Localization of a cruciform cutting endonuclease to yeast mitochondria

We have found a cruciform cutting endonuclease in the yeast, Saccharomyces cerevisiae, which localizes to the mitochondria. This activity apparently is associated with the mitochondrial inner membrane since the activity is not released into solution by osmolysis, in contrast to the matrix enzyme, isocitrate dehydrogenase. The cruciform cutting activity appears to be encoded by CCE1. This gene has been shown to encode one of the major cruciform cutting endonucleases present in a yeast cell. In ccel strains, which lack CCE1 endonuclease activity, the mitochondrial cruciform cutting endonucleolytic activity is also absent. Since CCE1 is allelic to MGT1, a gene required for the highly biased transmission of petite mitochondrial DNA in crosses between ⁺ and hypersuppressive ^– cells, it seems likely that the CCE1 endonuclease functions within mitochondria.     

The Triplo-Lethal Locus of Drosophila: Reexamination of Mutants and Discovery of a Second-Site Suppressor

In the genome of Drosophila melanogaster there is a single locus, Triplo-lethal (Tpl), that causes lethality when present in either one or three copies in an otherwise diploid animal. Previous attempts to mutagenize Tpl produced alleles that were viable over a chromosome bearing a duplication of Tpl, but were not lethal in combination with a wild-type chromosome, as deficiencies for Tpl are. These mutations were interpreted as hypomorphic alleles of Tpl. In this work, we show that these alleles are not mutations at Tpl; rather, they are dominant mutations in a tightly linked, but cytologically distant, locus that we have named Suppressor-of-Tpl (Su(Tpl)). Su(Tpl) mutations suppress the lethality associated with three copies of the Triplo-lethal locus and are recessive lethal. We have mapped Su(Tpl) to the approximate map position 3-46.5, within the cytological region 76B-76D.     

Analysis of lambda insertions in the fucose utilization region of Escherichia coli K-12: use of lambda fuc and lambda argA transducing bacteriophages to partially order the fucose utilization genes

Escherichia coli K-12 strains have deletions for the normal lambda integration site were lysogenized with bacteriophage lambda at a site within the L-fucose utilization system (fuc). The frequency of lambda integration at this site is approximately 2 X 10(-8) to 5 X 10(-7). Studies of the lytic properties of these strains indicated very infrequent cell lysis with a relatively low phage burst size. Transductional ability of the phage lysates was found to be normal, comparable to that found in conventional low-frequency transducing lysates. Two major classes of transducing phage were found. One carried the markers argA and fucA (a fucose utilization gene of unknown function previously referred to as fuc-1) and the gene for D-arabinose utilization (dar+). The other carried only fucC, the gene specifying L-fuculose-1-phosphate aldolase. A minor class of phage was found that carried fucA, but not argA or dar+. Upon consideration of the transductional nature of these phage classes, we are proposing that the gene order for the L-fucose utilization system is dar, fucA, (lambda), fucC.     

Stryspinolactone,an unusual monoterpene lactone from Strychnos spinosa

The structure of stryspinolactone, an unusual lactone from Strychnos spinosa, was determined from spectroscopic data.     

Binding and transport of SFPQ-RNA granules by KIF5A/KLC1 motors promotes axon survival

Complex neural circuitry requires stable connections formed by lengthy axons. To maintain these functional circuits, fast transport delivers RNAs to distal axons where they undergo local translation. However, the mechanism that enables long-distance transport of RNA granules is not yet understood. Here, we demonstrate that a complex containing RNA and the RNA-binding protein (RBP) SFPQ interacts selectively with a tetrameric kinesin containing the adaptor KLC1 and the motor KIF5A. We show that the binding of SFPQ to the KIF5A/KLC1 motor complex is required for axon survival and is impacted by KIF5A mutations that cause Charcot-Marie Tooth (CMT) disease. Moreover, therapeutic approaches that bypass the need for local translation of SFPQ-bound proteins prevent axon degeneration in CMT models. Collectively, these observations indicate that KIF5A-mediated SFPQ-RNA granule transport may be a key function disrupted in KIF5A-linked neurologic diseases and that replacing axonally translated proteins serves as a therapeutic approach to axonal degenerative disorders.     

Brucella melitensis Methionyl-tRNA-Synthetase (MetRS), a Potential Drug Target for Brucellosis

We investigated Brucella melitensis methionyl-tRNA-synthetase (BmMetRS) with molecular, structural and phenotypic methods to learn if BmMetRS is a promising target for brucellosis drug development. Recombinant BmMetRS was expressed, purified from wild type Brucella melitensis biovar Abortus 2308 strain ATCC/CRP #DD-156 and screened by a thermal melt assay against a focused library of one hundred previously classified methionyl-tRNA-synthetase inhibitors of the blood stage form of Trypanosoma brucei. Three compounds showed appreciable shift of denaturation temperature and were selected for further studies on inhibition of the recombinant enzyme activity and cell viability against wild type B. melitensis strain 16M. BmMetRS protein complexed with these three inhibitors resolved into three-dimensional crystal structures and was analyzed. All three selected methionyl-tRNA-synthetase compounds inhibit recombinant BmMetRS enzymatic functions in an aminoacylation assay at varying concentrations. Furthermore, growth inhibition of B. melitensis strain 16M by the compounds was shown. Inhibitor-BmMetRS crystal structure models were used to illustrate the molecular basis of the enzyme inhibition. Our current data suggests that BmMetRS is a promising target for brucellosis drug development. However, further studies are needed to optimize lead compound potency, efficacy and safety as well as determine the pharmacokinetics, optimal dosage, and duration for effective treatment.     

Biochemical Screening of Five Protein Kinases from Plasmodium falciparum against 14,000 Cell-Active Compounds

In 2010 the identities of thousands of anti-Plasmodium compounds were released publicly to facilitate malaria drug development. Understanding these compounds’ mechanisms of action—i.e., the specific molecular targets by which they kill the parasite—would further facilitate the drug development process. Given that kinases are promising anti-malaria targets, we screened ~14,000 cell-active compounds for activity against five different protein kinases. Collections of cell-active compounds from GlaxoSmithKline (the ~13,000-compound Tres Cantos Antimalarial Set, or TCAMS), St. Jude Children’s Research Hospital (260 compounds), and the Medicines for Malaria Venture (the 400-compound Malaria Box) were screened in biochemical assays of Plasmodium falciparum calcium-dependent protein kinases 1 and 4 (CDPK1 and CDPK4), mitogen-associated protein kinase 2 (MAPK2/MAP2), protein kinase 6 (PK6), and protein kinase 7 (PK7). Novel potent inhibitors (IC₅₀ < 1 μM) were discovered for three of the kinases: CDPK1, CDPK4, and PK6. The PK6 inhibitors are the most potent yet discovered for this enzyme and deserve further scrutiny. Additionally, kinome-wide competition assays revealed a compound that inhibits CDPK4 with few effects on ~150 human kinases, and several related compounds that inhibit CDPK1 and CDPK4 yet have limited cytotoxicity to human (HepG2) cells. Our data suggest that inhibiting multiple Plasmodium kinase targets without harming human cells is challenging but feasible.     

Pertussis Prevalence and Its Determinants among Children with Persistent Cough in Urban Uganda

Background

We determined prevalence of pertussis infection and its associated host and environmental factors to generate information that would guide strategies for disease control.

Methods

In a cross-sectional study, 449 children aged 3 months to 12 years with persistent cough lasting ≥14 days were enrolled and evaluated for pertussis using DNA polymerase chain reaction (PCR) and ELISA serology tests.

Results

Pertussis prevalence was 67 (15% (95% Confidence Interval (CI): 12–18)) and 81 (20% (95% CI: 16–24)) by PCR and ELISA, respectively among 449 participating children. The prevalence was highest in children with >59 months of age despite high vaccination coverage of 94% in this age group. Study demographic and clinical characteristics were similar between pertussis and non-pertussis cases. Of the 449 children, 133 (30%) had a coughing household member and 316 (70%) did not. Among 133 children that had a coughing household member, sex of child, sharing bed with a coughing household member and having a coughing individual in the neighborhood were factors associated with pertussis. Children that had shared a bed with a coughing household individual had seven-fold likelihood of having pertussis compared to children that did not (odds ratio (OR) 7.16 (95% CI: 1.24–41.44)). Among the 316 children that did not have a coughing household member, age <23 months, having or contact with a coughing individual in neighborhood, a residence with one room, and having a caretaker with >40 years of age were the factors associated with pertussis. Age <23months was three times more likely to be associated with pertussis compared to age 24–59 months (OR 2.97 (95% CI: 1.07–8.28)).

Conclusion

Findings suggest high prevalence of pertussis among children with persistent cough at a health facility and it was marked in children >59 months of age, suggesting the possibility of waning immunity. The factors associated with pertussis varied by presence or absence of a coughing household member.     

Improving Maternal Care through a State-Wide Health Insurance Program: A Cost and Cost-Effectiveness Study in Rural Nigeria

Background

While the Nigerian government has made progress towards the Millennium Development Goals, further investments are needed to achieve the targets of post-2015 Sustainable Development Goals, including Universal Health Coverage. Economic evaluations of innovative interventions can help inform investment decisions in resource-constrained settings. We aim to assess the cost and cost-effectiveness of maternal care provided within the new Kwara State Health Insurance program (KSHI) in rural Nigeria.

Methods and Findings

We used a decision analytic model to simulate a cohort of pregnant women. The primary outcome is the incremental cost effectiveness ratio (ICER) of the KSHI scenario compared to the current standard of care. Intervention cost from a healthcare provider perspective included service delivery costs and above-service level costs; these were evaluated in a participating hospital and using financial records from the managing organisations, respectively. Standard of care costs from a provider perspective were derived from the literature using an ingredient approach. We generated 95% credibility intervals around the primary outcome through probabilistic sensitivity analysis (PSA) based on a Monte Carlo simulation. We conducted one-way sensitivity analyses across key model parameters and assessed the sensitivity of our results to the performance of the base case separately through a scenario analysis. Finally, we assessed the sustainability and feasibility of this program’s scale up within the State’s healthcare financing structure through a budget impact analysis. The KSHI scenario results in a health benefit to patients at a higher cost compared to the base case. The mean ICER (US$46.4/disability-adjusted life year averted) is considered very cost-effective compared to a willingness-to-pay threshold of one gross domestic product per capita (Nigeria, US$ 2012, 2,730). Our conclusion was robust to uncertainty in parameters estimates (PSA: median US$49.1, 95% credible interval 21.9–152.3), during one-way sensitivity analyses, and when cost, quality, cost and utilization parameters of the base case scenario were changed. The sustainability of this program’s scale up by the State is dependent on further investments in healthcare.

Conclusions

This study provides evidence that the investment made by the KSHI program in rural Nigeria is likely to have been cost-effective; however, further healthcare investments are needed for this program to be successfully expanded within Kwara State. Policy makers should consider supporting financial initiatives to reduce maternal mortality tackling both supply and demand issues in the access to care.