Isolation and in vitro fusion of egg and sperm cells in Oryza sativa.

Protocols for isolation of gametes of Oryza sativa were developed and initial results on in vitro fusion of sperm and egg cells are reported. The best yield of viable sperm cells was obtained when pollen grains were cultured in a medium containing of 1.3 mM boric acid, 3.6 mM calcium chloride, 0.74 mM potassium phosphate, and 438 mM sucrose. Embryo sacs were isolated using cell wall degrading enzyme treatments for 2-5 h followed by mechanical manipulation. The maximum yield (38.2%) of egg cell was achieved when 2% cellulase and 0.55% pectinase were used in the medium. However, the optimum concentration of cellulase and pectinase was found to be 1% and 0.85%, respectively. Fluorescein diacetate (FDA) stain was used to determine the viable sperm and egg cells. The optimal procedures (fusion conditions) for gametes fusion occurred in a medium containing calcium chloride at a concentration of 7 mM (pH 7.5) and the best result obtained (55.5%) in terms of fused gametes, is reported.     

Role of the CXCR4-SDF1-HMGB1 pathway in the directional migration of cells and regeneration of affected organs

In recent years, several studies have reported positive outcomes of cell-based therapies despite insufficient engraftment of transplanted cells. These findings have created a huge interest in the regenerative potential of paracrine factors released from transplanted stem or progenitor cells. Interestingly, this notion has also led scientists to question the role of proteins in the secretome produced by cells, tissues or organisms under certain conditions or at a particular time of regenerative therapy. Further studies have revealed that the secretomes derived from different cell types contain paracrine factors that could help to prevent apoptosis and induce proliferation of cells residing within the tissues of affected organs. This could also facilitate the migration of immune, progenitor and stem cells within the body to the site of inflammation. Of these different paracrine factors present within the secretome, researchers have given proper consideration to stromal cell-derived factor-1 (SDF1) that plays a vital role in tissue-specific migration of the cells needed for regeneration. Recently researchers recognized that SDF1 could facilitate site-specific migration of cells by regulating SDF1-CXCR4 and/or HMGB1-SDF1-CXCR4 pathways which is vital for tissue regeneration. Hence in this study, we have attempted to describe the role of different types of cells within the body in facilitating regeneration while emphasizing the HMGB1-SDF1-CXCR4 pathway that orchestrates the migration of cells to the site where regeneration is needed.     

Effect of Rhizobium sp. BARIRGm901 inoculation on nodulation,nitrogen fixation and yield of soybean (Glycine max) genotypes in gray terrace soil

Soybean plants require high amounts of nitrogen, which are mainly obtained from biological nitrogen fixation. A field experiment was conducted by soybean (Glycine max) genotypes, growing two varieties (Shohag and BARI Soybean6) and two advanced lines (MTD10 and BGM02026) of soybean with or without Rhizobium sp. BARIRGm901 inoculation. Soybean plants of all genotypes inoculated with Rhizobium sp. BARIRGm901 produced greater nodule numbers, nodule weight, shoot and root biomass, and plant height than non-inoculated plants. Similarly, inoculated plants showed enhanced activity of nitrogenase (NA) enzyme, contributing to higher nitrogen fixation and assimilation, compared to non-inoculated soybean plants in both years. Plants inoculated with Rhizobium sp. BARIRGm901 also showed higher pod, stover, and seed yield than non-inoculated plants. Therefore, Rhizobium sp. BARIRGm901 established an effective symbiotic relationship with a range of soybean genotypes and thus increased the nodulation, growth, and yield of soybean grown in gray terrace soils in Bangladesh.     

Potential of mean force for separation of the repeating units in cellulose and hemicellulose

As a first step toward understanding the energetics of removal of cello-oligomers from the cellulose surface, we have performed umbrella sampling calculations to determine the free energy required for separation of repeating units of cellulose and hemicellulose from each other. Molecular dynamics (MD) simulations were performed for both the stacked and non-stacked arrangements of the cellobiose pair system and the xylobiose pair system. These stacked and non-stacked arrangements were taken as representative systems for the crystalline and amorphous domains in cellulose and hemicellulose. In addition, similar calculations were also carried out to determine the energetics involved in the separation of the cellobiose–xylobiose molecule pair in the non-stacked arrangement. The potential of mean force profiles exhibit a single minimum in all cases and are qualitatively similar. Our results show that the location of the minimum as well as the depth of the well can be correlated with the size of the disaccharide molecules.     

Overexpression of <Emphasis Type="Italic">AtATM3</Emphasis> in <Emphasis Type="Italic">Brassica juncea</Emphasis> confers enhanced heavy metal tolerance and accumulation

The eucalypt Corymbia torelliana × C. citriodora is planted widely in India, Brazil and Australia although plantation establishment has been limited by inadequate seed supply and low amenability to propagation via cuttings. This study optimised node culture and organogenic culture methods for in vitro propagation of Corymbia hybrids by identifying explant position (topophysic) effects on rooting, shoot elongation and shoot proliferation. Strong, negative morphogenic gradients in shoot elongation and proliferation capacity were evident from the cotyledonary node to the fourth or fifth node of seedlings when their nodes were transferred to node culture (without benzyladenine). These topophysic effects were related to differences in rooting capacity of individual nodes. Root formation in node culture was associated with formation of long multi-nodal axillary shoots, and so higher rooting of shoots from the cotyledonary node or first true-leaf node was associated with higher shoot proliferation. However, all nodes were equally capable of shoot proliferation in organogenic culture (with 2.2 μM benzyladenine), where rooting and rapid stem elongation did not occur. Most shoots (61–100%) from both node culture and organogenic culture were converted to plantlets, with plantlet conversion and primary root number not differing significantly among explant node positions. The strong topophysic effect in node culture, combined with the lack of a topophysic effect in organogenic culture, provides for an optimised clonal propagation system based on segregation of nodes from the same seedling into separate node and organogenic culture pathways.     

Evaluation of a Typhoid/Paratyphoid Diagnostic Assay (TPTest) Detecting Anti-Salmonella IgA in Secretions of Peripheral Blood Lymphocytes in Patients in Dhaka,Bangladesh

Background

Rapid and reliable diagnostic assays for enteric (typhoid and paratyphoid) fever are urgently needed. We report the characterization of novel approach utilizing lymphocyte secretions, for diagnosing patients with enteric fever by the TPTest procedure.

Methodology

TPTest detects Salmonella-specific IgA responses in lymphocyte culture supernatant. We utilized TPTest in patients with suspected enteric fever, patients with other illnesses, and healthy controls. We also evaluated simplified modifications of TPTest for adaptation in laboratories with limited facilities and equipment.

Principal Findings

TPTest was positive in 39 (27 typhoid and 12 paratyphoid A) patients confirmed by blood culture and was negative in 74 healthy individuals. Among 32 individuals with other illnesses, 29 were negative by TPTest. Of 204 individuals with suspected enteric fever who were negative by blood culture, 44 were positive by TPTest and the patients were clinically indistinguishable from patients with confirmed bacteremia, except they were more likely to be under 5 years of age. We evaluated simplifications in TPTest, including showing that lymphocytes could be recovered using lysis buffer or buffy coat method as opposed to centrifugation, that incubation of cells at 37°C did not require supplemental CO₂, and that results were available for majority of samples within 24 hours. Positive results by TPTest are transient and revert to negative during convalescence, supporting use of the test in endemic areas. The results can also be read using immunodot blot approach as opposed to ELISA. Since no true gold standard currently exists, we used a number of definitions of true positives and negatives. TPTest had sensitivity of 100% compared to blood culture, and specificity that ranged from 78–97% (73–100, 95% CI), depending on definition of true negative.

Conclusion

The TPTest is useful for identification of patients with enteric fever in an endemic area, and additional development of simplified TPTest is warranted.     

SMAD2 rs4940086 heterozygosity increases the risk of cervical cancer development among the women in Bangladesh

Molecular Biology Reports - SMAD2 is a critical signal transducer molecule in the TGFβ- SMAD pathway which is also known for its tumor suppressor role. Genetic variations in SMAD2 render cells...     

Th1 and Th17 Responses to Helicobacter pylori in Bangladeshi Infants,Children and Adults

Both Th1 and Th17 cells are important components of the immune response to Helicobacter pylori (Hp) in adults, but less is known about T cell responses to Hp during early childhood, when the infection is often acquired. We investigated Th1 and Th17 type responses to Hp in adults, children and infants in Bangladesh, where Hp is highly endemic. IL-17 and IFN-γ mRNA levels in gastric biopsies from Hp-infected Bangladeshi adults were analyzed and compared to levels in infected and uninfected Swedish controls. Since biopsies could not be collected from infants and children, cytokine responses in Bangladeshi infants (6–12 months), children (3–5 years) and adults (>19 years) were instead compared by stimulating peripheral blood mononuclear cells (PBMCs) with a Hp membrane preparation (MP) and analyzing culture supernatants by ELISA and cytometric bead array. We found significantly higher expression of IL-17 and IFN-γ mRNA in gastric mucosa of Hp-infected Bangladeshi and Swedish adults compared to uninfected Swedish controls. PBMCs from all age groups produced IL-17 and IFN-γ after MP stimulation, but little Th2 cytokines. IL-17 and IFN-γ were primarily produced by CD4⁺ T cells, since CD4⁺ T cell depleted PBMCs produced reduced amounts of these cytokines. Infant cells produced significantly more IL-17, but similar levels of IFN-γ, compared to adult cells after MP stimulation. In contrast, polyclonal stimulation induced lower levels IL-17 and IFN-γ in infant compared to adult PBMCs and CD4⁺ T cells. The strong IL-17 production in infants after MP stimulation was paralleled by significantly higher production of the IL-17 promoting cytokine IL-1β from infant compared to adult PBMCs and monocytes. In conclusion, these results show that T cells can produce high levels of IL-17 and IFN-γ in response to Hp from an early age and indicate a potential role for IL-1β in promoting Th17 responses to Hp during infancy.     

Metabolic cycles are linked to the cardiovascular diurnal rhythm in rats with essential hypertension

Background

The loss of diurnal rhythm in blood pressure (BP) is an important predictor of end-organ damage in hypertensive and diabetic patients. Recent evidence has suggested that two major physiological circadian rhythms, the metabolic and cardiovascular rhythms, are subject to regulation by overlapping molecular pathways, indicating that dysregulation of metabolic cycles could desynchronize the normal diurnal rhythm of BP with the daily light/dark cycle. However, little is known about the impact of changes in metabolic cycles on BP diurnal rhythm.

Methodology/Principal Findings

To test the hypothesis that feeding-fasting cycles could affect the diurnal pattern of BP, we used spontaneously hypertensive rats (SHR) which develop essential hypertension with disrupted diurnal BP rhythms and examined whether abnormal BP rhythms in SHR were caused by alteration in the daily feeding rhythm. We found that SHR exhibit attenuated feeding rhythm which accompanies disrupted rhythms in metabolic gene expression not only in metabolic tissues but also in cardiovascular tissues. More importantly, the correction of abnormal feeding rhythms in SHR restored the daily BP rhythm and was accompanied by changes in the timing of expression of key circadian and metabolic genes in cardiovascular tissues.

Conclusions/Significance

These results indicate that the metabolic cycle is an important determinant of the cardiovascular diurnal rhythm and that disrupted BP rhythms in hypertensive patients can be normalized by manipulating feeding cycles.     

Individuals with Le(a+b-) blood group have increased susceptibility to symptomatic vibrio cholerae O1 infection

Background

Human genetic factors such as blood group antigens may affect the severity of infectious diseases. Presence of specific ABO and Lewis blood group antigens has been shown previously to be associated with the risk of different enteric infections. The aim of this study was to determine the relationship of the Lewis blood group antigens with susceptibility to cholera, as well as severity of disease and immune responses to infection.

Methodology

We determined Lewis and ABO blood groups of a cohort of patients infected by Vibrio cholerae O1, their household contacts, and healthy controls, and analyzed the risk of symptomatic infection, severity of disease if infected and immune response following infection.

Principal Findings

We found that more individuals with cholera expressed the Le(a+b−) phenotype than the asymptomatic household contacts (OR 1.91, 95% CI 1.03–3.56) or healthy controls (OR 1.90, 95% CI 1.13–3.21), as has been seen previously for the risk of symptomatic ETEC infection. Le(a–b+) individuals were less susceptible to cholera and if infected, required less intravenous fluid replacement in hospital, suggesting that this blood group may be associated with protection against V. cholerae O1. Individuals with Le(a–b−) blood group phenotype who had symptomatic cholera had a longer duration of diarrhea and required higher volumes of intravenous fluid replacement. In addition, individuals with Le(a–b−) phenotype also had lessened plasma IgA responses to V. cholerae O1 lipopolysaccharide on day 7 after infection compared to individuals in the other two Lewis blood group phenotypes.

Conclusion

Individuals with Lewis blood type Le(a+b−) are more susceptible and Le(a–b+) are less susceptible to V. cholerae O1 associated symptomatic disease. Presence of this histo-blood group antigen may be included in evaluating the risk for cholera in a population, as well as in vaccine efficacy studies, as is currently being done for the ABO blood group antigens.