can represent a potential tool for investigation of M. tuberculosis gene expression.

Typing of human Mycobacterium avium isolates in Italy by IS1245-based restriction fragment length polymorphism analysis.

All but 2 of 63 Mycobacterium avium isolates from distinct geographic areas of Italy exhibited markedly polymorphic, multibanded IS1245 restriction fragment length polymorphism (RFLP) patterns; 2 isolates showed the low-number banding pattern typical of bird isolates. By computer analysis, 41 distinct IS1245 patterns and 10 clusters of essentially identical strains were detected; 40% of the 63 isolates showed genetic relatedness, suggesting the existence of a predominant AIDS-associated IS1245 RFLP pattern.

PCR ELISA for the quantitative detection of Epstein-Barr virus genome.

A highly sensitive and nonradioactive microplate hybridization assay for the detection of Epstein-Barr virus (EBV)-specific polymerase chain reaction (PCR) product was developed. The PCR product is labelled by adding digoxigenin-dUTP directly to the reaction mixture and, after denaturation, is captured by a microtitre plate coated with an extravidin-linked biotinylated probe. Captured products are reacted with a peroxidase-conjugated anti-digoxigenin antibody and detected using tetramethylbenzidine. The assay detected less than ten EBV genomes in a background EBV-negative DNA of 0.75 microg and, when tested on clinical samples, it was able to define the viral load in throat washings of patients with acute infectious mononucleosis, immunosuppressed patients with HIV infection, and rare normal individuals who shed the virus in the oropharynx.

Oligo(dT)-primed synthesis of cDNA by reverse transcriptase in mycobacteria.

The possibility that mRNA from Mycobacterium tuberculosis and M. bovis BCG may present polyadenylation at the 3' end was investigated. The total RNA, extracted from the bacterial cells and treated with DNase, was used as substrate for reverse transcriptase (RT)-dependent cDNA synthesis. The RT reaction was primed with oligo(dT) and with downstream specific primers for the genes of the antigens 65 KDa and 85-C. PCR probing of the reaction products for cDNAs of the two mycobacterial genes yielded the expected 225 and 307 bp bands when RT synthesis was primed by oligo(dT) and by downstream specific primers. Reaction products from oligo(dT)-primed RT of RNase-treated RNA and untranscribed RNA, probed by PCR, failed to generate the 225 and 307 bp specific bands. These findings support the existence of polyadenylated tracts in mRNA of mycobacteria that can be targeted by oligo(dT) primers to initiate RT-dependent cDNA synthesis. This may result in an advance in the study of gene expression in these and possibly other bacteria.

Potential role of the Epstein-Barr virus in systemic lupus erythematosus autoimmunity.

OBJECTIVE: To investigate the possibility that Epstein-Barr virus (EBV), the agent of infectious mononucleosis (IM), may play a role in systemic lupus erythematosus (SLE). METHODS: EBV was searched for by PCR and by culture isolation in oropharyngeal lavage fluids of 15 SLE patients and, as controls, in 13 IM patients and in 28 healthy individuals with past EBV infection. Computer analysis was performed to select an antigenic domain of the virus-encoded nuclear antigen EBNA-2, in order to set up a synthetic peptide-based immunoassay. IgG antibodies to a 20-amino acid synthetic peptide derived from the selected domain of EBNA-2 (354GRGKGKSRDKQRKPGGPWRP373) were titrated in the sera of 20 SLE patients, 24 IM patients and 12 healthy subjects. RESULTS: EBV type 1 DNA was demonstrated by PCR in the oropharyngeal secretions of 8 SLE patients and the virus was isolated from 6 DNA-positive specimens. Moreover, 50% of the patients with SLE and 100% of the patients in the acute phase of IM, but none of the EBV-seropositive normal individuals, produced IgG antibodies to the EBNA-2-derived synthetic peptide. Computer analysis revealed a high degree of homology between the EBNA-2 354GRGKGKSRDKQRKPGGPWRP373 sub-sequence and the antigenic C-terminal domain 101GRGRGRGRGRGRGRGGPRR119 of the SmD1 ribonucleoprotein, a target of autoantibodies in a portion of SLE patients. CONCLUSION: We suggest the possibility that EBV may establish a persistent infection at least in a certain number of SLE patients. The antibodies elicited by the viral antigen EBNA-2 may cross-react with SmD1, thus indicating a role of EBV-specific immune responses in the outcome of SmD1 autoantibodies in SLE patients.