Efficient screening of process variables inPenicillium janthinellum fermentations

Summary Several potentially important process variables involved in xylanase production byPenicillium janthinellum in large lab-scale fermentors were efficiently screened by employing a Plackett-Burman statistical design. Results illustrate the design's general applicability to experimentation with fermentation processes.     

Hypervariability of intronic simple (gt)n(ga)m repeats in HLA-DRB genes

We have investigated the extent of DNA variability in intronic simple (gt)_n(ga)_m repeat sequences and correlated this to sequence polymorphisms in the flanking exon 2 of HLA-DRB genes. The polymerase chain reaction (PCR) was used to amplify a DNA fragment containing exon 2 and the repeat region of intron 2. The PCR products were separated on sequencing gels in order to demonstrate length hypervariability of the (gt)_n(ga)_m repeats. In a parallel experiment, the PCR products were cloned and sequenced (each exon 2 plus adjacent simple repeats) to characterize the simple repeats in relation to the HLA-DRB sequences. In a panel of 25 DRB1, DRB4, and DRB5 alleles new sequences were not detected. Restriction fragment length polymorphism (RFLP) subtyping of serologically defined haplotypes corresponds to translated DNA sequences in 85% of the cases, the exceptions involving unusual DR/DQ combinations. Many identical DRB1 alleles can be distinguished on the basis of their adjacent simple repeats. We found group-specific organization of the repeats: the DRw52 supergroup repeats differ from those of DRB1*0101, DRB4*0101, and DRB5*0101 alleles and from those of pseudogenes. Finally, we amplified baboon DNA and found a DRB allele with extensive similarity to DRB1 sequences of the DRw52 supergroup. The simple repeat of the baboon gene, however, resembles that of human pseudogenes. In addition to further subtyping, the parallel study of polymorphic protein and hypervariable DNA alleles may allow conclusions to be drawn on the relationships between the DRB genes and perhaps also on the theory of trans-species evolution.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M 34258.     

Characterization of inositolpolyphosphate binding to myocardial membranes

Although it is well-accepted that the phosphatidylinositol signalling transduction pathway, producing inositol-1,4,5-P3 (InsP₃) and inositol-1,3,4,5-P₄ (InsP4) as second messengers, functions in heart muscle, virtually nothing is known about the roles of the higher inositol polyphosphates such as inositolhexakisphosphate (InsP₆). This study demonstrates that InSP₆ has the ability to bind intracellularly, with different binding characteristics, to different myocardial membranes. Binding to purified sarcoplasmic reticulum (SR) membranes, purified sarcolemmal (SL) membranes as well as to viable mitochondria were characterized. Binding to all these membranes display high as well as low affinity binding sites, with differing affinities. Kd values of binding to SR were 32 and 383 nM, to SL 61 and 1312 nM, while those of mitochondrial binding were 230 and 2200 nM respectively.InsP₄ binding was also investigated and displayed the following characteristics: to SR, one low affinity binding site (Kd = 203 nM) and to SL, a high as well as a low affinity binding site with Kd values of 41 and 2075 nM respectively. Presence of InsP₃, the second messenger for SR calcium release, at concentrations of 1 nM, elevated the binding of InsP₄ to SR and SL by a mean of 30% and 20% respectively.Fractionation of SR and SL membranes on sucrose density gradients, after solubilization with CHAPS, indicated that InsP₆ bound to two separate protein peaks in both these membranes, while InsP₄ bound to only one. In SR membranes, InsP₄ bound preferentially to a protein separating at high sucrose density while it bound to a protein separating at low sucrose density in SL membranes.     

Early electrochemical events in lectin-lymphocyte interaction

Concanavalin A, at extremely low concentrations, will produce significant increases in the electrophoretic mobility of murine splenic T lymphocytes. It has been established that the alteration in cellular surface charge is mediated by a factor produced by those lymphocytes that have reacted directly with con A. We originally conjectured that the mobility change might be the consequence of an alteration in the distribution of the charged moieties of membrane glycoproteins. The results of experiments conducted at low temperature raise some questions about this mechanism. Further experiments have been performed to establish the nature of the physicochemical alterations in the peripheral zone of the factor-stimulated lymphocytes that are manifest as changes in cellular surface charge. The results of these studies indicate that, subsequent to the interaction of T lymphocytes with con A, there is a reduction in the number of positively charged amino groups effective at the electrophoretic surface of the cells.     

Concentration by ultrafiltration and stabilization of phytase produced by solid-state fermentation

Ultrafiltration is an attractive downstream processing technique for concentrating enzymes and could be considered the primary step of purification. However, the efficiency of this process is often limited by protein fouling and shear-induced enzyme inactivation, which decreases permeate flux and results in the loss of enzyme activity. Although the rejection of phytase was higher than 99%, the loss of the enzyme activity was 14% during operation, indicating that the shear forces generated in the filter have significant influences on the enzyme activity. Two preparations using glycerol (25% and 35%, v/v) as a cryo-protecting agent at different temperatures were studied. The preparation containing 35% glycerol retained 70% of the initial enzyme activity at 70 °C after 1 h and had more than 3 and 6 months storage half-life at 29 °C and 4 °C, respectively.     

HIV Testing and Tolerance to Gender Based Violence: A Cross-Sectional Study in Zambia

This paper explores the effect of social relations and gender-based conflicts on the uptake of HIV testing in the South and Central provinces of Zambia. We conducted a community-based cross-sectional study of 1716 randomly selected individuals. Associations were examined using mixed-effect multivariable logistic regression. A total of 264 men (64%) and 268 women (56%) had never tested for HIV. The strongest determinants for not being tested were disruptive couple relationships (OR = 2.48 95% CI = 1.00–6.19); tolerance to gender-based violence (OR = 2.10 95% CI = 1.05–4.32) and fear of social rejection (OR = 1.48 95% CI = 1.23–1.80). In the Zambian context, unequal power relationships within the couple and the community seem to play a pivotal role in the decision to test which until now have been largely underestimated. Policies, programs and interventions to rapidly increase HIV testing need to urgently address gender-power inequity in relationships and prevent gender-based violence to reduce the negative impact on the lives of couples and families.     

Young Children's Probability of Dying Before and After Their Mother's Death: A Rural South African Population-Based Surveillance Study

Background

There is evidence that a young child''s risk of dying increases following the mother''s death, but little is known about the risk when the mother becomes very ill prior to her death. We hypothesized that children would be more likely to die during the period several months before their mother''s death, as well as for several months after her death. Therefore we investigated the relationship between young children''s likelihood of dying and the timing of their mother''s death and, in particular, the existence of a critical period of increased risk.

Methods and Findings

Data from a health and socio-demographic surveillance system in rural South Africa were collected on children 0–5 y of age from 1 January 1994 to 31 December 2008. Discrete time survival analysis was used to estimate children''s probability of dying before and after their mother''s death, accounting for moderators. 1,244 children (3% of sample) died from 1994 to 2008. The probability of child death began to rise 6–11 mo prior to the mother''s death and increased markedly during the 2 mo immediately before the month of her death (odds ratio [OR] 7.1 [95% CI 3.9–12.7]), in the month of her death (OR 12.6 [6.2–25.3]), and during the 2 mo following her death (OR 7.0 [3.2–15.6]). This increase in the probability of dying was more pronounced for children whose mothers died of AIDS or tuberculosis compared to other causes of death, but the pattern remained for causes unrelated to AIDS/tuberculosis. Infants aged 0–6 mo at the time of their mother''s death were nine times more likely to die than children aged 2–5 y. The limitations of the study included the lack of knowledge about precisely when a very ill mother will die, a lack of information about child nutrition and care, and the diagnosis of AIDS deaths by verbal autopsy rather than serostatus.

Conclusions

Young children in lower income settings are more likely to die not only after their mother''s death but also in the months before, when she is seriously ill. Interventions are urgently needed to support families both when the mother becomes very ill and after her death. Please see later in the article for the Editors'' Summary     

Morphological study for understanding the sexual pattern in ghost shrimp Callichirus major (Crustacea: Axiidea)

Callichirus major is a species of ghost shrimp that has burrowing habits, building underground galleries in the intertidal region of beaches. This study aimed to analyse some morphological parameters to verify its type of sexual pattern. A sample of 79 individuals, collected at the beach of Corujão, Espírito Santo, Brazil, was used. Data on the external morphology, presence of gonopores, carapace length (CL) and cheliped lengths (Lch) were obtained. In addition, the gonads were processed for histological analysis. The animals were divided into three categories: A, individuals with male characteristics; B, those presenting exclusively female characteristics; C, animals with characteristics of intersex, which presented supernumerary gonopores. Positive allometries were found for Lch versus CL only for category A, characterizing the group's marked heterochely. Histologically, it was possible to observe the presence of oocytes in category A and one case of male tissue in an intersex individual (category C). However, no genital ducts of both sexes were found in that individual. Callichirus major presents controversial sexual characters, which makes it difficult to understand the reproductive parameters and sexual pattern. However, maybe that species can develop a sexual system of hermaphroditism.     

Parasite load evaluation by qPCR and blood culture in Chagas disease and HIV co-infected patients under antiretroviral therapy

Chagas disease also known as American trypanosomiasis, is caused by Trypanosoma cruzi and transmitted by triatominae-contaminated feces. It is considered a neglected tropical disease that affects 6 to 7 million people worldwide. The reactivation of Chagas disease occurs when the chronically infected hosts are not able to control T. cruzi infection, generating recurrence of the acute phase. HIV is the main immunosuppressive infection that can lead to the reactivation of chronic Chagas disease in AIDS conditions. In co-infected patients, the reactivation of Chagas disease is related to their high parasite load, high HIV viral load, and CD4 T-cell counting less than 200/mm³, which may evolve to meningoencephalitis and myocarditis. Eight T. cruzi/HIV co-infected patients under antiretroviral therapy (ART) and ten Chagas disease patients without HIV infection that attended at Study Group of Chagas Disease, Hospital de Clínicas, University of Campinas (GEdoCh/HC/UNICAMP-SP) and Pontifical Catholic University of Campinas SP (PUCC/SP) were evaluated. Tests for Chagas disease were performed, such as qPCR and T. cruzi blood culture. The patient’s medical records were analyzed to verify clinical and epidemiological data, viral load, and CD4 T-cell counting since the outset of ART. For both groups, we found no statically significant differences between parasite load via blood culture and qPCR. In T. cruzi/HIV co-infected subjects, we observed a significant increase of CD4 T-cells counting and viral load decrease, which became undetectable over the years after ART. Parasites isolated from the patient’s blood culture were genotyped, being the majority of them infected with TcII and one case of mixed infection (TcII and TcV/TcVI). These results were expected according to the region of origin of the patients. We suggest that the parasite load be monitored through qPCR in T.cruzi/HIV co-infected patients. We conclude that ART in people living with HIV improves infection and immunosuppression control, enabling the natural evolution of the American trypanosomiasis.