Impaired opsonization with complement and phagocytosis of Streptococcus pyogenes in sera from subjects with inherited C2 deficiency

Although subjects with inherited defects of the classical complement pathway component C2 are at increased risk of infection, there are few experimental data available on which bacterial pathogens they might be susceptible to. In order to investigate whether patients with inherited C2 deficiency may have increased susceptibility to Streptococcus pyogenes infection we have analysed opsonization with C3b/iC3b and phagocytosis of three different strains of S. pyogenes in serum from 8 C2^?/? subjects using flow cytometry assays. Sera from patients with C2 deficiency had a markedly reduced ability to opsonise S. pyogenes with C3b/iC3b. In addition, phagocytosis of all three S. pyogenes strains was impaired in sera from C2^?/? subjects. Both the reduced opsonisation with C3b/iC3b and phagocytosis in C2^?/? sera were markedly improved by addition of exogenous C2 protein. Neutrophil dependent killing was also reduced, confirming the functional importance of C2 deficiency for immunity to S. pyogenes. Impaired opsonisation with C3b/iC3b and phagocytosis was not related to reduced recognition of the bacteria by antibody. These data suggest that patients with C2 deficiency are at increased risk of S. pyogenes infections.     

Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectral Analysis of Marine Lipopeptides with Potential Therapeutic Implications

Marine Bacillus circulans DMS-2 (MTCC 8281) was found to produce lipopeptides, which reduced the surface tension of the medium from 69 to 28 mNm⁻¹. The methanol extracts of the lipopeptides were resolved in RP-HPLC, which resulted in the presence of two major surface active fractions corresponding to the peaks E and F were eluted at the retention times of 16.8 and 18 min, respectively. Fourier transform infrared spectroscopy and matrix assisted laser desorption ionization time of flight (MALDI-ToF) mass spectral analysis showed that the produced lipopeptides belonged to surfactin family. In the MALDI-ToF spectrum, the major molecular mass of the HPLC purified isoforms were identified at m/z 1,044 and 1,058 Da, respectively. In addition, this marine strain also produced new variants of surfactins with molecular weights ranging from m/z 1,066 to 1,098 Da, which have not been reported earlier. Both surface-active fractions were found to have potent antimicrobial activity against the Gram-positive and Gram-negative bacterial strains.     

Optimizing Seed Water Content: Relevance to Storage Stability and Molecular Mobility

This research was conducted to determine the optimum moisture content (MC) that gave maximum longevity to seeds. Three species were used to represent seeds with different dry matter reserves, which gives them different sorption properties: maize (Zea mays L.), elm (Ulmus pumila L.) and safflower (Carthamus tinctorius L.). The seeds of elm, safflower, and maize embryos with MC ranging from 0.00–0.15 g H₂O/g dry weight (DW) were stored at 35 °C for different periods of time. The results showed that the optimum MC for seed and embryo storage varied between species (0.057 g H₂O/g DW for maize embryos, 0.045 g H₂O/g DW for elm, and 0.02 g H₂O/g DW for safflower). Drying below this optimum MC increased the aging rate and there were detrimental effects of drying. The relative humidity corresponding to optimum MC in embryos of maize, elms and safflower was about 15%, 12% and 7% respectively, according to the lipid composition of the embryos. The data provided confirmatory evidence that molecular mobility (ΔAzz) in elms, maize and safflower embryos was compatible with the optimum moisture content.     

p38α MAPK is a MTOC-associated protein regulating spindle assembly,spindle length and accurate chromosome segregation during mouse oocyte meiotic maturation

P38αMAPK (p38α) is usually activated in response to various stresses and plays a role in the inhibition of cell proliferation and tumor progression, but little is known about its roles in meiotic spindle assembly. In this study, we characterized the dynamic localization of p38α and explored its function in mouse oocyte meiotic maturation. P38α specifically colocalized with γ-tubulin and Plk1 at the center of MTOCs and spindle poles. Depletion of p38α by specific morpholino injection resulted in severely defective spindles and misaligned chromosomes probably via MK2 dephosphorylation. Notably, depletion of p38α led to significant spindle pole defects, spindle elongation, non-tethered kinetochore microtubules and increased microtubule tension. The disruption of spindle stability was coupled with decreased γ-tubulin and Plk1 at MTOCs. Overexpression of Eg5, a conserved motor protein, also caused spindle elongation and its morpholino injection almost completely rescued spindle elongation caused by p38α depletion. In addition, p38α-depletion decreased BubR1 and interfered with spindle assembly checkpoint (SAC), which resulted in aneuploid oocytes. Together, these data indicate that p38α is an important component of MTOCs, which regulates spindle assembly and spindle length, as well as stabilizes the spindle and spindle poles. Perturbed SAC and abnormal microtubule tension may be responsible for the misaligned chromosomes and high aneuploidy in p38α-depleted mouse oocytes.Key words: p38α, meiosis, mouse oocyte, spindle assembly, microtubule organization center (MTOC), Eg5, spindle assembly checkpoint     

Conserved genes act as modifiers of invertebrate SMN loss of function defects

Spinal Muscular Atrophy (SMA) is caused by diminished function of the Survival of Motor Neuron (SMN) protein, but the molecular pathways critical for SMA pathology remain elusive. We have used genetic approaches in invertebrate models to identify conserved SMN loss of function modifier genes. Drosophila melanogaster and Caenorhabditis elegans each have a single gene encoding a protein orthologous to human SMN; diminished function of these invertebrate genes causes lethality and neuromuscular defects. To find genes that modulate SMN function defects across species, two approaches were used. First, a genome-wide RNAi screen for C. elegans SMN modifier genes was undertaken, yielding four genes. Second, we tested the conservation of modifier gene function across species; genes identified in one invertebrate model were tested for function in the other invertebrate model. Drosophila orthologs of two genes, which were identified originally in C. elegans, modified Drosophila SMN loss of function defects. C. elegans orthologs of twelve genes, which were originally identified in a previous Drosophila screen, modified C. elegans SMN loss of function defects. Bioinformatic analysis of the conserved, cross-species, modifier genes suggests that conserved cellular pathways, specifically endocytosis and mRNA regulation, act as critical genetic modifiers of SMN loss of function defects across species.     

Biosurfactant production and growth kinetics of bacteria in a designer marine medium: improved physiochemical properties

A combinatorial screening strategy was adopted for the development of a suitable medium for enhanced biosurfactant production by a marine strain. As a result, a modified marine medium (MMM) was developed, which contained urea and strontium chloride besides other salts important for the growth of marine bacteria. This medium supported growth, evident from a higher maximum growth rate value of 0.42 h(-1) and an enhanced biosurfactant production of 2.58 g/L. The critical micelle concentration (CMC) was determined for the biosurfactants obtained from all tested media combinations. The biosurfactant produced with this medium was stable at high temperature (100 °C), a wide range of pH (5-11) and salt concentration of 5-35%. The emulsifying activity and stability of the biosurfactant obtained using MMM was better than the biosurfactant obtained using conventional media. This biosurfactant with improved physiochemical properties is suitable for a wide range of applications in industry and for marine environmental cleaning.     

Distinct Genetic Diversity of Oncomelania hupensis,Intermediate Host of Schistosoma japonicum in Mainland China as Revealed by ITS Sequences

Background

Oncomelania hupensis is the unique intermediate host of Schistosoma japonicum, which causes schistosomiasis endemic in the Far East, and especially in mainland China. O. hupensis largely determines the parasite''s geographical range. How O. hupensis''s genetic diversity is distributed geographically in mainland China has never been well examined with DNA sequence data.

Methodology/Principal Findings

In this study we investigate the genetic variation among O. hupensis from different geographical origins using the combined complete internal transcribed spacer 1 (ITS1) and ITS2 regions of nuclear ribosomal DNA. 165 O. hupensis isolates were obtained in 29 localities from 7 provinces across mainland China: lake/marshland and hill regions in Anhui, Hubei, Hunan, Jiangxi and Jiangsu provinces, located along the middle and lower reaches of Yangtze River, and mountainous regions in Sichuan and Yunnan provinces. Phylogenetic and haplotype network analyses showed distinct genetic diversity and no shared haplotypes between populations from lake/marshland regions of the middle and lower reaches of the Yangtze River and populations from mountainous regions of Sichuan and Yunnan provinces. The genetic distance between these two groups is up to 0.81 based on Fst, and branch time was estimated as 2–6 Ma. As revealed in the phylogenetic tree, snails from Sichuan and Yunnan provinces were also clustered separately. Geographical separation appears to be an important factor accounting for the diversification of the two groups of O. hupensis in mainland China, and probably for the separate clades between snails from Sichuan and Yunnan provinces. In lake/marshland and hill regions along the middle and lower reaches of the Yangtze River, three clades were identified in the phylogenetic tree, but without any obvious clustering of snails from different provinces.

Conclusions

O. hupensis in mainland China may have considerable genetic diversity, and a more complex population structure than expected. It will be of significant importance to consider the genetic diversity of O. hupensis when assessing co-evolutionary interactions with S. japonicum.     

Assessment of the N-PCR Assay in Diagnosis of Pleural Tuberculosis: Detection of M.tuberculosis in Pleural Fluid and Sputum Collected in Tandem

Background

The nonspecific clinical presentation and paucibacillary nature of tuberculous pleuritis remains a challenge for diagnosis. Diagnosis of tuberculous pleural effusion depends on the demonstration of the presence of tubercle bacilli in the sputum, pleural fluid, or pleural biopsy specimen, or demonstration of granuloma in pleura by histological examination. We examined the clinical utility of the diagnosis of pleural tuberculosis using the in house N-PCR assay, AFB smear microscopy and culture. Besides pleural fluid the inclusion of sputum in the efficacy of diagnosis of pleural tuberculosis was scrutinized.

Methodology/Principal Findings

Pleural fluid and sputum samples of 58 tuberculous and 42 non-tuberculous pleural effusion patients were processed for AFB smear microscopy, culture and the N-PCR assay. Mycobacteria were detected exclusively in tuberculous pleural effusion samples. None of the non-tuberculous pleural effusion samples were positive for mycobacteria. Comparative analysis showed that the N-PCR assay had the highest sensitivity. Inclusion of sputum along with pleural fluid increased N-PCR sensitivity from 51.7 to 70.6% (p<0.0001).This improved sensitivity was reflected in AFB smear microscopy and isolation by culture. The sensitivity enhanced on inclusion of sputum from 3.4 (p = 0.50) to 10.3% (p = 0.038) for AFB smear microscopy and for isolation of mycobacteria from 10.3(p = 0.03) to 22.4% (p = 0.0005). Thirteen isolates were obtained from 58 pleural tuberculosis patients. Eleven mycobacterial isolates were identified as M.tuberculosis and two as M.fortuitum and M.chelonae. Complete concordance was seen between the biochemical identification of isolates and the N-PCR identification of mycobacterial species prior to isolation.

Conclusions/Significance

To the best of our knowledge this is the first PCR based report on utility of sputum for diagnosis of pleural tuberculosis. The present study demonstrates that a combination of pleural fluid with sputum sample and N-PCR improved the diagnosis of pleural tuberculosis.     

Dual-mode Imaging of Cutaneous Tissue Oxygenation and Vascular Function

Accurate assessment of cutaneous tissue oxygenation and vascular function is important for appropriate detection, staging, and treatment of many health disorders such as chronic wounds. We report the development of a dual-mode imaging system for non-invasive and non-contact imaging of cutaneous tissue oxygenation and vascular function. The imaging system integrated an infrared camera, a CCD camera, a liquid crystal tunable filter and a high intensity fiber light source. A Labview interface was programmed for equipment control, synchronization, image acquisition, processing, and visualization. Multispectral images captured by the CCD camera were used to reconstruct the tissue oxygenation map. Dynamic thermographic images captured by the infrared camera were used to reconstruct the vascular function map. Cutaneous tissue oxygenation and vascular function images were co-registered through fiduciary markers. The performance characteristics of the dual-mode image system were tested in humans.