Structure and function of the Si3 insertion integrated into the trigger loop/helix of cyanobacterial RNA polymerase.

Cyanobacteria and evolutionarily related chloroplasts of algae and plants possess unique RNA polymerases (RNAPs) with characteristics that distinguish them from canonical bacterial RNAPs. The largest subunit of cyanobacterial RNAP (cyRNAP) is divided into two polypeptides, ß'1 and ß'2, and contains the largest known lineage-specific insertion domain, Si3, located in the middle of the trigger loop and spanning approximately half of the ß'2 subunit. In this study, we present the X-ray crystal structure of Si3 and the cryo-EM structures of the cyRNAP transcription elongation complex plus the NusG factor with and without incoming nucleoside triphosphate (iNTP) bound at the active site. Si3 has a well-ordered and elongated shape that exceeds the length of the main body of cyRNAP, fits into cavities of cyRNAP in the absence of iNTP bound at the active site and shields the binding site of secondary channel-binding proteins such as Gre and DksA. A small transition from the trigger loop to the trigger helix upon iNTP binding results in a large swing motion of Si3; however, this transition does not affect the catalytic activity of cyRNAP due to its minimal contact with cyRNAP, NusG, or DNA. This study provides a structural framework for understanding the evolutionary significance of these features unique to cyRNAP and chloroplast RNAP and may provide insights into the molecular mechanism of transcription in specific environment of photosynthetic organisms and organelle.

Structure and function of the Si3 insertion integrated into the trigger loop/helix of cyanobacterial RNA polymerase.

Cyanobacteria and evolutionarily related chloroplasts of algae and plants possess unique RNA polymerases (RNAPs) with characteristics that distinguish from canonical bacterial RNAPs. The largest subunit of cyanobacterial RNAP (cyRNAP) is divided into two polypeptides, ß'1 and ß'2, and contains the largest known lineage-specific insertion domain, Si3, located in the middle of the trigger loop and spans approximately half of the ß'2 subunit. In this study, we present the X-ray crystal structure of Si3 and the cryo-EM structures of the cyRNAP transcription elongation complex plus the NusG factor with and without incoming nucleoside triphosphate (iNTP) bound at the active site. Si3 has a well-ordered and elongated shape that exceeds the length of the main body of cyRNAP, fits into cavities of cyRNAP and shields the binding site of secondary channel-binding proteins such as Gre and DksA. A small transition from the trigger loop to the trigger helix upon iNTP binding at the active site results in a large swing motion of Si3; however, this transition does not affect the catalytic activity of cyRNAP due to its minimal contact with cyRNAP, NusG or DNA. This study provides a structural framework for understanding the evolutionary significance of these features unique to cyRNAP and chloroplast RNAP and may provide insights into the molecular mechanism of transcription in specific environment of photosynthetic organisms.

The carboxy terminus causes interfacial assembly of oleate hydratase on a membrane bilayer.

The soluble flavoprotein oleate hydratase (OhyA) hydrates the 9-cis double bond of unsaturated fatty acids. OhyA substrates are embedded in membrane bilayers; OhyA must remove the fatty acid from the bilayer and enclose it in the active site. Here, we show that the positively charged helix-turn-helix motif in the carboxy terminus (CTD) is responsible for interacting with the negatively charged phosphatidylglycerol (PG) bilayer. Super-resolution microscopy of Staphylococcus aureus cells expressing green fluorescent protein fused to OhyA or the CTD sequence shows subcellular localization along the cellular boundary, indicating OhyA is membrane-associated and the CTD sequence is sufficient for membrane recruitment. Using cryo-electron microscopy, we solved the OhyA dimer structure and conducted 3D variability analysis of the reconstructions to assess CTD flexibility. Our surface plasmon resonance experiments corroborated that OhyA binds the PG bilayer with nanomolar affinity and we found the CTD sequence has intrinsic PG binding properties. We determined that the nuclear magnetic resonance structure of a peptide containing the CTD sequence resembles the OhyA crystal structure. We observed intermolecular NOE from PG liposome protons next to the phosphate group to the CTD peptide. The addition of paramagnetic MnCl2 indicated the CTD peptide binds the PG surface but does not insert into the bilayer. Molecular dynamics simulations, supported by site-directed mutagenesis experiments, identify key residues in the helix-turn-helix that drive membrane association. The data show that the OhyA CTD binds the phosphate layer of the PG surface to obtain bilayer-embedded unsaturated fatty acids.

Allosteric mechanism of transcription inhibition by NusG-dependent pausing of RNA polymerase.

NusG is a transcription elongation factor that stimulates transcription pausing in Gram+ bacteria including B. subtilis by sequence-specific interaction with a conserved pause-inducing -11TTNTTT-6 motif found in the non-template DNA (ntDNA) strand within the transcription bubble. To reveal the structural basis of NusG-dependent pausing, we determined a cryo-EM structure of a paused transcription complex (PTC) containing RNA polymerase (RNAP), NusG, and the TTNTTT motif in the ntDNA strand. The interaction of NusG with the ntDNA strand rearranges the transcription bubble by positioning three consecutive T residues in a cleft between NusG and the ß-lobe domain of RNAP. We revealed that the RNAP swivel module rotation (swiveling), which widens (swiveled state) and narrows (non-swiveled state) a cleft between NusG and the ß-lobe, is an intrinsic motion of RNAP and is directly linked to trigger loop (TL) folding, an essential conformational change of all cellular RNAPs for the RNA synthesis reaction. We also determined cryo-EM structures of RNAP escaping from the paused transcription state. These structures revealed the NusG-dependent pausing mechanism by which NusG-ntDNA interaction inhibits the transition from swiveled to non-swiveled states, thereby preventing TL folding and RNA synthesis allosterically. This motion is also reduced by the formation of an RNA hairpin within the RNA exit channel. Thus, the pause half-life can be modulated by the strength of the NusG-ntDNA interaction and/or the stability of the RNA hairpin. NusG residues that interact with the TTNTTT motif are widely conserved in bacteria, suggesting that NusG-dependent pausing is widespread.

The structure and activities of the archaeal transcription termination factor Eta detail vulnerabilities of the transcription elongation complex.

Transcription must be properly regulated to ensure dynamic gene expression underlying growth, development, and response to environmental cues. Regulation is imposed throughout the transcription cycle, and while many efforts have detailed the regulation of transcription initiation and early elongation, the termination phase of transcription also plays critical roles in regulating gene expression. Transcription termination can be driven by only a few proteins in each domain of life. Detailing the mechanism(s) employed provides insight into the vulnerabilities of transcription elongation complexes (TECs) that permit regulated termination to control expression of many genes and operons. Here, we describe the biochemical activities and crystal structure of the superfamily 2 helicase Eta, one of two known factors capable of disrupting archaeal transcription elongation complexes. Eta retains a twin-translocase core domain common to all superfamily 2 helicases and a well-conserved C terminus wherein individual amino acid substitutions can critically abrogate termination activities. Eta variants that perturb ATPase, helicase, single-stranded DNA and double-stranded DNA translocase and termination activities identify key regions of the C terminus of Eta that, when combined with modeling Eta-TEC interactions, provide a structural model of Eta-mediated termination guided in part by structures of Mfd and the bacterial TEC. The susceptibility of TECs to disruption by termination factors that target the upstream surface of RNA polymerase and potentially drive termination through forward translocation and allosteric mechanisms that favor opening of the clamp to release the encapsulated nucleic acids emerges as a common feature of transcription termination mechanisms.

On the stability of stalled RNA polymerase and its removal by RapA.

Stalling of the transcription elongation complex formed by DNA, RNA polymerase (RNAP) and RNA presents a serious obstacle to concurrent processes due to the extremely high stability of the DNA-bound polymerase. RapA, known to remove RNAP from DNA in an ATP-dependent fashion, was identified over 50 years ago as an abundant binding partner of RNAP; however, its mechanism of action remains unknown. Here, we use single-molecule magnetic trapping assays to characterize RapA activity and begin to specify its mechanism of action. We first show that stalled RNAP resides on DNA for times on the order of 106 seconds and that increasing positive torque on the DNA reduces this lifetime. Using stalled RNAP as a substrate we show that the RapA protein stimulates dissociation of stalled RNAP from positively supercoiled DNA but not negatively supercoiled DNA. We observe that RapA-dependent RNAP dissociation is torque-sensitive, is inhibited by GreB and depends on RNA length. We propose that stalled RNAP is dislodged from DNA by RapA via backtracking in a supercoiling- and torque-dependent manner, suggesting that RapA's activity on transcribing RNAP in vivo is responsible for resolving conflicts between converging polymerase molecular motors.

Structural basis of RNA polymerase recycling by the Swi2/Snf2 family of ATPase RapA in Escherichia coli.

After transcription termination, cellular RNA polymerases (RNAPs) are occasionally trapped on DNA, impounded in an undefined post-termination complex (PTC), limiting the free RNAP pool and subsequently leading to inefficient transcription. In Escherichia coli, a Swi2/Snf2 family of ATPase called RapA is known to be involved in countering such inefficiency through RNAP recycling; however, the precise mechanism of this recycling is unclear. To better understand its mechanism, here we determined the structures of two sets of E. coli RapA-RNAP complexes, along with the RNAP core enzyme and the elongation complex, using cryo-EM. These structures revealed the large conformational changes of RNAP and RapA upon their association that has been implicated in the hindrance of PTC formation. Our results along with DNA-binding assays reveal that although RapA binds RNAP away from the DNA-binding main channel, its binding can allosterically close the RNAP clamp, thereby preventing its nonspecific DNA binding and PTC formation. Taken together, we propose that RapA acts as a guardian of RNAP by which RapA prevents nonspecific DNA binding of RNAP without affecting the binding of promoter DNA recognition σ factor, thereby enhancing RNAP recycling.

Seroepizootiological investigation on Goat Warble Fly Infestation (Przhevalskiana silenus) in Pothwar Plateau, Pakistan.

Goat Warble Fly Infestation (GWFI) is also known as subcutaneous myiasis caused by Przhevalskiana silenus (Diptera: Oestridae). It is widely distributed in tropical and sub-tropical areas of the world. In goats, WFI is usually detected through conventional procedure which underestimated the infestation. The current study was designed to determine the serodiagonsis of GWFI (through IDEXX Hypodermosis serum antibody test) and also aimed to investigate its seroepizootiological profile in Pothwar region, Pakistan from 2013-14. The results showed that average seropositivity (ELISA kit) of GWFI was 18.5% whereas, it was 11% by using conventional procedure (Palpation method) depicting a significant difference (p<0.05). Higher seropositivity (30.8%) was observed in Jhelum district as compared to e Attock district (6%). The L1 larvae were found in September, while nodules start appearing in October to December and last until the end of February. The month wise peaks of optical density (OD) was higher in December which gradually decrease along with the end of winter season. The prevalence of GWFI revealed no significant difference among three host breeds (Jattal, Beetal and Tedy). According to the results, high infestation rate (28%) was observed in young animals of age group < 1 year as compared to old animals (> 2 years). Topographically, hilly areas (33%) provide favourable climatic conditions for the propagating of larval stages. Sex difference showed no significant difference. The seroprevalence varied significantly with respect to age, month, districts and topographical location. The current study proved that serologic diagnosis (commercial ELISA kit) as more sensitive and accurate for timely diagnosis of GWFI than traditional method. The information on the epizootiology of P. silenus in goats of Pothwar region would help in devising effective control strategies.

Structural basis of ribosomal RNA transcription regulation.

Ribosomal RNA (rRNA) is most highly expressed in rapidly growing bacteria and is drastically downregulated under stress conditions by the global transcriptional regulator DksA and the alarmone ppGpp. Here, we determined cryo-electron microscopy structures of the Escherichia coli RNA polymerase (RNAP) σ70 holoenzyme during rRNA promoter recognition with and without DksA/ppGpp. RNAP contacts the UP element using dimerized α subunit carboxyl-terminal domains and scrunches the template DNA with the σ finger and ß' lid to select the transcription start site favorable for rapid promoter escape. Promoter binding induces conformational change of σ domain 2 that opens a gate for DNA loading and ejects σ1.1 from the RNAP cleft to facilitate open complex formation. DksA/ppGpp binding also opens the DNA loading gate, which is not coupled to σ1.1 ejection and impedes open complex formation. These results provide a molecular basis for the exceptionally active rRNA transcription and its vulnerability to DksA/ppGpp.

Seroepizootiological investigation on Goat Warble Fly Infestation (Przhevalskiana silenus) in Pothwar Plateau, Pakistan

@#Goat Warble Fly Infestation (GWFI) is also known as subcutaneous myiasis caused by Przhevalskiana silenus (Diptera: Oestridae). It is widely distributed in tropical and sub-tropical areas of the world. In goats, WFI is usually detected through conventional procedure which underestimated the infestation. The current study was designed to determine the serodiagonsis of GWFI (through IDEXX Hypodermosis serum antibody test) and also aimed to investigate its seroepizootiological profile in Pothwar region, Pakistan from 2013-14. The results showed that average seropositivity (ELISA kit) of GWFI was 18.5% whereas, it was 11% by using conventional procedure (Palpation method) depicting a significant difference (p<0.05). Higher seropositivity (30.8%) was observed in Jhelum district as compared to e Attock district (6%). The L1 larvae were found in September, while nodules start appearing in October to December and last until the end of February. The month wise peaks of optical density (OD) was higher in December which gradually decrease along with the end of winter season. The prevalence of GWFI revealed no significant difference among three host breeds (Jattal, Beetal and Tedy). According to the results, high infestation rate (28%) was observed in young animals of age group < 1 year as compared to old animals (> 2 years). Topographically, hilly areas (33%) provide favourable climatic conditions for the propagating of larval stages. Sex difference showed no significant difference. The seroprevalence varied significantly with respect to age, month, districts and topographical location. The current study proved that serologic diagnosis (commercial ELISA kit) as more sensitive and accurate for timely diagnosis of GWFI than traditional method. The information on the epizootiology of P. silenus in goats of Pothwar region would help in devising effective control strategies.

Screening marine algae metabolites as high-affinity inhibitors of SARS-CoV-2 main protease (3CLpro): an in silico analysis to identify novel drug candidates to combat COVID-19 pandemic.

The recent dissemination of SARS-CoV-2 from Wuhan city to all over the world has created a pandemic. COVID-19 has cost many human lives and created an enormous economic burden. Although many drugs/vaccines are in different stages of clinical trials, still none is clinically available. We have screened a marine seaweed database (1110 compounds) against 3CLpro of SARS-CoV-2 using computational approaches. High throughput virtual screening was performed on compounds, and 86 of them with docking score < - 5.000 kcal mol-1 were subjected to standard-precision docking. Based on binding energies (< - 6.000 kcal mol-1), 9 compounds were further shortlisted and subjected to extra-precision docking. Free energy calculation by Prime-MM/GBSA suggested RC002, GA004, and GA006 as the most potent inhibitors of 3CLpro. An analysis of ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) properties of RC002, GA004, and GA006 indicated that only RC002 (callophysin A, from red alga Callophycus oppositifolius) passed Lipinski's, Veber's, PAINS and Brenk's filters and displayed drug-like and lead-like properties. Analysis of 3CLpro-callophysin A complex revealed the involvement of salt bridge, hydrogen bonds, and hydrophobic interactions. callophysin A interacted with the catalytic residues (His41 and Cys145) of 3CLpro; hence it may act as a mechanism-based competitive inhibitor. Docking energy and docking affinity of callophysin A towards 3CLpro was - 8.776 kcal mol-1 and 2.73 × 106 M-1, respectively. Molecular dynamics simulation confirmed the stability of the 3CLpro-callophysin A complex. The findings of this study may serve as the basis for further validation by in vitro and in vivo studies.

Electron number density conservation model combined with a self-absorption correction methodology for analysis of nanostructure plasma using laser-induced breakdown spectroscopy.

We studied laser ablation and plasma property evolution for a nickel (Ni) doped tin (Sn) oxide nanostructures target using laser-induced breakdown spectroscopy (LIBS). The transition metal Ni doped tin oxide nanostructures were synthesized by co-precipitation and hydrothermal methodologies. The size of prepared nanoparticles was verified by X-ray diffraction and transmission electron microscopy techniques. A frequency-doubled pulsed Nd:YAG laser with a wavelength of 532 nm was used to produce ablated plasma nanostructures. Ablation of doped and undoped nanostructures revealed salient-enhanced spectral emissions compared with their bulky counterparts. The emission lines of the constituent elements of doped material were used to find plasma parameters. The plasma temperature was estimated from a Boltzmann plot, and the electron number density was determined from the Saha-Boltzmann equation. The self-absorption effect has been observed in tiny plasma of nanostructures. The affected profiles of spectral lines of Ni and Sn nanoparticles due to self-absorption in LIBS spectra were corrected by the internal reference self-absorption correction (IRSAC) methodology. After correction of emitted line intensities by IRSAC, the electron number density (END) conservation approach was applied for quantitative analysis of doped nanostructures. In the END conservation approach, quantitative analysis of samples was carried out using electron number densities. Quantitative results derived from the END conservation approach at high and low concentrations exhibited good correlation when these were compared and validated with results from a conventional calibration free approach and the standard recognized energy dispersive X-ray technique.

An evaluation of a tuberculosis case-finding and treatment program among Syrian refugees-Jordan and Lebanon, 2013-2015.

BACKGROUND: The displacement crisis in Syria poses challenges for tuberculosis (TB) control across the region. Since 2012 in Jordan and 2013 in Lebanon, the International Organization for Migration (IOM) has supported the National TB Program (NTP) in detecting and treating TB among Syrian refugees. In December 2016, IOM asked US Centers for Disease Control and Prevention (CDC) staff to evaluate its program of support to Jordan and Lebanon's NTPs for TB control among Syrian refugees. This manuscript focuses on case-finding, including contact investigations, and treatment components of the IOM program during 2013-2015 in Jordan and 2015 in Lebanon. METHODS: The evaluation consisted of a retrospective review of de-identified Jordan and Lebanon line lists of TB cases and of investigated contacts (Lebanon only). Syrian refugee TB cases were categorized by sex, age group (age < 5 years, 5-14 years, ≥15 years), TB type (pulmonary versus extra-pulmonary), and additionally in Jordan, by refugee camp status (residence in versus outside a refugee camp), to evaluate differences in treatment completion and contact investigation. RESULTS: In Jordan, Syrian refugee cases represented 24.4% of TB cases in 2013, when Syrian refugees made up 6.8% of the country's population, and 13.8% of TB cases in 2015, when Syrians made up 8.3% of the total population. In Lebanon in 2015, Syrian refugee cases represented 21.4% of TB cases, when Syrians made up 20.1% of the total population. In Jordan, the proportion of Syrian TB cases residing in refugee camps (29.3%) was higher than the proportion of Syrians refugees residing in camps (17.1%). Of Syrian TB cases in 2015, 94.8% in Jordan and 87.8% in Lebanon completed treatment. In Lebanon, among Syrian TB cases with household contacts listed, contact investigation was completed for 77.8% of cases. CONCLUSION: IOM's program of NTP support provides critical TB services for Syrian refugees with high treatment completion rates. More community and health practitioner outreach for enhanced active case finding among community-based Syrian refugees in Jordan may improve TB case detection in populations outside of refugee camps. Thorough contact investigations need continued emphasis, including completely recording investigations in both countries, to find active TB cases.

Cryo-EM structure of Escherichia coli σ70 RNA polymerase and promoter DNA complex revealed a role of σ non-conserved region during the open complex formation.

First step of gene expression is transcribing the genetic information stored in DNA to RNA by the transcription machinery including RNA polymerase (RNAP). In Escherichia coli, a primary σ70 factor forms the RNAP holoenzyme to express housekeeping genes. The σ70 contains a large insertion between the conserved regions 1.2 and 2.1, the σ non-conserved region (σNCR), but its function remains to be elucidated. In this study, we determined the cryo-EM structures of the E. coli RNAP σ70 holoenzyme and its complex with promoter DNA (open complex, RPo) at 4.2 and 5.75 Å resolutions, respectively, to reveal native conformations of RNAP and DNA. The RPo structure presented here found an interaction between the σNCR and promoter DNA just upstream of the -10 element, which was not observed in a previously determined E. coli RNAP transcription initiation complex (RPo plus short RNA) structure by X-ray crystallography because of restraint of crystal packing effects. Disruption of the σNCR and DNA interaction by the amino acid substitutions (R157A/R157E) influences the DNA opening around the transcription start site and therefore decreases the transcription activity of RNAP. We propose that the σNCR and DNA interaction is conserved in proteobacteria, and RNAP in other bacteria replaces its role with a transcription factor.

Measurement of Ascaris lumbricoides IgG antibody, associated risk factors and identification of serum biochemical parameters as biomarkers of pathogenicity: among patients with gastrointestinal complains in Pakistan.

Soil transmitted helminths are causing significant morbidity worldwide and the most common infection is Ascaris lumbricoides in developing countries. The present study aimed to determine the immuno-epidemiological status of ascariasis among patients with gastrointestinal complaints and to identify the associated risk factors and eventual changes in serum biochemical parameters to reflect its pathogenicity. This study was conducted on 700 respondents aged between 5-45 years. A total of 356 patients participated in an enzymelinked immunosorbent assay (ELISA) study to determine anti-Ascaris IgG levels and biochemical parameters. The overall seroprevalence was 58.4%, with 100% sensitivity and 84.4% specificity of the assay. The infection was highest among the 21-28 year age group (14.0%), and ascariasis was found to be not significantly (P>0.05) different between the age groups. The results showed that the risk of ascariasis was significantly (P<0.05) increased in individuals who had no contact with soil (OR=4.6, 95% CI: 1.9-10.8), eating unwashed vegetables one month prior to the study (OR=2.7, 95% CI: 1.4-5.2), eating mixed food (OR=2.4, 95% CI: 1.2-4.7), drinking pressure pump water (OR=3.4, 95% CI: 1.9-6.1), and those who had no complain of vomiting (OR=3.1, 95% CI: 1.6-5.8) and nausea (OR=1.9, 95% CI: 1.1-3.2). The results showed significantly (P<0.05) elevated level of serum alanine aminotransferase, alkaline phosphatase, serum cholesterol, total protein and globulin in anti-Ascaris IgG positive cases than the control group. The study concluded that patients who visited health care centres with gastrointestinal complain were at higher risk of ascariasis as compared to other diseases. In conclusion epidemiological studies are needed to establish baseline data for public health authorities in order to plan and implement health education programs to reduce the impact of the disease.

Prevalence and risk factors associated with intestinal parasitic infections among schoolchildren in Punjab, Pakistan.

Intestinal parasitic infections (IPIs) are a major cause of morbidity worldwide and have been described as an important public health concern. The present study aimed to determine the prevalence and identification of risk factors associated with IPIs among 3-15 years old school age children residing in Mandi Bahauddin, Pakistan from 2011- 2013. A cross sectional school-based study was conducted using a structured pre-tested questionnaire. Anthropometric tools and stool tests were used to obtain epidemiological and disease data. The direct wet mount preperation in saline/iodine/haematoxylin stain and Kato-Katz methods were used for stool examination. Data were analysed using appropriate descriptive, univariate and multivariable logistic regression methods. Of the 1,434 children studied (mean age of 8.6±3.6 years) the overall prevalence rate for intestinal parasitic infections was found to be 33.3%. Children infected with single parasite accounted for 27.6% and 5.7% were detected with poly-parasitism. The study showed that helminths (21.4%) were more prevalent than protozoans (17.9%). Ascaris lumbricoides (17.5%), Giardia lamblia (9.8%), Entamoeba histolytica (8.2%), Hymenolepis nana (2.0%), Trichuris trichiura (1.3%) and Taenia saginata (0.7%) were identified in children living in irrigated areas. The multiple logistic regression model indicated that age of the child, gender, family size, source of drinking water, type of milk used, house condition, feeding habit, personal hygiene and socioeconomic status were significantly (p<0.05) associated with the IPIs. Intestinal parasites were prevalent in varying magnitude among the schoolchildren located in irrigated areas. We conclude that there is a need for mass scale campaigns to create awareness regarding health and hygiene in children, and the need for development of effective poverty control programmes because deworming alone is not adequate to control parasitic infections.

The application of One Health concept to an outdoor problem-based learning activity for veterinary students.

BACKGROUND: The One Health (OH) approach, which seeks to bring together human and animal health, is particularly suited to the effective management of zoonotic diseases across both sectors. To overcome professional silos, OH needs to be taught at the undergraduate level. Here, we describe a problem-based learning activity using the OH approach that was conducted outdoors for 3rd-year veterinary students in Malaysia. MATERIALS AND METHODS: A total of 118 students, divided into two groups, completed the activity which spanned 1½ days at a deer park adjacent to a wilderness area. Students were asked to evaluate the activity using an online survey that had quantitative and qualitative components. RESULTS: Response rate was 69.5%. The activity was rated excellent by 69.5% and good by 30.4%. Levels of satisfaction were high on a range of criteria. 97.5% of students intended to take action in their studies as a result of what they had learned. CONCLUSIONS: Delivery of an outdoor problem-based learning activity using OH approach was very successful in terms of participation, knowledge delivery and understanding, and the willingness of students to integrate OH into their future practice. For the improvement of future programs, the involvement of other disciplines (such as Medical, Biology, Biotechnology, Biomedical, and Public Health) is being considered.

Transcription Elongation Factor NusA Is a General Antagonist of Rho-dependent Termination in Escherichia coli.

NusA is an essential protein that binds to RNA polymerase and also to the nascent RNA and influences transcription by inducing pausing and facilitating the process of transcription termination/antitermination. Its participation in Rho-dependent transcription termination has been perceived, but the molecular nature of this involvement is not known. We hypothesized that, because both Rho and NusA are RNA-binding proteins and have the potential to target the same RNA, the latter is likely to influence the global pattern of the Rho-dependent termination. Analyses of the nascent RNA binding properties and consequent effects on the Rho-dependent termination functions of specific NusA-RNA binding domain mutants revealed an existence of Rho-NusA direct competition for the overlappingnut(NusA-binding site) andrut(Rho-binding site) sites on the RNA. This leads to delayed entry of Rho at therutsite that inhibits the latter's RNA release process. High density tiling microarray profiles of these NusA mutants revealed that a significant number of genes, together with transcripts from intergenic regions, are up-regulated. Interestingly, the majority of these genes were also up-regulated when the Rho function was compromised. These results provide strong evidence for the existence of NusA-binding sites in different operons that are also the targets of Rho-dependent terminations. Our data strongly argue in favor of a direct competition between NusA and Rho for the access of specific sites on the nascent transcripts in different parts of the genome. We propose that this competition enables NusA to function as a global antagonist of the Rho function, which is unlike its role as a facilitator of hairpin-dependent termination.

Anthelmintic properties of extracts from Artemisia plants against nematodes.

Artemisia plant genus, natural inhabitant of northern Punjab Pakistan, is well known for its anthelmintic properties; many Artemisia species have not been so far scientifically proved. The aim of this study was to assess in vitro anthelmintic activity of Artemisia indica and Artemisia roxburghiana against mixed infection of gastrointestinal nematodes in small ruminants. This study is first scientifically proven study on anthelmintic activity of A. indica and A. roxburghiana. Five different concentrations (50, 25, 12.5, 6.25 and 3.75 mg/mL) accompanied by negative control (PBS) and positive control (albendazole, 10%) were used to carry out the egg hatch inhibition assay, larval mortality assay and adult worm mortality assay. The Baermann technique was used first time in larval mortality assay and proved to be effective. The results revealed that methanolic extracts of both A. indica and A. roxburghiana, showed maximum anthelmintic activity at concentration of 50 mg/ml by egg hatch inhibition (85±21.2; 80±28.3), larvae mortality (18±2.8; 17±4.2) and adult worm mortality (8.5±2.1; 8±2.8) assays. However, at concentration of 50 mg/ml both plant extracts in comparison to albendazole showed statistically insignificant (p≤0.05) results. The A. indica showed higher anthelmintic activity at all concentrations as compared to A. roburghiana. It has been concluded both plants exhibit anthelmintic activity and further evaluation of these plants should be carried out to purify the active ingredients for anthelmintic activity. Moreover, the decoctions of these plants could be used to GINs after confirming anthelmintic properties through in vivo.

Redundancy of primary RNA-binding functions of the bacterial transcription terminator Rho.

The bacterial transcription terminator, Rho, terminates transcription at half of the operons. According to the classical model derived from in vitro assays on a few terminators, Rho is recruited to the transcription elongation complex (EC) by recognizing specific sites (rut) on the nascent RNA. Here, we explored the mode of in vivo recruitment process of Rho. We show that sequence specific recognition of the rut site, in majority of the Rho-dependent terminators, can be compromised to a great extent without seriously affecting the genome-wide termination function as well as the viability of Escherichia coli. These terminators function optimally only through a NusG-assisted recruitment and activation of Rho. Our data also indicate that at these terminators, Rho-EC-bound NusG interaction facilitates the isomerization of Rho into a translocase-competent form by stabilizing the interactions of mRNA with the secondary RNA binding site, thereby overcoming the defects of the primary RNA binding functions.