The DNA relaxation activity and covalent complex accumulation of Mycobacterium tuberculosis topoisomerase I can be assayed in Escherichia coli: application for identification of potential FRET-dye labeling sites

<p>Abstract</p> <p>Background</p> <p><it>Mycobacterium tuberculosis </it>topoisomerase I (MtTOP1) and <it>Escherichia coli </it>topoisomerase I have highly homologous transesterification domains, but the two enzymes have distinctly different C-terminal domains. To investigate the structure-function of MtTOP1 and to target its activity for development of new TB therapy, it is desirable to have a rapid genetic assay for its catalytic activity, and potential bactericidal consequence from accumulation of its covalent complex.</p> <p>Results</p> <p>We show that plasmid-encoded recombinant MtTOP1 can complement the temperature sensitive <it>topA </it>function of <it>E. coli </it>strain AS17. Moreover, expression of MtTOP1-G116 S enzyme with the TOPRIM mutation that inhibits DNA religation results in SOS induction and loss of viability in <it>E. coli</it>. The absence of cysteine residues in the MtTOP1 enzyme makes it an attractive system for introduction of potentially informative chemical or spectroscopic probes at specific positions via cysteine mutagenesis. Such probes could be useful for development of high throughput screening (HTS) assays. We employed the AS17 complementation system to screen for sites in MtTOP1 that can tolerate cysteine substitution without loss of complementation function. These cysteine substitution mutants were confirmed to have retained the relaxation activity. One such mutant of MtTOP1 was utilized for fluorescence probe incorporation and fluorescence resonance energy transfer measurement with fluorophore-labeled oligonucleotide substrate.</p> <p>Conclusions</p> <p>The DNA relaxation and cleavage complex accumulation of <it>M. tuberculosis </it>topoisomerase I can be measured with genetic assays in <it>E. coli</it>, facilitating rapid analysis of its activities, and discovery of new TB therapy targeting this essential enzyme.</p

SARS-CoV 9b protein diffuses into nucleus, undergoes active Crm1 mediated nucleocytoplasmic export and triggers apoptosis when retained in the nucleus

10.1371/journal.pone.0019436PLoS ONE65

Stock Assessment of Small Pelagic Fishes Caught by Ring Net Fishery in the Camotes Sea, Central Visayas, Philippines (2003-2012)

An analysis of the ten-year data on catch, effort, and length frequency of small pelagic caught from ring net fishery in the Camotes Sea was made. Length frequency data were used to estimate growth, mortality (total, natural, and fishing mortalities) and exploitation levels using the FAO-ICLARM Fish Stock Assessment Tools (FISAT) program developed by FAO/ICLARM. Generally, the sizes of the major species caught from ring net fishery are smaller than the maximum size they can attain and were harvested before reaching maturity lengths. The probability of capture at L50, however, was higher in 2003-2012 than in 1983-1987 assessment. Analysis of the recruitment pattern in the Camotes Sea reveals a dual recruitment mode per year starting from April to June which conforms to the findings of Jabat and Dalzell (1988) of which bimodal pattern of recruitment was observed for most of the small pelagic species in the catch of the ring net fishery from the Camotes Sea and in the Philippines in general. Exploitation rate (E=F/Z) estimates of three dominant pelagic species, Decapterus macrosoma (L∞=27.56 and present E=0.71), Selar crumenophthalmus (L∞=30.03 cm and present E=0.68), and Decapterus kurroides (L∞= 41.64 and present E=0.51) revealed they are vulnerable to ring net fishery as exhibited by high E values exceeding the 0.5 threshold. The present exploitation levels suggest growth overfishing of these pelagic species which will eventually lead to unsustainable ring net fishery in the Camotes Sea

Patient journey during and after a pre-eclampsia-complicated pregnancy:a cross-sectional patient registry study

OBJECTIVES: To gain insight into the patient journey through a pre-eclampsia-complicated pregnancy. DESIGN: Cross-sectional patient registry study. SETTING: Online patient registry initiated by the Preeclampsia Foundation. PARTICIPANTS: Women with a history of pre-eclampsia enrolled in The Preeclampsia Registry (TPR). PRIMARY AND SECONDARY OUTCOME MEASURES: Retrospective patient-reported experience measures concerning awareness of pre-eclampsia, timing and type of information on pre-eclampsia received, involvement in decision making regarding medical care, mental/emotional impact of the pre-eclampsia-complicated pregnancy and impact on future pregnancy planning. RESULTS: Of 3618 TPR-participants invited to complete the Patient Journey questionnaire, data from 833 (23%) responders were available for analysis. Most responders were white (n=795, 95.4%) and lived in the USA (n=728, 87.4%). Before their pre-eclampsia diagnosis, 599 (73.9%) responders were aware of the term ‘pre-eclampsia’, but only 348 (43.7%) were aware of its associated symptoms. Women with a lower level of education were less likely to have heard of pre-eclampsia (OR 0.36, 95% CI 0.21 to 0.62). Around the time of diagnosis, 29.2% of responders did not feel involved in the decision making, which was associated with reporting a serious mental/emotional impact of the pre-eclampsia experience (OR 2.46, 95% CI 1.58 to 3.84). Over time, there was an increase in the proportion of women who were aware of the symptoms of pre-eclampsia (32.2% before 2011 to 52.5% after 2016; p<0.001) and in the proportion of responders stating they received counselling about the later-life health risks associated with pre-eclampsia (14.2% before 2011 to 25.6% after 2016; p=0.005). CONCLUSIONS: This study demonstrates that improved patient education regarding pre-eclampsia is needed, that shared decision making is of great importance to patients to enhance their healthcare experience, and that healthcare providers should make efforts to routinely incorporate counselling about the later-life health risks associated with pre-eclampsia. TRIAL REGISTRATION NUMBER: NCT02020174

Inhibition of Mg2+ binding and DNA religation by bacterial topoisomerase I via introduction of an additional positive charge into the active site region

Among bacterial topoisomerase I enzymes, a conserved methionine residue is found at the active site next to the nucleophilic tyrosine. Substitution of this methionine residue with arginine in recombinant Yersinia pestis topoisomerase I (YTOP) was the only substitution at this position found to induce the SOS response in Escherichia coli. Overexpression of the M326R mutant YTOP resulted in ∼4 log loss of viability. Biochemical analysis of purified Y. pestis and E. coli mutant topoisomerase I showed that the Met to Arg substitution affected the DNA religation step of the catalytic cycle. The introduction of an additional positive charge into the active site region of the mutant E. coli topoisomerase I activity shifted the pH for optimal activity and decreased the Mg2+ binding affinity. This study demonstrated that a substitution outside the TOPRIM motif, which binds Mg2+directly, can nonetheless inhibit Mg2+ binding and DNA religation by the enzyme, increasing the accumulation of covalent cleavage complex, with bactericidal consequence. Small molecules that can inhibit Mg2+ dependent religation by bacterial topoisomerase I specifically could be developed into useful new antibacterial compounds. This approach would be similar to the inhibition of divalent ion dependent strand transfer by HIV integrase in antiviral therapy

[Thriving transitions in celebration of the College of Education Week 1998]

Dr. Alexa Abrenica, Chair of the Psychology Department gave a talk on Thriving transitions in celebration of the College of Education Week 1998

Etching and passivation of Group IV semiconductors: Ge and SiGe

status: publishe

[Dr. Alexa Abrenica delivers lecture on womanhood]

Dr. Alexa Abrenica, faculty member of the Psychology Dept. and holder of the John Gokongwei Professorial Chair for Asian Studies, delivered her lecture entitled Womanhood at the crossroads: the Lumad perspective