Purification and biological characterization of bacterially expressed recombinant buffalo prolactin

Recombinant prolactin (PRL) from water buffalo (Bubalus bubalis) has been cloned and expressed in a prokaryotic expression system. The hormone was also successfully refolded into a biologically active form. Total RNA was purified from buffalo pituitaries and the buPRL cDNA was synthesized using primers designed on bovine PRL sequence. This prolactin cDNA was cloned in a pET 28a vector and expressed in Escherichia coli strain BL21(DE3)pLysS. Most of the expressed protein was present as insoluble inclusion bodies. The inclusion bodies were solubilized and buPRL was purified by Ni-NTA column. The purified protein was refolded by gradually decreasing the concentration of denaturant during dialysis. Total yield of the refolded and soluble prolactin was 22?mg/L from 100?mL bacterial culture in LB medium. The recombinant prolactin was as active as native prolactin in stimulating growth of Nb2 lymphoma cells.     

Geographic and ecologic heterogeneity in elimination thresholds for the major vector-borne helminthic disease,lymphatic filariasis

Background

Large-scale intervention programmes to control or eliminate several infectious diseases are currently underway worldwide. However, a major unresolved question remains: what are reasonable stopping points for these programmes? Recent theoretical work has highlighted how the ecological complexity and heterogeneity inherent in the transmission dynamics of macroparasites can result in elimination thresholds that vary between local communities. Here, we examine the empirical evidence for this hypothesis and its implications for the global elimination of the major macroparasitic disease, lymphatic filariasis, by applying a novel Bayesian computer simulation procedure to fit a dynamic model of the transmission of this parasitic disease to field data from nine villages with different ecological and geographical characteristics. Baseline lymphatic filariasis microfilarial age-prevalence data from three geographically distinct endemic regions, across which the major vector populations implicated in parasite transmission also differed, were used to fit and calibrate the relevant vector-specific filariasis transmission models. Ensembles of parasite elimination thresholds, generated using the Bayesian fitting procedure, were then examined in order to evaluate site-specific heterogeneity in the values of these thresholds and investigate the ecological factors that may underlie such variability

Results

We show that parameters of density-dependent functions relating to immunity, parasite establishment, as well as parasite aggregation, varied significantly between the nine different settings, contributing to locally varying filarial elimination thresholds. Parasite elimination thresholds predicted for the settings in which the mosquito vector is anopheline were, however, found to be higher than those in which the mosquito is culicine, substantiating our previous theoretical findings. The results also indicate that the probability that the parasite will be eliminated following six rounds of Mass Drug Administration with diethylcarbamazine and albendazole decreases markedly but non-linearly as the annual biting rate and parasite reproduction number increases.

Conclusions

This paper shows that specific ecological conditions in a community can lead to significant local differences in population dynamics and, consequently, elimination threshold estimates for lymphatic filariasis. These findings, and the difficulty of measuring the key local parameters (infection aggregation and acquired immunity) governing differences in transmission thresholds between communities, mean that it is necessary for us to rethink the utility of the current anticipatory approaches for achieving the elimination of filariasis both locally and globally.

     

Targeting antibiotics to households for trachoma control

Background

Mass drug administration (MDA) is part of the current trachoma control strategy, but it can be costly and results in many uninfected individuals receiving treatment. Here we explore whether alternative, targeted approaches are effective antibiotic-sparing strategies.

Methodology/Principal Findings

We analysed data on the prevalence of ocular infection with Chlamydia trachomatis and of active trachoma disease among 4,436 individuals from two communities in The Gambia (West Africa) and two communities in Tanzania (East Africa). An age- and household-structured mathematical model of transmission was fitted to these data using maximum likelihood. The presence of active inflammatory disease as a marker of infection in a household was, in general, significantly more sensitive (between 79% [95%CI: 60%–92%] and 86% [71%–95%] across the four communities) than as a marker of infection in an individual (24% [16%–33%]–66% [56%–76%]). Model simulations, under the best fit models for each community, showed that targeting treatment to households has the potential to be as effective as and significantly more cost-effective than mass treatment when antibiotics are not donated. The cost (2007US$) per incident infection averted ranged from 1.5 to 3.1 for MDA, from 1.0 to 1.7 for household-targeted treatment assuming equivalent coverage, and from 0.4 to 1.7 if household visits increased treatment coverage to 100% in selected households. Assuming antibiotics were donated, MDA was predicted to be more cost-effective unless opportunity costs incurred by individuals collecting antibiotics were included or household visits improved treatment uptake. Limiting MDA to children was not as effective in reducing infection as the other aforementioned distribution strategies.

Conclusions/Significance

Our model suggests that targeting antibiotics to households with active trachoma has the potential to be a cost-effective trachoma control measure, but further work is required to assess if costs can be reduced and to what extent the approach can increase the treatment coverage of infected individuals compared to MDA in different settings.     

The role of reduced iron powder in the fermentative production of tetanus toxin

When tetanus toxin is made by fermentation with Clostridium tetani, the traditional source of iron is an insoluble preparation called reduced iron powder. This material removes oxygen from the system by forming FeO₂ (rust). When inoculated in a newly developed medium lacking animal and dairy products and containing glucose, soy-peptone, and inorganic salts, growth and toxin production were poor without reduced iron powder. The optimum concentration of reduced iron powder for toxin production was found to be 0.5 g/l. Growth was further increased by higher concentrations, but toxin production decreased. Inorganic iron sources failed to replace reduced iron powder for growth or toxin formation. The iron source that came closest was ferrous ammonium sulfate. The organic iron sources ferric citrate and ferrous gluconate were more active than the inorganic compounds but could not replace reduced iron powder. Insoluble iron sources, such as iron wire, iron foil, and activated charcoal, were surprisingly active. Combinations of activated charcoal with soluble iron sources such as ferrous sulfate, ferric citrate, and ferrous gluconate showed increased activity, and the ferrous gluconate combination almost replaced reduced iron powder. It thus appears that the traditional iron source, reduced iron powder, plays a double role in supporting tetanus toxin formation, i.e., releasing soluble sources of iron and providing an insoluble surface.     

Influence of extrinsic factors on granulation in UASB reactor

The aim of this mini-review is to synthesize and analyze information on how the process of granulation is affected by environmental and operational conditions in the reactor. The factors reviewed are temperature, pH, alkalinity, organic loading rate, upflow velocity, nature and strength of substrate, nutrients, multivalent cations and heavy metals, microbial ecology of seed sludge, exo-cellular polymer, and addition of natural and synthetic polymers. Careful temperature control and adequate alkalinity is required for generation and maintenance of granules. Nature and strength of substrate in conjunction with intra-granular diffusion to a large extent determines the microstructure of the granules. The divalent cations such as calcium and iron may enhance granulation by ionic bridging and linking exo-cellular polymers. However, their presence in excess may lead to cementation due to precipitation leading to increased ash content and mass transfer limitation. The addition of external additives such as ionic polymers may enhance granulation in the upflow anaerobic sludge blanket reactors.     

Substrate mapping and inhibitor profiling of falcipain-2, falcipain-3 and berghepain-2: implications for peptidase anti-malarial drug discovery

The Plasmodium falciparum cysteine peptidases FP-2 (falcipain-2) and FP-3 (falcipain-3), members of the papain-like CAC1 family, are essential haemoglobinases and are therefore potential anti-malarial drug targets. To facilitate a rational drug discovery programme, in the current study we analysed the synthetic substrate and model inhibitor profiles of FP-2 and FP-3 as well as BP-2 (berghepain-2), an orthologue from the rodent parasite Plasmodium berghei. With respect to substrate catalysis, FP-2 exhibited a promiscuous substrate profile based around a consensus non-primeside motif, FP-3 was somewhat more restricted and BP-2 was comparatively specific. Substrate turnover for FP-2 was driven by a basic or acidic P1 residue, whereas for FP-3 turnover occurred predominately through a basic P1 residue only, and for BP-2, turnover was again mainly through a basic P1 residue for some motifs and surprisingly a glycine in the P1 position for other motifs. Within these P1 binding elements, additional recognition motifs were observed with subtle nuances that switched substrate turnover on or off through specific synergistic combinations. The peptidases were also profiled against reversible and irreversible cysteine peptidase inhibitors. The results re-iterated the contrasting kinetic behaviour of each peptidase as observed through the substrate screens. The results showed that the substrate and inhibitor preferences of BP-2 were markedly different from those of FP-2 and FP-3. When FP-2 and FP-3 were compared to each other they also displayed similarities and some significant differences. In conclusion, the in vitro data highlights the current difficulties faced by a peptidase directed anti-malarial medicinal chemistry programme where compounds need to be identified with potent activity against at least three peptidases, each of which displays distinct biochemical traits.     

Role of anion gap and different electrolytes in hypertension during pregnancy (preeclampsia)

The present study was designed to determine the changes in serum sodium, anion gap, different antioxidants and free radicals in preeclamptic patients and control subjects. Serum sodium, chloride, bicarbonate, calcium, potassium and magnesium were estimated and anion gap was determined in 100 proteinuric hypertensive and 100 normotensive pregnant women. Mean serum sodium, chloride and bicarbonate level (133.26 ± 13.1, 104.97 ± 11.37, and 22.01 ± 4.66 mEq/l, respectively) were significantly higher in proteinuric hypertensive women as compared to controls (125.85 ± 10.4, 101.90 ± 6.3, 19.34 ± 3.21 mEq/l, respectively) whereas anion gap level (6.28 ± 16.147) was nonsignificantly higher in proteinuric hypertensive as compared to normotensive (4.61 ± 11.84). Total serum sodium concentration increases in preeclamptic subjects, the exact distribution of serum sodium in various compartments of the body are not clear and correlation of serum sodium and anion gap with proteinuria is also not known. The levels of different antioxidants were decreased in preeclamptic patients as compared to the controls while the level of free radicals elevated in preeclamptic subjects as compared to controls. In our study, anion gap level was found to be rather non-significantly higher in proteinuric hypertensive women as compared to normotensive women.     

Differential usage of carbohydrate co-receptors influences cellular tropism of Theiler's murine encephalomyelitis virus infection of the central nervous system

Theiler's murine encephalomyelitis viruses (TMEV) are ubiquitous pathogens of mice, producing either rapidly fatal encephalitis (high-neurovirulence strains) or persistent central nervous system infection and inflammatory demyelination (low-neurovirulence strains). Although a protein entry receptor has not yet been identified, carbohydrate co-receptors that effect docking and concentration of the virus on the cell surface are known for both TMEV neurovirulence groups. Low-neurovirulence TMEV use α2,3-linked N-acetylneuramic acid (sialic acid) on an N-linked glycoprotein, whereas high-neurovirulence TMEV use the proteoglycan heparan sulfate (HS) as a co-receptor. While the binding of low-neurovirulence TMEV to sialic acid can be inhibited completely, only a third of the binding of high-neurovirulence TMEV to HS is inhibitable, suggesting that high-neurovirulence strains use another co-receptor or bind directly to the putative protein entry receptor. Four amino acids on the surface (VP2 puff B) of low-neurovirulence strains make contact with sialic acid through non-covalent hydrogen bonds. Since these virus residues are conserved in all TMEV strains, the capsid conformation of this region is probably responsible for sialic acid binding. A persistence determinant that maps within the virus coat using recombinant TMEV is also conformational in nature. Low-neurovirulence virus variants that do not bind to sialic acid fail to persist in the central nervous system of mice, indicating a role for sialic acid binding in TMEV persistence. Analysis of high-neurovirulence variants that do not bind HS demonstrates that HS co-receptor usage influences neuronal tropism in brain, whereas, the HS co-receptor use is not required for the infection of spinal cord anterior horn cells associated with poliomyelitis.