High Sequence Variability of the ppE18 Gene of Clinical Mycobacterium tuberculosis Complex Strains Potentially Impacts Effectivity of Vaccine Candidate M72/AS01E

The development of an effective vaccine is urgently needed to fight tuberculosis (TB) which is still the leading cause of death from a single infectious agent worldwide. One of the promising vaccine candidates M72/AS01E consists of two proteins subunits PepA and PPE18 coded by Rv0125 and Rv1196. However, preliminary data indicate a high level of sequence variability among clinical Mycobacterium tuberculosis complex (MTBC) strains that might have an impact on the vaccine efficacy. To further investigate this finding, we determined ppE18 sequence variability in a well-characterized reference collection of 71 MTBC strains from 23 phylogenetic lineages representing the global MTBC diversity. In total, 100 sequence variations consisting of 96 single nucleotide polymorphisms (SNPs), three insertions and one deletion were detected resulting in 141 variable positions distributed over the entire gene. The majority of SNPs detected were non-synonymous (n = 68 vs. n = 28 synonymous). Strains from animal adapted lineages, e.g., M. bovis, showed a significant higher diversity than the human pathogens such as M. tuberculosis Haarlem. SNP patterns specific for different lineages as well as for deeper branches in the phylogeny could be identified. The results of our study demonstrate a high variability of the ppE18 gene even in the N-terminal domains that is normally highly conserved in ppe genes. As the N-terminal region interacts with TLR2 receptor inducing a protective anti-inflammatory immune response, genetic heterogeneity has a potential impact on the vaccine efficiency, however, this has to be investigated in future studies.     

Rediscovery of Nebela ansata (Amoebozoa: Arcellinida) in eastern North America: biogeographical implications

Aim The question whether free‐living protists are generally cosmopolitan is currently a matter of debate. In this study we investigate the geographical distribution of a distinctive testate amoeba species, Nebela ansata, and use our data to assess the potential for highly restricted distribution patterns in some protist species. Location Global. Methods We analysed (1) 3400 testate amoeba publications from North America and other continents, (2) unpublished slides of the Penard Collection of the Natural History Museum, London, UK, and (3) 104 Sphagnum samples from eastern North America. Non‐metric multidimensional scaling (NMDS) was used to visualize the similarities in testate amoeba community composition among 1012 North American samples, including two communities that contained N. ansata. Results We rediscovered N. ansata at a site in New Jersey located close to its type locality, and in Nova Scotia. We also report the existence of an apparently unpublished museum specimen originally collected from New Jersey. Our extensive literature survey confirmed the presence of this species only in the temperate part of eastern North America. The NMDS revealed that communities with N. ansata were less similar to each other than to communities from other parts of North America, suggesting that favourable habitats for N. ansata occur in other Sphagnum‐dominated peatlands, a habitat type that has been extensively sampled in North America and elsewhere. Main conclusions These data provide an unusually convincing case of a free‐living microorganism with a very limited distribution range in the temperate part of eastern North America. The remarkably restricted distribution of N. ansata highlights the extent of our ignorance about the natural history of free‐living microorganisms, and raises questions about the lack of attention to microbial diversity in conservation biology.     

Determination of ovulation and pregnancy in the marmoset (Callithrix jacchus) by monitoring of urinary hydroxypregnanolone excretion

In Callithrix jacchus the urinary excretion of hydroxypregnanolone (5 alpha-pregnane-3 alpha, 7 alpha-diol-20-one) shows a luteal rise during the ovulatory cycle. This progesterone metabolite can therefore be used as an indication of ovulation, implantation, and subsequently for the persistence of the pregnancy. The excretion can be monitored by high performance thin-layer chromatography (HPTLC) and postchromatographic derivatization after enzymatic hydrolysis of the conjugate. Profiles of urinary hydroxypregnanolone levels are presented and correlated with luteinizing hormone excretion. The method is noninvasive and therefore suited for long-term studies in these monkeys.     

Directed mutagenesis of Mycobacterium smegmatis 16S rRNA to reconstruct the in vivo evolution of aminoglycoside resistance in Mycobacterium tuberculosis

Drug resistance in Mycobacterium tuberculosis is a global problem, with major consequences for treatment and public health systems. As the emergence and spread of drug‐resistant tuberculosis epidemics is largely influenced by the impact of the resistance mechanism on bacterial fitness, we wished to investigate whether compensatory evolution occurs in drug‐resistant clinical isolates of M. tuberculosis. By combining information from molecular epidemiology studies of drug‐resistant clinical M. tuberculosis isolates with genetic reconstructions and measurements of aminoglycoside susceptibility and fitness in Mycobacterium smegmatis, we have reconstructed a plausible pathway for how aminoglycoside resistance develops in clinical isolates of M. tuberculosis. Thus, we show by reconstruction experiments that base changes in the highly conserved A‐site of 16S rRNA that: (i) cause aminoglycoside resistance, (ii) confer a high fitness cost and (iii) destabilize a stem‐loop structure, are associated with a particular compensatory point mutation that restores rRNA secondary structure and bacterial fitness, while maintaining to a large extent the drug‐resistant phenotype. The same types of resistance and associated mutations can be found in M. tuberculosis in clinical isolates, suggesting that compensatory evolution contributes to the spread of drug‐resistant tuberculosis disease.     

Investigation of a New Electrode Array Technology for a Central Auditory Prosthesis

Ongoing clinical studies on patients recently implanted with the auditory midbrain implant (AMI) into the inferior colliculus (IC) for hearing restoration have shown that these patients do not achieve performance levels comparable to cochlear implant patients. The AMI consists of a single-shank array (20 electrodes) for stimulation along the tonotopic axis of the IC. Recent findings suggest that one major limitation in AMI performance is the inability to sufficiently activate neurons across the three-dimensional (3-D) IC. Unfortunately, there are no currently available 3-D array technologies that can be used for clinical applications. More recently, there has been a new initiative by the European Commission to fund and develop 3-D chronic electrode arrays for science and clinical applications through the NeuroProbes project that can overcome the bulkiness and limited 3-D configurations of currently available array technologies. As part of the NeuroProbes initiative, we investigated whether their new array technology could be potentially used for future AMI patients. Since the NeuroProbes technology had not yet been tested for electrical stimulation in an in vivo animal preparation, we performed experiments in ketamine-anesthetized guinea pigs in which we inserted and stimulated a NeuroProbes array within the IC and recorded the corresponding neural activation within the auditory cortex. We used 2-D arrays for this initial feasibility study since they were already available and were sufficient to access the IC and also demonstrate effective activation of the central auditory system. Based on these encouraging results and the ability to develop customized 3-D arrays with the NeuroProbes technology, we can further investigate different stimulation patterns across the ICC to improve AMI performance.     

Effects of Iron and Nitrogen Limitation on Sulfur Isotope Fractionation during Microbial Sulfate Reduction

Sulfate-reducing microbes utilize sulfate as an electron acceptor and produce sulfide that is depleted in heavy isotopes of sulfur relative to sulfate. Thus, the distribution of sulfur isotopes in sediments can trace microbial sulfate reduction (MSR), and it also has the potential to reflect the physiology of sulfate-reducing microbes. This study investigates the relationship between the availability of iron and reduced nitrogen and the magnitude of S-isotope fractionation during MSR by a marine sulfate-reducing bacterium, DMSS-1, a Desulfovibrio species, isolated from salt marsh in Cape Cod, MA. Submicromolar levels of iron increase sulfur isotope fractionation by about 50% relative to iron-replete cultures of DMSS-1. Iron-limited cultures also exhibit decreased cytochrome c-to-total protein ratios and cell-specific sulfate reduction rates (csSRR), implying changes in the electron transport chain that couples carbon and sulfur metabolisms. When DMSS-1 fixes nitrogen in ammonium-deficient medium, it also produces larger fractionation, but it occurs at faster csSRRs than in the ammonium-replete control cultures. The energy and reducing power required for nitrogen fixation may be responsible for the reverse trend between S-isotope fractionation and csSRR in this case. Iron deficiency and nitrogen fixation by sulfate-reducing microbes may lead to the large observed S-isotope effects in some euxinic basins and various anoxic sediments.