Intranuclear localization of apoptosis-inducing factor (AIF) and large scale DNA fragmentation after traumatic brain injury in rats and in neuronal cultures exposed to peroxynitrite

Programmed cell death occurs after ischemic, excitotoxic, and traumatic brain injury (TBI). Recently, a caspase-independent pathway involving intranuclear translocation of mitochondrial apoptosis-inducing factor (AIF) has been reported in vitro; but whether this occurs after acute brain injury was unknown. To address this question adult rats were sacrificed at various times after TBI. Western blot analysis on subcellular protein fractions demonstrated intranuclear localization of AIF in ipsilateral cortex and hippocampus at 2-72 h. Immunocytochemical analysis showed AIF labeling in neuronal nuclei with DNA fragmentation in the ipsilateral cortex and hippocampus. Immunoelectronmicroscopy verified intranuclear localization of AIF in hippocampal neurons after TBI, primarily in regions of euchromatin. Large-scale DNA fragmentation ( approximately 50 kbp), a signature event in AIF-mediated cell death, was detected in ipsilateral cortex and hippocampi by 6 h. Neuron-enriched cultures exposed to peroxynitrite also demonstrated intranuclear AIF and large-scale DNA fragmentation concurrent with impaired mitochondrial respiration and cell death, events that are inhibited by treatment with a peroxynitrite decomposition catalyst. Intranuclear localization of AIF and large-scale DNA fragmentation occurs after TBI and in neurons under conditions of oxidative/nitrosative stress, providing the first evidence of this alternative mechanism by which programmed cell death may proceed in neurons after brain injury.     

The Gel Electrophoresis Markup Language (GelML) from the Proteomics Standards Initiative

The Human Proteome Organisation's Proteomics Standards Initiative has developed the GelML (gel electrophoresis markup language) data exchange format for representing gel electrophoresis experiments performed in proteomics investigations. The format closely follows the reporting guidelines for gel electrophoresis, which are part of the Minimum Information About a Proteomics Experiment (MIAPE) set of modules. GelML supports the capture of metadata (such as experimental protocols) and data (such as gel images) resulting from gel electrophoresis so that laboratories can be compliant with the MIAPE Gel Electrophoresis guidelines, while allowing such data sets to be exchanged or downloaded from public repositories. The format is sufficiently flexible to capture data from a broad range of experimental processes, and complements other PSI formats for MS data and the results of protein and peptide identifications to capture entire gel‐based proteome workflows. GelML has resulted from the open standardisation process of PSI consisting of both public consultation and anonymous review of the specifications.     

Using Health Extension Workers for Monitoring Child Mortality in Real-Time: Validation against Household Survey Data in Rural Ethiopia

Background

Ethiopia has scaled up its community-based programs over the past decade by training and deploying health extension workers (HEWs) in rural communities throughout the country. Consequently, child mortality has declined substantially, placing Ethiopia among the few countries that have achieved the United Nations’ fourth Millennium Development Goal. As Ethiopia continues its efforts, results must be assessed regularly to provide timely feedback for improvement and to generate further support for programs. More specifically the expansion of HEWs at the community level provides a unique opportunity to build a system for real-time monitoring of births and deaths, linked to a civil registration and vital statistics system that Ethiopia is also developing. We tested the accuracy and completeness of births and deaths reported by trained HEWs for monitoring child mortality over 15 -month periods.

Methods and Findings

HEWs were trained in 93 randomly selected rural kebeles in Jimma and West Hararghe zones of the Oromia region to report births and deaths over a 15-month period from January, 2012 to March, 2013. Completeness of number of births and deaths, age distribution of deaths, and accuracy of resulting under-five, infant, and neonatal mortality rates were assessed against data from a large household survey with full birth history from women aged 15–49. Although, in general HEWs, were able to accurately report events that they identified, the completeness of number of births and deaths reported over twelve-month periods was very low and variable across the two zones. Compared to household survey estimates, HEWs reported only about 30% of births and 21% of under-five deaths occurring in their communities over a twelve-month period. The under-five mortality rate was under-estimated by around 30%, infant mortality rate by 23% and neonatal mortality by 17%. HEWs reported disproportionately higher number of deaths among the very young infants than among the older children.

Conclusion

Birth and death data reported by HEWs are not complete enough to support the monitoring of changes in childhood mortality. HEWs can significantly contribute to the success of a CRVS in Ethiopia, but cannot be relied upon as the sole source for identification of vital events. Further studies are needed to understand how to increase the level of completeness.     

Optima TB: A tool to help optimally allocate tuberculosis spending

Approximately 85% of tuberculosis (TB) related deaths occur in low- and middle-income countries where health resources are scarce. Effective priority setting is required to maximise the impact of limited budgets. The Optima TB tool has been developed to support analytical capacity and inform evidence-based priority setting processes for TB health benefits package design. This paper outlines the Optima TB framework and how it was applied in Belarus, an upper-middle income country in Eastern Europe with a relatively high burden of TB. Optima TB is a population-based disease transmission model, with programmatic cost functions and an optimisation algorithm. Modelled populations include age-differentiated general populations and higher-risk populations such as people living with HIV. Populations and prospective interventions are defined in consultation with local stakeholders. In partnership with the latter, demographic, epidemiological, programmatic, as well as cost and spending data for these populations and interventions are then collated. An optimisation analysis of TB spending was conducted in Belarus, using program objectives and constraints defined in collaboration with local stakeholders, which included experts, decision makers, funders and organisations involved in service delivery, support and technical assistance. These analyses show that it is possible to improve health impact by redistributing current TB spending in Belarus. Specifically, shifting funding from inpatient- to outpatient-focused care models, and from mass screening to active case finding strategies, could reduce TB prevalence and mortality by up to 45% and 50%, respectively, by 2035. In addition, an optimised allocation of TB spending could lead to a reduction in drug-resistant TB infections by 40% over this period. This would support progress towards national TB targets without additional financial resources. The case study in Belarus demonstrates how reallocations of spending across existing and new interventions could have a substantial impact on TB outcomes. This highlights the potential for Optima TB and similar modelling tools to support evidence-based priority setting.     

Uptake hydrogenase system in urd bean (Vigna mungo) Rhizobium in relation to nitrogen fixation

Twenty-five isolates of urd bean ( Vigna mungo ) Rhizobium were tested for the presence of an H₂-uptake system using triphenyl tetrazolium chloride reduction as the screening procedure. The isolates which reduced the dye rapidly at early stages of growth were found to recycle H₂ both in culture as well as in nodules. H₂-uptake positive, H₂-uptake negative strains and H₂-uptake negative mutants were compared on the basis of their effect on dry matter accumulation and N₂-fixation. Greenhouse experiments have shown the beneficial effect of the H₂-uptake positive system in nodules on N₂-fixation. Plants inoculated with H₂-uptake positive strains produced higher N content and dry matter over the plants inoculated with H₂-uptake negative strains.     

High-Efficiency Transformation of Rhizobium leguminosarum by Electroporation

Electrotransformation of Rhizobium leguminosarum was successfully carried out with a 15.1-kb plasmid, pMP154 (Cm^r), containing a nodABC-lacZ fusion by electroporation. The maximum transformation efficiency, 10⁸ transformants/μg of DNA, was achieved at a field strength of 14 kV/cm with a pulse of 7.3 ms (186 Ω). The number of transformants was found to increase with increasing cell density, with no sign of saturation. In relation to DNA dosage, the maximum transformation efficiency (5.8 × 10⁸ transformants/μg of DNA) was obtained with 0.5 μg of DNA/ml of cell suspension, and a further increase in the DNA concentration resulted in a decline in transformation efficiency.     

Covasim: An agent-based model of COVID-19 dynamics and interventions

The COVID-19 pandemic has created an urgent need for models that can project epidemic trends, explore intervention scenarios, and estimate resource needs. Here we describe the methodology of Covasim (COVID-19 Agent-based Simulator), an open-source model developed to help address these questions. Covasim includes country-specific demographic information on age structure and population size; realistic transmission networks in different social layers, including households, schools, workplaces, long-term care facilities, and communities; age-specific disease outcomes; and intrahost viral dynamics, including viral-load-based transmissibility. Covasim also supports an extensive set of interventions, including non-pharmaceutical interventions, such as physical distancing and protective equipment; pharmaceutical interventions, including vaccination; and testing interventions, such as symptomatic and asymptomatic testing, isolation, contact tracing, and quarantine. These interventions can incorporate the effects of delays, loss-to-follow-up, micro-targeting, and other factors. Implemented in pure Python, Covasim has been designed with equal emphasis on performance, ease of use, and flexibility: realistic and highly customized scenarios can be run on a standard laptop in under a minute. In collaboration with local health agencies and policymakers, Covasim has already been applied to examine epidemic dynamics and inform policy decisions in more than a dozen countries in Africa, Asia-Pacific, Europe, and North America.     

Purification and characterization of Escherichia coli xanthine-guanine phosphoribosyltransferase produced by plasmid pSV2gpt

The enzyme xanthine-guanine phosphoribosyltransferase from scherichia coli cells harboring the plasmid pSV2gpt has been purified 30-fold to near homogeneity by single-step GMP-agarose affinity chromatography. It has a K_m value of 2.5, 42 and 182 μM for the substrates guanine, xanthine and hypoxanthine, respectively, with guanine being the most preferred substrate. The enzyme exhibits a K_m value of 38.5 μM for PRib-PP with guanine as second substrate and of 100 μM when xanthine is used as the second substrate. It is markedly inhibited by 6-thioguanine, GMP and to a lesser extent by some other purine analogues. Thioguanine has been found to be the most potent inhibitor. The subunit molecular weight of xanthine-guanine phosphoribosyltransferase was determined to be 19 000. The in situ activity assay on a nondenaturing polyacrylamide gel electrophoresis gel has indicated that a second E. coli phosphoribosyltransferase preferentially uses hypoxanthine as opposed to guanine as a substrate, and it does not use xanthine.     

Influence of TRPV1 on diabetes-induced alterations in thermal pain sensitivity

Animals detect environmental changes through sensory neural mechanisms that enable them to differentiate the quality, intensity and temporal characteristics of stimuli. The 'doctrine of specific nervous energies' postulates that the different sensory modalities experienced by humans result of the activation of specific nervous pathways. Identification of functional classes of sensory receptors provided scientific support to the concept that somatosensory modalities (touch, pain, temperature, kinesthesis) are subserved by separate populations of sensory receptor neurons specialized in detecting innocuous and injurious stimuli of different quality (mechanical forces, temperature, chemical compounds). The identification of receptor proteins activated by different physicochemical stimuli, in particular ion channels of the Transient Receptor Potential (TRP) superfamily, has put forward the concept that specificity of peripheral sensory receptor neurons is determined by their expression of a particular "molecular sensor" that confers to each functional type its selectivity to respond with a discharge of nerve impulses to stimuli of a given quality. Nonetheless, recent experimental data suggest that the various molecular sensors proposed as specific transducer molecules for stimuli of different quality are not as neatly associated with the distinct functional types of sensory receptors as originally proposed. First, many ion channel molecules initially associated to the transduction of only one particular form of energy are also activated by stimuli of different quality, implying a limited degree of specificity in their transducing capacities. Second, molecular sensors associated with a stimulus quality and hence to a sensory receptor type and ultimately to a sensory modality may be concomitantly expressed in sensory receptor neurons functionally defined as specific for another stimulus quality. Finally, activation of voltage gated channels involved primarily in nerve impulse generation can also influence the gating of transducing channels, dramatically modifying their activation profile. Thus, we propose that the capacity exhibited by the different functional types of somatosensory receptor neurons to preferentially detect and encode specific stimuli into a discharge of nerve impulses, appears to result of a characteristic combinatorial expression of different ion channels in each neuronal type that finally determines their transduction and impulse firing properties. Transduction channels don't operate in isolation and their cellular context should also be taken into consideration to fully understand their function. Moreover, the inhomogeneous distribution of transduction and voltage-gated channels at soma, axonal branches and peripheral endings of primary sensory neurons influences the characteristics of the propagated impulse discharge that encodes the properties of the stimulus. Alteration of this concerted operation of ion channels in pathological conditions may underlie the changes in excitability accompanying peripheral sensory neuron injuries.