Subfossil 16S rRNA Gene Sequences of Green Sulfur Bacteria in the Black Sea and Their Implications for Past Photic Zone Anoxia

The Black Sea is the largest extant anoxic water body on Earth. Its oxic-anoxic boundary is located at a depth of 100 m and is populated by a single phylotype of marine green sulfur bacteria. This organism, Chlorobium sp. strain BS-1, is extraordinarily low light adapted and can therefore serve as an indicator of deep photic zone anoxia (A. K. Manske, J. Glaeser, M. M. M. Kuypers, and J. Overmann, Appl. Environ. Microbiol. 71:8049-8060, 2005). In the present study, two sediment cores were retrieved from the bottom of the Black Sea at depths of 2,006 and 2,162 m and were analyzed for the presence of subfossil DNA sequences of BS-1 using ancient-DNA methodology. Using optimized cultivation media, viable cells of the BS-1 phylotype were detected only at the sediment surface and not in deeper layers. In contrast, green sulfur bacterial 16S rRNA gene fragments were amplified from all the sediment layers investigated, including turbidites. After separation by denaturing gradient gel electrophoresis and sequencing, 14 different sequence types were distinguished. The sequence of BS-1 represented only a minor fraction of the amplification products and was found in 6 of 22 and 4 of 26 samples from the 2,006- and 2,162-m stations, respectively. Besides the sequences of BS-1, three additional phylotypes of the marine clade of green sulfur bacteria were detected. However, the majority of sequences clustered with groups from freshwater habitats. Our results suggest that a considerable fraction of green sulfur bacterial chemofossils did not originate in a low-light marine chemocline environment and therefore were likely to have an allochthonous origin. Thus, analysis of subfossil DNA sequences permits a more differentiated interpretation and reconstruction of past environmental conditions if specific chemofossils of stenoec species, like Chlorobium sp. strain BS-1, are employed.     

Antigenic modulation by anti-CD5 immunotoxins

We evaluated the modulation of T101 immunotoxins (IT) and free T101 antibody from the surface of normal and leukemic cells to determine whether the presence of toxin on antibody affected antigenic modulation. Reagents were made by conjugating T101, which binds to the T cell antigen CD5, to either intact ricin or purified ricin A chain. We found that T101-A chain modulated CD5 more efficiently than T101-ricin, which modulated CD5 more efficiently than T101 alone. Kinetic studies showed that maximal modulation of IT was reached within 3 hr. When toxicity of the reagents was tested in protein synthesis inhibition assays, T101-ricin in the presence of lactose inhibited 99% of the protein synthesis of CEM cells. T101-A chain was less toxic, inhibiting protein synthesis only 23 to 43%. The addition of the potentiating agent monensin nearly doubled the toxicity of T101-A chain, but did not affect T101-A chain modulation. To determine the fate of bound IT, T101 and T101-ricin were labeled with 125I. Cells were incubated under modulating conditions in the presence of radiolabeled reagents. T101 and T101-ricin were internalized into CEM cells. In contrast, T101, but not T101-ricin, appeared to be shed from peripheral blood mononuclear cells. Our findings show clearly that: 1) the presence of toxin on antibody does not inhibit--and may actually enhance--modulation; 2) T101-IT are internalized, not shed from the cell surface; 3) the lack of toxicity of T101-A chain is not attributed to inability to modulate; 4) there is no correlation between enhancement of T101-A chain toxicity by monensin and antigenic modulation by A chain reagents; and 5) modulation, which is undesirable in monoclonal antibody therapy, may be advantageous in the therapeutic use of IT.     

Laminin synthesized by stationary and migrating rat liver epithelial cells lacks the A chain

The expression of laminin chains was analyzed in normal and Ha-ras1-transformed rat liver epithelial cells. The normal, nontumorigenic cells were induced to migrate by epidermal growth factor, whereas the Ha-ras1-transformed, malignant derivatives migrate constitutively. None of these cells express a typical (EHS-like) laminin A chain. Immunoprecipitation of [35S]-methionine-labeled liver cell lysates with an antibody against EHS-laminin revealed B1 and B2 chains and, in addition, two high Mr polypeptides. These polypeptides were not recognized by the antibody in immunoblots, suggesting that they might constitute alternative laminin A chains. Analysis of the expression of all three laminin chains at the RNA and protein level revealed that the pattern of expression of the stationary cells does not differ from that of the migratory ones and is also not influenced by epidermal growth factor. These results indicate that expression of a typical laminin A chain by rat liver epithelial cells is not required for the secretion and deposition of the protein in the extracellular matrix. The data also indicate that an EHS-like laminin A chain is not required for the migration of these epithelial cells.     

Effective Preparation of Plasmodium vivax Field Isolates for High-Throughput Whole Genome Sequencing

Whole genome sequencing (WGS) of Plasmodium vivax is problematic due to the reliance on clinical isolates which are generally low in parasitaemia and sample volume. Furthermore, clinical isolates contain a significant contaminating background of host DNA which confounds efforts to map short read sequence of the target P. vivax DNA. Here, we discuss a methodology to significantly improve the success of P. vivax WGS on natural (non-adapted) patient isolates. Using 37 patient isolates from Indonesia, Thailand, and travellers, we assessed the application of CF11-based white blood cell filtration alone and in combination with short term ex vivo schizont maturation. Although CF11 filtration reduced human DNA contamination in 8 Indonesian isolates tested, additional short-term culture increased the P. vivax DNA yield from a median of 0.15 to 6.2 ng µl⁻¹ packed red blood cells (pRBCs) (p = 0.001) and reduced the human DNA percentage from a median of 33.9% to 6.22% (p = 0.008). Furthermore, post-CF11 and culture samples from Thailand gave a median P. vivax DNA yield of 2.34 ng µl⁻¹ pRBCs, and 2.65% human DNA. In 22 P. vivax patient isolates prepared with the 2-step method, we demonstrate high depth (median 654X coverage) and breadth (≥89%) of coverage on the Illumina GAII and HiSeq platforms. In contrast to the A+T-rich P. falciparum genome, negligible bias was observed in coverage depth between coding and non-coding regions of the P. vivax genome. This uniform coverage will greatly facilitate the detection of SNPs and copy number variants across the genome, enabling unbiased exploration of the natural diversity in P. vivax populations.     

Inflammatory thresholds and the species-specific effects of colonising bacteria in stable chronic obstructive pulmonary disease

Background

There has been increasing interest in the use of newer, culture-independent techniques to study the airway microbiome of COPD patients. We investigated the relationships between the three common potentially pathogenic microorganisms (PPMs) Haemophilus influenzae, Streptococcus pneumoniae and Moraxella catarrhalis, as detected by quantitative PCR (qPCR), and inflammation and health status in stable patients in the London COPD cohort.

Methods

We prospectively collected sputum, serum and plasma samples for analysis of airway bacterial presence and load, and airway and systemic inflammation from 99 stable COPD patients between January 2011 and October 2012. Health status was measured with St George’s Respiratory Questionnaire and COPD Assessment Test.

Results

Airway inflammation and plasma fibrinogen, but not C-reactive protein, were greater in samples with PPM detection (p < 0.001, p = 0.049 and p = 0.261, respectively). Increasing total bacterial load was associated with increasing airway (p < 0.01) but not systemic inflammation (p > 0.05). Samples with high total bacterial loads had significantly higher airway inflammation than both samples without PPM detection and those with lower loads. Haemophilus influenzae presence was associated with significantly higher levels of airway but not systemic inflammation for all given pathogen loads (p < 0.05), and was significantly greater than with other PPMs. No association was observed between inflammation and health status (p > 0.05).

Conclusions

Airway and systemic inflammation, as measured by fibrinogen, is greater in stable COPD patients with PPMs detected using the culture-independent qPCR technique. The airway, but not systemic inflammatory response, appears to have a total pathogen-load threshold and appears attributable to Haemophilus influenzae. However, discordance between inflammation and health status was observed.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0114-1) contains supplementary material, which is available to authorized users.     

A Genome Wide Association Study of Plasmodium falciparum Susceptibility to 22 Antimalarial Drugs in Kenya

Background

Drug resistance remains a chief concern for malaria control. In order to determine the genetic markers of drug resistant parasites, we tested the genome-wide associations (GWA) of sequence-based genotypes from 35 Kenyan P. falciparum parasites with the activities of 22 antimalarial drugs.

Methods and Principal Findings

Parasites isolated from children with acute febrile malaria were adapted to culture, and sensitivity was determined by in vitro growth in the presence of anti-malarial drugs. Parasites were genotyped using whole genome sequencing techniques. Associations between 6250 single nucleotide polymorphisms (SNPs) and resistance to individual anti-malarial agents were determined, with false discovery rate adjustment for multiple hypothesis testing. We identified expected associations in the pfcrt region with chloroquine (CQ) activity, and other novel loci associated with amodiaquine, quinazoline, and quinine activities. Signals for CQ and primaquine (PQ) overlap in and around pfcrt, and interestingly the phenotypes are inversely related for these two drugs. We catalog the variation in dhfr, dhps, mdr1, nhe, and crt, including novel SNPs, and confirm the presence of a dhfr-164L quadruple mutant in coastal Kenya. Mutations implicated in sulfadoxine-pyrimethamine resistance are at or near fixation in this sample set.

Conclusions/Significance

Sequence-based GWA studies are powerful tools for phenotypic association tests. Using this approach on falciparum parasites from coastal Kenya we identified known and previously unreported genes associated with phenotypic resistance to anti-malarial drugs, and observe in high-resolution haplotype visualizations a possible signature of an inverse selective relationship between CQ and PQ.     

Dietary use and conservation concern of edible wetland plants at indo-burma hotspot: a case study from northeast India

Background

The wetlands of the North East India fall among the global hotspots of biodiversity. However, they have received very little attention with relation to their intrinsic values to human kind; therefore their conservation is hardly addressed. These wetlands are critical for the sustenance of the tribal communities.

Methods

Field research was conducted during 2003 to 2006 in seven major wetlands of four districts of Manipur state, Northeast India (viz. Imphal-East, Imphal-West, Thoubal, and Bishnupur). A total of 224 wetland-plant-collectors were interviewed for the use and economics of species using semi-structured questionnaires and interview schedules. Imphal, Bishenpur and Thoubal markets were investigated in detail for influx and consumption pattern of these plants. The collectors were also inquired for medicinal use of wetland species. Nutritive values of 21 species were analyzed in laboratory. The vouchers were collected for all the species and deposited in the CSIR-NEIST (Formerly Regional Research Laboratory), Substation, Lamphelpat, Imphal, Manipur, India.

Results

We recorded 51 edible wetland species used by indigenous people for food and medicinal purposes. Thirty eight species had high medicinal values and used in the traditional system to treat over 22 diseases. At least 27 species were traded in three markets studied (i.e. Imphal, Thoubal and Bishenpur), involving an annual turnover of 113 tons of wetland edible plants and a gross revenue of Rs. 907, 770/- (US$1 = Rs. 45/-). The Imphal market alone supplies 60% of the total business. Eighty per cent of the above mentioned species are very often used by the community. The community has a general opinion that the availability of 45% species has depleted in recent times, 15 species need consideration for conservation while another 7 species deserved immediate protection measures. The nutrient analysis showed that these species contribute to the dietary balance of tribal communities.

Conclusions

Considering the importance of wild wetland plants in local sustenance, it is suggested to protect their habitats, develop domestication protocols of selected species, and build programs for the long-term management of wetland areas by involving local people. Some medicinal plants may also be used to develop into modern medicines.