Identification of a Caldariomyces fumago Mutant Secreting an Inactive Form of Chloroperoxidase Lacking the Heme Group and N-Glycans

By mutant colony screening of Caldariomyces fumago a mutant was isolated which was slightly greenish on fructose minimal medium and grew slower in comparison to the wild type. The supernatant samples lacked the Soret band typical for the heme group of the CPO and nearly no CPO activity was detected. SDS-PAGE analysis of mutant culture supernatant samples showed production of a 38–40 kDa protein while wild type samples contain the 42 kDa CPO protein. Protein identification using nanoLC-ESI-MS/MS was performed and based on three peptides the protein in the mutant culture was identified as CPO. No differences in the CPO gene sequences of wild type and mutant were found indicating a post-translational defect in protein maturation. Deglycosylation experiments using CPO from wild type and mutant were carried out. After removing N-linked oligosaccharides from wild type CPO a protein band at 38–40 kDa was detected. Our results reveal that the mutant protein lacks the heme group as well as the N-glycans.     

GST genotypes and lung cancer susceptibility in Asian populations with indoor air pollution exposures: a meta-analysis

About half of the world's population is exposed to smoke from heating or cooking with coal, wood, or biomass. These exposures, and fumes from cooking oil use, have been associated with increased lung cancer risk. Glutathione S-transferases play an important role in the detoxification of a wide range of human carcinogens in these exposures. Functional polymorphisms have been identified in the GSTM1, GSTT1, and GSTP1 genes, which may alter the risk of lung cancer among individuals exposed to coal, wood, and biomass smoke, and cooking oil fumes. We performed a meta-analysis of 6 published studies (912 cases; 1063 controls) from regions in Asia where indoor air pollution makes a substantial contribution to lung cancer risk, and evaluated the association between the GSTM1 null, GSTT1 null, and GSTP1 105Val polymorphisms and lung cancer risk. Using a random effects model, we found that carriers of the GSTM1 null genotype had a borderline significant increased lung cancer risk (odds ratio (OR), 1.31; 95% confidence interval (CI), 0.95-1.79; p=0.10), which was particularly evident in the summary risk estimate for the four studies carried out in regions of Asia that use coal for heating and cooking (OR, 1.64; 95% CI, 1.25-2.14; p=0.0003). The GSTT1 null genotype was also associated with an increased lung cancer risk (OR, 1.49; 95% CI, 1.17-1.89; p=0.001), but no association was observed for the GSTP1 105Val allele. Previous meta- and pooled-analyses suggest at most a small association between the GSTM1 null genotype and lung cancer risk in populations where the vast majority of lung cancer is attributed to tobacco, and where indoor air pollution from domestic heating and cooking is much less than in developing Asian countries. Our results suggest that the GSTM1 null genotype may be associated with a more substantial risk of lung cancer in populations with coal exposure.     

The influence of life history and climate driven diversification on the mtDNA phylogeographic structures of two southern African Mastomys species (Rodentia: Muridae: Murinae)

The phylogeographic patterns of small mammals in southern Africa are frequently disjunct. This pattern is predominately attributed to vicariant geographical barriers coupled to climate driven diversification. To gain further insights into this hypothesis, we embarked on a comparative mtDNA phylogeographic study of two common rodent species in southern Africa, Mastomys natalensis and Mastomys coucha. Parsimony haplotype networks and SplitsTrees of mtDNA cytochrome oxidase I data showed a large degree of haplotype sharing throughout the sampling range. Within southern Africa, we found no conclusive evidence to support geographic vicariance as a contributing factor towards Mastomys speciation. We proposed that the regional phylogeographic structures detected for M. natalensis and M. coucha are the result of weak isolation by distance coupled to repeated expansions and contractions of suitable habitat. Both species probably survived in multiple refugia during unfavourable periods and mismatch distributions show signs of population expansion. Mitochondrial DNA nucleotide diversity values (π) show marked differences between the two species (M. natalensis: 0.003 and M. coucha: 0.468), and M. coucha also shows a higher level of population differentiation in AMOVA analyses. These differences are most likely due to life history discrepancies between the two species. Mastomys coucha is regarded to be more of a habitat specialist when compared to M. natalensis, and this probably places a higher constraint on M. coucha dispersal abilities. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 114 , 58–68.     

Identification of the minimal protein domain required for priming activity of Munc13-1

Most nerve cells communicate with each other through synaptic transmission at chemical synapses. The regulated exocytosis of neurotransmitters, hormones, and peptides occurs at specialized membrane areas through Ca2+-triggered fusion of secretory vesicles with the plasma membrane . Prior to fusion, vesicles are docked at the plasma membrane and must then be rendered fusion-competent through a process called priming. The molecular mechanism underlying this priming process is most likely the formation of the SNARE complex consisting of Syntaxin 1, SNAP-25, and Synaptobrevin 2. Members of the Munc13 protein family consisting of Munc13-1, -2, -3, and -4 were found to be absolutely required for this priming process . In the present study, we identified the minimal Munc13-1 domain that is responsible for its priming activity. Using Munc13-1 deletion constructs in an electrophysiological gain-of-function assay of chromaffin-granule secretion, we show that priming activity is mediated by the C-terminal residues 1100-1735 of Munc13-1, which contains both Munc13-homology domains and the C-terminal C2 domain. Priming by Munc13-1 appears to require its interaction with Syntaxin 1 because point mutants that do not bind Syntaxin 1 do not prime chromaffin granules.     

Do Strategies to Improve Quality of Maternal and Child Health Care in Lower and Middle Income Countries Lead to Improved Outcomes? A Review of the Evidence

Objectives

Efforts to scale-up maternal and child health services in lower and middle income countries will fail if services delivered are not of good quality. Although there is evidence of strategies to increase the quality of health services, less is known about the way these strategies affect health system goals and outcomes. We conducted a systematic review of the literature to examine this relationship.

Methods

We undertook a search of MEDLINE, SCOPUS and CINAHL databases, limiting the results to studies including strategies specifically aimed at improving quality that also reported a measure of quality and at least one indicator related to health system outcomes. Variation in study methodologies prevented further quantitative analysis; instead we present a narrative review of the evidence.

Findings

Methodologically, the quality of evidence was poor, and dominated by studies of individual facilities. Studies relied heavily on service utilisation as a measure of strategy success, which did not always correspond to improved quality. The majority of studies targeted the competency of staff and adequacy of facilities. No strategies addressed distribution systems, public-private partnership or equity. Key themes identified were the conflict between perceptions of patients and clinical measures of quality and the need for holistic approaches to health system interventions.

Conclusion

Existing evidence linking quality improvement strategies to improved MNCH outcomes is extremely limited. Future research would benefit from the inclusion of more appropriate indicators and additional focus on non-facility determinants of health service quality such as health policy, supply distribution, community acceptability and equity of care.     

Heterologous expression of the plant cysteine protease bromelain and its inhibitor in Pichia pastoris

Expression of proteases in heterologous hosts remains an ambitious challenge due to severe problems associated with digestion of host proteins. On the other hand, proteases are broadly used in industrial applications and resemble promising drug candidates. Bromelain is an herbal drug that is medicinally used for treatment of oedematous swellings and inflammatory conditions and consists in large part of proteolytic enzymes. Even though various experiments underline the requirement of active cysteine proteases for biological activity, so far no investigation succeeded to clearly clarify the pharmacological mode of action of bromelain. The potential role of proteases themselves and other molecules of this multi‐component extract currently remain largely unknown or ill defined. Here, we set out to express several bromelain cysteine proteases as well as a bromelain inhibitor molecule in order to gain defined molecular entities for subsequent studies. After cloning the genes from its natural source Ananas comosus (pineapple plant) into Pichia pastoris and subsequent fermentation and purification, we obtained active protease and inhibitor molecules which were subsequently biochemically characterized. Employing purified bromelain fractions paves the way for further elucidation of pharmacological activities of this natural product. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:54–65, 2017