Role of Intracellular Calcium Stores on the Effect of Metabotropic Glutamate Receptors on Phosphorylation of Glial Fibrillary Acidic Protein in Hippocampal Slices from Immature Rats

Phosphorylation of glial fibrillary acidic protein (GFAP) in slices from immature rats is stimulated by glutamate via a group II metabotropic glutamate receptor (mGluR II) and by absence of external Ca2+ in reactions that are not additive (Wofchuk and Rodnight, Neurochem. Int. 24:517-523, 1994). These observations suggested that glutamate, via an mGluR, inhibits Ca(2+)-entry through L-type Ca2+ channels and down-regulates a Ca(2+)-dependent dephosphorylation event coupled to GFAP. Because ryanodine receptors are present on internal Ca2+ stores and are associated with L-type Ca(2+)-channels, we investigated the possibility that the glutamatergic modulation of GFAP phosphorylation involves internal Ca2+ stores regulated by ryanodine receptors and whether the Ca2+ originating from these stores acts in a similar manner to external Ca2+. The results showed that the ryanodine receptor-agonists, caffeine and ryanodine and thapsigargin, all of which in appropriate doses increase cytoplasmic Ca2+, reversed the stimulation of GFAP phosphorylation given by 1S,3R-ACPD, an mGluR II agonist.     

HOXA10 and HOXA13 sequence variations in human female genital malformations including congenital absence of the uterus and vagina

Congenital genital malformations occurring in the female population are estimated to be 5 per 1000 and associate with infertility, abortion, stillbirth, preterm delivery and other organ abnormalities. Complete aplasia of the uterus, cervix and upper vagina (Mayer–Rokitansky–Küster–Hauser (MRKH) syndrome) has an incidence of 1 per 4000 female live births. The molecular etiology of congenital genital malformations including MRKH is unknown up to date. The homeobox (HOX) genes HOXA10 and HOXA13 are involved in the development of human genitalia. In this investigation, HOXA10 and HOXA13 genes of 20 patients with the MRKH syndrome, 7 non-MRKH patients with genital malformations and 53 control women were sequenced to assess for DNA variations. A total of 14 DNA sequence variations (10 novel and 4 known) within exonic and untranslated regions were detected in HOXA10 and HOXA13 among our cohorts. Four HOXA10 and two HOXA13 DNA sequence variations were found solely in patients with genital malformations. In addition to mutations resulting in synonymous amino acid substitutions, in the HOXA10 gene a missense mutation was identified and predicted by computer analysis as probably damaging to protein function in two non-MRKH patients, one with a bicornate and the other patient with a septated uterus. A novel exonic HOXA10 cytosine deletion was also identified in a non-MRKH patient with a septate uterus and renal malformations resulting in a premature stop codon and loss of the homeodomain helix 3/4. This cytosine deletion and the missense mutation in HOXA10 were analysed by real time PCR and sequencing, respectively, in two additional larger cohorts of 103 patients with MRKH and 109 non-MRKH patients with genital malformations. No other patients were found with the cytosine deletion however one additional patient was identified regarding the missense mutation. Rare DNA sequence variations in the HOXA10 gene could contribute to the misdevelopment of female internal genitalia.     

The Hydrogenase Chip: a tiling oligonucleotide DNA microarray technique for characterizing hydrogen-producing and -consuming microbes in microbial communities

We developed a broad-ranging method for identifying key hydrogen-producing and consuming microorganisms through analysis of hydrogenase gene content and expression in complex anaerobic microbial communities. The method is based on a tiling hydrogenase gene oligonucleotide DNA microarray (Hydrogenase Chip), which implements a high number of probes per gene by tiling probe sequences across genes of interest at 1.67 × –2 × coverage. This design favors the avoidance of false positive gene identification in samples of DNA or RNA extracted from complex microbial communities. We applied this technique to interrogate interspecies hydrogen transfer in complex communities in (i) lab-scale reductive dehalogenating microcosms enabling us to delineate key H₂-consuming microorganisms, and (ii) hydrogen-generating microbial mats where we found evidence for significant H₂ production by cyanobacteria. Independent quantitative PCR analysis on selected hydrogenase genes showed that this Hydrogenase Chip technique is semiquantitative. We also determined that as microbial community complexity increases, specificity must be traded for sensitivity in analyzing data from tiling DNA microarrays.     

Genetic diversity of Leptospira isolates in Lao PDR and genome analysis of an outbreak strain

BackgroundAlthough Southeast Asia is one of the most leptospirosis afflicted regions, little is known about the diversity and molecular epidemiology of the causative agents of this widespread and emerging zoonotic disease.Methodology/Principal findingsWe used whole genome sequencing to examine genetic variation in 75 Leptospira strains isolated from patients in the Lao PDR (Laos) between 2006 and 2017.Eleven serogroups from 4 Leptospira species and 43 cgMLST-defined clonal groups (CGs) were identified. The most prevalent CG was CG272 (n = 18, 26.8%), composed of L. interrogans serogroup Autumnalis isolates. This genotype was recovered throughout the 12-year period and was associated with deaths, and with a large outbreak in neighbouring Thailand. Genome analysis reveals that the CG272 strains form a highly clonal group of strains that have, for yet unknown reasons, recently spread in Laos and Thailand. Additionally, accessory genes clearly discriminate CG272 strains from the other Leptospira strains.Conclusions/SignificanceThe present study reveals a high diversity of Leptospira genotypes in Laos, thus extending our current knowledge of the pan- and core-genomes of these life-threatening pathogens. Our results demonstrate that the CG272 strains belong to a unique clonal group, which probably evolved through clonal expansion following niche adaptation. Additional epidemiological studies are required to better evaluate the spread of this genotype in Southeast Asia. To further investigate the key factors driving the virulence and spread of these pathogens, more intense genomic surveillance is needed, combining detailed clinical and epidemiological data.     

Production of neosolaniol monoacetate by an undescribedFusarium species resemblingF camptoceras

Cultures of 12 South African isolates of an undescribedFusarium species resembling but distinct fromF camptoceras were analysed for the presence of diacetoxyscirpenol (DAS), neosolaniol monoacetate (NMA), and T-2 toxin, by capillary gas chromatography utilizing electron capture detection. No DAS or T-2 toxin could be detected in any of the cultures of the isolates. NMA was, however, detected in 10 of the 12 isolates at levels ranging from 310 to 2060 ng/g. The method used, was primarily developed for the determination of DAS and T-2 toxin in fungal cultures and grain samples but was found to be suitable for the coextraction of NMA at an average recovery of 80.8%, with a detection limit in the order of 100 ng/g. Supportive evidence for the presence of the NMA was obtained by capillary gas chromatography / mass spectrometry. Regarded as a relatively rare trichothecene, NMA has never been reported to occur naturally and has previously been shown to be produced by only a fewFusarium strains.     

The sensitivity of Afromontane tarns in the Maloti-Drakensberg region of South Africa and Lesotho to acidic deposition

Despite their remoteness from sources of atmospheric pollutant emissions, the Afromontane tarns in the Maloti-Drakensberg region are perfect candidates to study the negative effects of acidifying atmospheric pollution, because mountain lakes are widely recognised as sentinel ecosystems, unimpacted by direct human disturbance within their catchments. Thirty-four tarns were sampled in the Maloti-Drakensberg region and most were found to be extremely sensitive to acidic deposition, as indicated by their low acid neutralising capacity. There are very few studies of freshwater critical loads for any region within South Africa. The steady-state water chemistry model (SSWC) was adapted and used to determine critical loads, whereas exceedance was estimated relative to modelled regional deposition data, in order to understand the risk of harmful effects to aquatic ecosystems. Seventy-six percent of sampled sites across the Maloti-Drakensberg would exceed critical loads even at the lowest modelled deposition levels, but there are no current measured deposition data for the region. The sensitivity of the Maloti-Drakensberg tarns needs to be considered in future policy formulation regarding acceptable levels of acidifying atmospheric pollution from South Africa’s energy sector and indicates the need for assessing aquatic ecosystem impacts in other regions of South Africa.     

Antimicrobial susceptibility patterns,emm type distribution and genetic diversity of Streptococcus pyogenes recovered in Brazil

Streptococcus pyogenes is responsible for a variety of infectious diseases and immunological complications. In this study, 91 isolates of S. pyogenes recovered from oropharynx secretions were submitted to antimicrobial susceptibility testing, emm typing and pulsed-field gel electrophoresis (PFGE) analysis. All isolates were susceptible to ceftriaxone, levofloxacin, penicillin G and vancomycin. Resistance to erythromycin and clindamycin was 15.4%, which is higher than previous reports from this area, while 20.9% of the isolates were not susceptible to tetracycline. The macrolide resistance phenotypes were cMLSB (10) and iMLSB (4). The ermB gene was predominant, followed by the ermA gene. Thirty-two emm types and subtypes were found, but five (emm1, emm4, emm12, emm22, emm81) were detected in 48% of the isolates. Three new emm subtypes were identified (emm1.74, emm58.14, emm76.7). There was a strong association between emm type and PFGE clustering. A variety of PFGE profiles as well as emm types were found among tetracycline and erythromycin-resistant isolates, demonstrating that antimicrobial resistant strains do not result from the expansion of one or a few clones. This study provides epidemiological data that contribute to the development of suitable strategies for the prevention and treatment of such infections in a poorly studied area.