Role of arginine-163 and the 163REEK166 motif in the oligomerization of truncated alpha A-crystallins

To gain insight into the mechanism by which Arg-163 influences oligomerization of alphaA-crystallin, we prepared a series of truncated alphaA-crystallins with or without mutation of the Arg-163 residue. Expression of the proteins was achieved in Escherichia coli BL21 (DE3) pLysS cells, and alphaA-crystallin was purified by size-exclusion chromatography. Molecular mass was determined by molecular sieve HPLC, chaperone activity was assayed with alcohol dehydrogenase as the target protein, and structural changes were ascertained by circular dichroism (CD) measurements. With an increasing number of residues deleted, there was about a 3% decrease in oligomeric size per residue, until 10 residues were deleted. When 11 residues, including Arg-163, were deleted, the oligomeric size decreased 85%. Mutation of Arg-163 to Gly (R163G) did not affect the molecular mass in the full-length alphaA-crystallin. However, R163G mutants of all the truncated alphaA-crystallins showed a decrease in oligomeric size, those lacking 8, 9, and 10 residues showing 60-80% decrease and those lacking 5, 6, and 7 residues showing only a 7-14% decrease as compared to the corresponding truncated alphaA-crystallin. These data suggest that R163, E164, E165, and K166 in the REEK motif are also relevant to alphaA-crystallin oligomerization. The molecular masses of alphaA1-163 and alphaA1-163 (R163K) were nearly the same, which suggests that the role of Arg-163 is to provide a positive charge for intersubunit electrostatic interactions in the C-terminal domain. In alphaA1-162 (S162R), recovery of the molecular mass to the level in alphaA1-163 has not occurred; this shows that the actual position of R163 is important.     

Cell damage and recovery in cryopreserved microphytobenthic diatoms

Two pennate microphytobenthic diatoms, Amphora coffeaeformis (Agardh) Kutzing and Navicula transitans var. derasa f. delicatula Heimdal, were cryopreserved and monitored on thawing to track the mechanical injuries and their post-preservation recovery. Cells were subjected to (1) direct freezing in liquid nitrogen and (2) two-step cooling with and without the cryoprotectant, dimethyl sulfoxide (Me(2)SO). Mechanical injury due to exposure to low temperature differed between the two species. While A. coffeaeformis cells were intact and could survive even direct freezing without a cryoprotectant, N. delicatula cell chloroplasts were damaged. However, the two-step cooling along with a cryoprotectant minimized the mechanical injury to cells of both species thereby enhancing the post-thaw viability.     

Relevance of biofilm bacteria in modulating the larval metamorphosis of Balanus amphitrite

Natural microbial communities found on different substrata exposed to the marine environment, including barnacle shell surfaces, are reported to have varying influences on the settlement and metamorphosis of competent cypris larvae. Experiments were carried out to compare the influence of settlement-inducing compounds from the bacteria isolated from the shell surface of Balanus amphitrite on its larval metamorphosis. The effect of multispecies bacterial film was also assessed. The production of different molecules by the bacteria was influenced by the nutrient media under which they were grown. It was observed that the promotory multispecies bacterial film turned to inhibition mode in the presence of the adult extract of the barnacle, indicating that bacteria-adult extract interactions alter the synthesis of different compounds produced by bacteria. The studies also show that the waterborne and the surface-associated cues from the bacteria function differentially in mediating larval metamorphosis. Understanding the complexities involved in such interactions and identification of the factors governing them would be a step forward.     

Sex-based differences in the tensile properties of the human anterior cruciate ligament

After immense amounts of research, the root cause for the significantly higher rates of anterior cruciate ligament (ACL) failure incidents in females as compared to males still remains unknown and the existing sex-based disparity has not diminished. To date, the possibility that the female ACL is mechanically weaker than the male ACL has not been directly investigated. Although it has been established in the literature that the female ACL is smaller in size, the differences in the structural and material properties of the ACL between sexes have not been studied. The aim of this cadaveric study was to determine if any sex-based differences in the tensile properties of the human ACL exist when considering age as well as ACL and body anthropometric measurements as covariates. Ten male and 10 female unpaired cadaveric knees (mean age 36.75 years) were used for this study. The geometry of the ACL (including length, cross-sectional area, and volume) was analyzed using a 3-D scanning system. The femur-ACL tibia complex was tested to failure along the longitudinal axis of the ligament in a tensile testing machine. The structural properties of the ACL as well as its mechanical properties were determined. Analysis of covariance was performed to assess the effect of sex on tensile properties. The female ACL was found to have a lower mechanical properties (8.3% lower strain at failure; 14.3% lower stress at failure, 9.43% lower strain energy density at failure, and most importantly, 22.49% lower modulus of elasticity) when considering age, ACL, and body anthropometric measurements as covariates.     

Oxidative alterations induced by D-aspartic acid in prepubertal rat testis in vitro: a mechanistic study

The objective of the present study was to investigate the oxidative induction response following in vitro treatment with D-aspartic acid (DA) in prepubertal rat testis (homogenates, explants, and cell suspensions). In all three preparations, DA enhanced (P<0.001) lipid peroxidation, manifest as increased reactive oxygen species (ROS) and malondialdehyde (MDA). Further, DA-induced oxidative induction was potentiated (P<0.001) in the presence of iron (5 microM) and 3-amino triazole and mercaptosuccinate (P<0.001), known inhibitors of the peroxide metabolizing enzymes, catalase and glutathione peroxidase, respectively. Testis homogenates exposed to L-arginine (LA) per se had reduced (P<0.001) endogenous levels of ROS and MDA; furthermore, pre-incubation with L-arginine markedly suppressed (P<0.001) DA-induced oxidative induction, suggesting an antagonistic action, perhaps due to LA-derived nitric oxide. In conclusion, DA caused significant oxidative induction in prepubertal rat testis, but this action was abrogated by L-arginine. The relevance of this phenomenon in vivo merits further study, as both of these molecules have specific physiological functions in the testis.     

Glucose-induced activation of H+-ATPase in Dunaliella salina and its role in hygromycin resistance

Dunaliella salina, a eukaryotic microalga, is known for its highly halophilic nature. The high level of salts in growth medium for this alga has made its genetic transformation a comparatively difficult procedure, particularly during the selection stage. The high salt content decreases the efficiency of most antibiotics which are being used as selection markers. Studies pertaining to the interrelationship between salt concentration and antibiotic sensitivity are scarce in Dunaliella. During our previous experiment at genetic transformation of Dunaliella, an inverse relationship between the amount of antibiotic hygromycin and sodium chloride in the medium was revealed. A possible link between plasma membrane activity and the hygromycin sensitivity was investigated in the present study by modulating plasma membrane H⁺-ATPase activity using glucose. Glucose-induced activation of H⁺-ATPase, reduced the tolerance of D. salina to the antibiotic hygromycin. Hygromycin concentration required for selection during genetic transformation of Dunaliella was lowered from 100 to 25 mg L^?1 in the presence of 10 mM glucose. Conversely, the inhibitors of the plasma membrane H⁺-ATPase, orthovanadate and diethylstilbestrol were found to inhibit the glucose activation at concentrations of 10 and 15 μM, respectively. The activation of H⁺-ATPase by glucose was further confirmed through H⁺-ATPase assay and medium acidification experiments. The results indicated that the sensitivity of Dunaliella to antibiotic is related to H⁺-ATPase and the possible involvement of pH gradient, created through H⁺-ATPase activation during drug transport.     

Integration of trichoderma pseudokoningii with agrochemicals for disease management and plant development in mulberry

Vegetative propagation of mulberry (Morus spp.) is greatly afflicted by soilborne diseases. Stem canker (Botryodiplodia theobromae) and cutting rot (Fusarium solani) are the major diseases which cause high rate of mortality of stem‐cuttings (vegetative clones), thus affecting the initial establishment of mulberry. Apart from diseases, the poor rooting ability of stem‐cuttings of many promising mulberry varieties also leads to unproductive propagation. An effective biocontrol agent (Trichoderma pseudokoningii), chemical fungicide (Dithane M‐45) and phytohormones (Indole‐3‐acetic acid, IAA and 1‐Naphthalene acetic acid, NAA) were evaluated individually and in combination for their effects on disease control and plant development. In plates, T. pseudokoningii was found to be compatible with Dithane M‐45 at 0.1%, and IAA and NAA at 20 ppm concentrations. Under field conditions, the integration of soil application of T. pseudokoningii (in the form of its formulated product, ‘NURSERY‐GUARD') and dipping of stem‐cuttings in 0.1% solution of Dithane M‐45, or 20 ppm solution of IAA showed better results than their individual application. NURSERY‐GUARD with Dithane M‐45 (0.1%) or IAA (20 ppm) provided 47.6 to 52.4% disease control to stem‐cuttings which resulted in significant increase (over check) in sprouting of stem‐cuttings (53.1–63.3%) and plant survival (52.6–57.8%).     

Optimal Allocation of Resources in Female Sex Worker Targeted HIV Prevention Interventions: Model Insights from Avahan in South India

Background

The Avahan programme has provided HIV prevention activities, including condom promotion, to female sex workers (FSWs) in southern India since 2004. Evidence suggests Avahan averted 202,000 HIV infections over 4 years. For replicating this intervention elsewhere, it is essential to understand how the intervention’s impact could have been optimised for different budget levels.

Methods

Behavioural data were used to determine how condom use varied for FSWs with different levels of intervention intensity. Cost data from 64 Avahan districts quantified how district-level costs related to intervention scale and intensity. A deterministic model for HIV transmission amongst FSWs and clients projected the impact and cost of intervention strategies for different scale and intensity, and identified the optimal strategies that maximise impact for different budget levels.

Results

As budget levels increase, the optimal intervention strategy is to first increase intervention intensity which achieves little impact, then scale-up coverage to high levels for large increases in impact, and lastly increase intensity further for small additional gains. The cost-effectiveness of these optimal strategies generally improves with increasing resources, while straying from these strategies can triple costs for the same impact. Projections suggest Avahan was close to being optimal, and moderate budget reductions (≥20%) would have reduced impact considerably (>40%).

Discussion

Our analysis suggests that tailoring the design of HIV prevention programmes for FSWs can improve impact, and that a certain level of resources are required to achieve demonstrable impact. These insights are critical for optimising the use of limited resources for preventing HIV.     

Community Mobilisation and Empowerment Interventions as Part of HIV Prevention for Female Sex Workers in Southern India: A Cost-Effectiveness Analysis

Background

Most HIV prevention for female sex workers (FSWs) focuses on individual behaviour change involving peer educators, condom promotion and the provision of sexual health services. However, there is a growing recognition of the need to address broader societal, contextual and structural factors contributing to FSW risk behaviour. We assess the cost-effectiveness of adding community mobilisation (CM) and empowerment interventions (eg. community mobilisation, community involvement in programme management and services, violence reduction, and addressing legal policies and police practices), to core HIV prevention services delivered as part of Avahan in two districts (Bellary and Belgaum) of Karnataka state, Southern India.

Methods

An ingredients approach was used to estimate economic costs in US$ 2011 from an HIV programme perspective of CM and empowerment interventions over a seven year period (2004–2011). Incremental impact, in terms of HIV infections averted, was estimated using a two-stage process. An ‘exposure analysis’ explored whether exposure to CM was associated with FSW’s empowerment, risk behaviours and HIV/STI prevalence. Pathway analyses were then used to estimate the extent to which behaviour change may be attributable to CM and to inform a dynamic HIV transmission model.

Findings

The incremental costs of CM and empowerment were US$ 307,711 in Belgaum and US$ 592,903 in Bellary over seven years (2004–2011). Over a 7-year period (2004–2011) the mean (standard deviation, sd.) number of HIV infections averted through CM and empowerment is estimated to be 1257 (308) in Belgaum and 2775 (1260) in Bellary. This translates in a mean (sd.) incremental cost per disability adjusted life year (DALY) averted of US$ 14.12 (3.68) in Belgaum and US$ 13.48 (6.80) for Bellary - well below the World Health Organisation recommended willingness to pay threshold for India. When savings from ART are taken into account, investments in CM and empowerment are cost saving.

Conclusions

Our findings suggest that CM and empowerment is, at worst, highly cost-effective and, at best, a cost-saving investment from an HIV programme perspective. CM and empowerment interventions should therefore be considered as core components of HIV prevention programmes for FSWs.