Exposure of mycobacteria to cell wall-inhibitory drugs decreases production of arabinoglycerolipid related to Mycolyl-arabinogalactan-peptidoglycan metabolism

The "cell wall core" consisting of a mycolyl-arabinogalactan-peptidoglycan (mAGP) complex represents the hallmark of the mycobacterial cell envelope. It has been the focus of intense research at both structural and biosynthetic levels during the past few decades. Because it is essential, mAGP is also regarded as a target for several antitubercular drugs. Herein, we demonstrate that exposure of Mycobacterium bovis Bacille Calmette-Guérin or Mycobacterium marinum to thiacetazone, a second line antitubercular drug, is associated with a severe decrease in the level of a major apolar glycolipid. This inhibition requires MmaA4, a methyltransferase reported to participate in the activation process of thiacetazone. Following purification, this glycolipid was subjected to detailed structural analyses, combining gas-liquid chromatography, mass spectrometry, and nuclear magnetic resonance. This allowed to identify it as a 5-O-mycolyl-β-Araf-(1→2)-5-O-mycolyl-α-Araf-(1→1)-Gro, designated dimycolyl diarabinoglycerol (DMAG). The presence of DMAG was subsequently confirmed in other slow growing pathogenic species, including Mycobacterium tuberculosis. DMAG production was stimulated in the presence of exogenous glycerol. Interestingly, DMAG appears structurally identical to the terminal portion of the mycolylated arabinosyl motif of mAGP, and the metabolic relationship between these two components was provided using antitubercular drugs such as ethambutol or isoniazid known to inhibit the biosynthesis of arabinogalactan or mycolic acid, respectively. Finally, DMAG was identified in the cell wall of M. tuberculosis. This opens the possibility of a potent biological function for DMAG that may be important to mycobacterial pathogenesis.     

Evidence of Cryptosporidium transmission between cattle and humans in northern New South Wales

Cryptosporidium is an enteric parasite of public health significance that causes diarrhoeal illness through faecal oral contamination and via water. Zoonotic transmission is difficult to determine as most species of Cryptosporidium are morphologically identical and can only be differentiated by molecular means. Transmission dynamics of Cryptosporidium in rural populations were investigated through the collection of 196 faecal samples from diarrheic (scouring) calves on 20 farms and 63 faecal samples from humans on 14 of these farms. The overall prevalence of Cryptosporidium in cattle and humans by PCR and sequence analysis of the 18S rRNA was 73.5% (144/196) and 23.8% (15/63), respectively. Three species were identified in cattle; Cryptosporidium parvum, Cryptosporidium bovis and Cryptosporidium ryanae, and from humans, C. parvum and C. bovis. This is only the second report of C. bovis in humans. Subtype analysis at the gp60 locus identified C. parvum subtype IIaA18G3R1 as the most common subtype in calves. Of the seven human C. parvum isolates successfully subtyped, five were IIaA18G3R1, one was IIdA18G2 and one isolate had a mix of IIaA18G3R1 and IIdA19G2. These findings suggest that zoonotic transmission may have occurred but more studies involving extensive sampling of both calves and farm workers are needed for a better understanding of the sources of Cryptosporidium infections in humans from rural areas of Australia.     

Plasma proteome dynamics: analysis of lipoproteins and acute phase response proteins with 2H2O metabolic labeling

Understanding the pathologies related to the regulation of protein metabolism requires methods for studying the kinetics of individual proteins. We developed a (2)H(2)O metabolic labeling technique and software for protein kinetic studies in free living organisms. This approach for proteome dynamic studies requires the measurement of total body water enrichments by GC-MS, isotopic distribution of the tryptic peptide by LC-MS/MS, and estimation of the asymptotical number of deuterium incorporated into a peptide by software. We applied this technique to measure the synthesis rates of several plasma lipoproteins and acute phase response proteins in rats. Samples were collected at different time points, and proteins were separated by a gradient gel electrophoresis. (2)H labeling of tryptic peptides was analyzed by ion trap tandem mass spectrometry (LTQ MS/MS) for measurement of the fractional synthesis rates of plasma proteins. The high sensitivity of LTQ MS in zoom scan mode in combination with (2)H label amplification in proteolytic peptides allows detection of the changes in plasma protein synthesis related to animal nutritional status. Our results demonstrate that fasting has divergent effects on the rate of synthesis of plasma proteins, increasing synthesis of ApoB 100 but decreasing formation of albumin and fibrinogen. We conclude that this technique can effectively measure the synthesis of plasma proteins and can be used to study the regulation of protein homeostasis under physiological and pathological conditions.     

The phylogeny of halichondrid demosponges: past and present re-visited with DNA-barcoding data

Halichondrid sponges play a pivotal role in the classification of demosponges as changes in their classification has had direct consequences for the classification of Demospongiae. Historically, the systematics of halichondrids has been unstable. During the 1950s, the order was divided into two subclasses, which were based on empirical and assumed reproductive data. Subsequent morphological and biochemical analyses postulated the re-merging of halichondrid families, but recent molecular data indicate their polyphyly. Here we review the classification history of halichondrid taxa, compare it with the current and predominantly ribosomal molecular data, and support the new phylogenetic hypotheses with mitochondrial data from DNA barcoding.     

3-Fluorotyrosine as a Complementary Probe of Hemoglobin Structure and Dynamics: A (19) F-NMR Study of Synechococcus sp. PCC 7002 GlbN

The hemoglobin from the cyanobacterium Synechococcus sp. PCC 7002 (GlbN) contains three tyrosines (Tyr5, Tyr22, and Tyr53), each of which undergoes a structural rearrangement when the protein binds an exogenous ligand such as cyanide. We explored the use of 3-fluorotyrosine and (19) F-NMR spectroscopy for the characterization of GlbN. Assignment of (19) F resonances in fluorinated GlbN (GlbN*) was achieved with individual Tyr5Phe and Tyr53Phe replacements. We observed marked variations in chemical shift and linewidth reflecting the dependence of structural and dynamic properties on oxidation state, ligation state, and covalent attachment of the heme group. The isoelectronic complexes of ferric GlbN* with cyanide and ferrous GlbN* with carbon monoxide gave contrasting spectra, the latter exhibiting heterogeneity and enhanced internal motions on a microsecond-to-millisecond time scale. The strength of the H-bond network involving Tyr22 (B10) and bound cyanide was tested at high pH. 3-Fluorotyrosine at position 22 had a pK(a) value at least 3 units higher than its intrinsic value, 8.5. In addition, evidence was found for long-range communication among the tyrosine sites. These observations demonstrated the utility of the 3-fluorotyrosine approach to gain insight in hemoglobin properties.     

The discovery of new deep-sea hydrothermal vent communities in the southern ocean and implications for biogeography

Since the first discovery of deep-sea hydrothermal vents along the Galápagos Rift in 1977, numerous vent sites and endemic faunal assemblages have been found along mid-ocean ridges and back-arc basins at low to mid latitudes. These discoveries have suggested the existence of separate biogeographic provinces in the Atlantic and the North West Pacific, the existence of a province including the South West Pacific and Indian Ocean, and a separation of the North East Pacific, North East Pacific Rise, and South East Pacific Rise. The Southern Ocean is known to be a region of high deep-sea species diversity and centre of origin for the global deep-sea fauna. It has also been proposed as a gateway connecting hydrothermal vents in different oceans but is little explored because of extreme conditions. Since 2009 we have explored two segments of the East Scotia Ridge (ESR) in the Southern Ocean using a remotely operated vehicle. In each segment we located deep-sea hydrothermal vents hosting high-temperature black smokers up to 382.8°C and diffuse venting. The chemosynthetic ecosystems hosted by these vents are dominated by a new yeti crab (Kiwa n. sp.), stalked barnacles, limpets, peltospiroid gastropods, anemones, and a predatory sea star. Taxa abundant in vent ecosystems in other oceans, including polychaete worms (Siboglinidae), bathymodiolid mussels, and alvinocaridid shrimps, are absent from the ESR vents. These groups, except the Siboglinidae, possess planktotrophic larvae, rare in Antarctic marine invertebrates, suggesting that the environmental conditions of the Southern Ocean may act as a dispersal filter for vent taxa. Evidence from the distinctive fauna, the unique community structure, and multivariate analyses suggest that the Antarctic vent ecosystems represent a new vent biogeographic province. However, multivariate analyses of species present at the ESR and at other deep-sea hydrothermal vents globally indicate that vent biogeography is more complex than previously recognised.     

Genomic hypomethylation in the human germline associates with selective structural mutability in the human genome

The hotspots of structural polymorphisms and structural mutability in the human genome remain to be explained mechanistically. We examine associations of structural mutability with germline DNA methylation and with non-allelic homologous recombination (NAHR) mediated by low-copy repeats (LCRs). Combined evidence from four human sperm methylome maps, human genome evolution, structural polymorphisms in the human population, and previous genomic and disease studies consistently points to a strong association of germline hypomethylation and genomic instability. Specifically, methylation deserts, the ~1% fraction of the human genome with the lowest methylation in the germline, show a tenfold enrichment for structural rearrangements that occurred in the human genome since the branching of chimpanzee and are highly enriched for fast-evolving loci that regulate tissue-specific gene expression. Analysis of copy number variants (CNVs) from 400 human samples identified using a custom-designed array comparative genomic hybridization (aCGH) chip, combined with publicly available structural variation data, indicates that association of structural mutability with germline hypomethylation is comparable in magnitude to the association of structural mutability with LCR-mediated NAHR. Moreover, rare CNVs occurring in the genomes of individuals diagnosed with schizophrenia, bipolar disorder, and developmental delay and de novo CNVs occurring in those diagnosed with autism are significantly more concentrated within hypomethylated regions. These findings suggest a new connection between the epigenome, selective mutability, evolution, and human disease.     

Defective membrane remodeling in neuromuscular diseases: insights from animal models

Proteins involved in membrane remodeling play an essential role in a plethora of cell functions including endocytosis and intracellular transport. Defects in several of them lead to human diseases. Myotubularins, amphiphysins, and dynamins are all proteins implicated in membrane trafficking and/or remodeling. Mutations in myotubularin, amphiphysin 2 (BIN1), and dynamin 2 lead to different forms of centronuclear myopathy, while mutations in myotubularin-related proteins cause Charcot-Marie-Tooth neuropathies. In addition to centronuclear myopathy, dynamin 2 is also mutated in a dominant form of Charcot-Marie-Tooth neuropathy. While several proteins from these different families are implicated in similar diseases, mutations in close homologues or in the same protein in the case of dynamin 2 lead to diseases affecting different tissues. This suggests (1) a common molecular pathway underlying these different neuromuscular diseases, and (2) tissue-specific regulation of these proteins. This review discusses the pathophysiology of the related neuromuscular diseases on the basis of animal models developed for proteins of the myotubularin, amphiphysin, and dynamin families. A better understanding of the common mechanisms between these neuromuscular disorders will lead to more specific health care and therapeutic approaches.