Broadening The Substrate Scope of Aldolases Through Metagenomic Enzyme Discovery.

Bio-processes based on enzymatic catalysis play a major role in the development of green, sustainable processes, and the discovery of new enzymes is key to this approach. In this work, we analysed ten metagenomes and retrieved 48 genes coding for deoxyribose-5-phosphate aldolases (DERAs, EC 4.1.2.4) using a sequence-based approach. These sequences were recombinantly expressed in Escherichia coli and screened for activity towards a range of aldol additions. Among these, one enzyme, DERA-61, proved to be particularly interesting and catalysed the aldol addition of furfural or benzaldehyde with acetone, butanone and cyclobutanone with unprecedented activity. The product of these reactions, aldols, can find applications as building blocks in the synthesis of biologically active compounds. Screening was carried out to identify optimized reaction conditions targeting temperature, pH, and salt concentrations. Lastly, the kinetics and the stereochemistry of the products were investigated, revealing that DERA-61 and other metagenomic DERAs have superior activity and stereoselectivity when they are provided with non-natural substrates, compared to well-known DERAs.

Mesocellular Silica Foam as Immobilization Carrier for Production of Statin Precursors.

The employment of 2-deoxyribose-5-phosphate aldolase (DERA) stands as a prevalent biocatalytic route for synthesizing statin side chains. The main problem with this pathway is the low stability of the enzyme. In this study, mesocellular silica foam (MCF) with different pore sizes was used as a carrier for the covalent immobilization of DERA. Different functionalizing and activating agents were tested and kinetic modeling was subsequently performed. The use of succinic anhydride as an activating agent resulted in an enzyme hyperactivation of approx. 140%, and the stability almost doubled compared to that of the free enzyme. It was also shown that the pore size of MCF has a decisive influence on the stability of the DERA enzyme.

Simple Enzyme Immobilization for Flow Chemistry? An Assessment of Available Strategies for an Acetaldehyde-Dependent Aldolase.

Enzyme immobilization is a technology that enables (bio-)catalysts to be applied in continuous-flow systems. However, there is a plethora of immobilization methods available with individual advantages and disadvantages. Here, we assessed the influence of simple and readily available methods with respect to the performance of 2-deoxy-d-ribose-5-phosphate aldolase (DERA) in continuous-flow conditions. The investigated immobilization strategies cover the unspecific attachment to carriers via epoxides, affinity-based attachment via metal ion affinity, StrepTag™-StrepTactin™ interaction as well as the covalent affinity attachment of an enzyme to a matrix tethered by the HaloTag®. The metal-ion-affinity-based approach outperformed the other methods in terms of immobilized activity and stability under applied conditions. As most enzymes examined today already have a HisTag for purification purposes, effective immobilization may be applied, as simple as a standard purification, if needed.

Rational engineering of 2-deoxyribose-5-phosphate aldolases for the biosynthesis of (R)-1,3-butanediol.

Carbon-carbon bond formation is one of the most important reactions in biocatalysis and organic chemistry. In nature, aldolases catalyze the reversible stereoselective aldol addition between two carbonyl compounds, making them attractive catalysts for the synthesis of various chemicals. In this work, we identified several 2-deoxyribose-5-phosphate aldolases (DERAs) having acetaldehyde condensation activity, which can be used for the biosynthesis of (R)-1,3-butanediol (1,3BDO) in combination with aldo-keto reductases (AKRs). Enzymatic screening of 20 purified DERAs revealed the presence of significant acetaldehyde condensation activity in 12 of the enzymes, with the highest activities in BH1352 from Bacillus halodurans, TM1559 from Thermotoga maritima, and DeoC from Escherichia coli The crystal structures of BH1352 and TM1559 at 1.40-2.50 Å resolution are the first full-length DERA structures revealing the presence of the C-terminal Tyr (Tyr224 in BH1352). The results from structure-based site-directed mutagenesis of BH1352 indicated a key role for the catalytic Lys155 and other active-site residues in the 2-deoxyribose-5-phosphate cleavage and acetaldehyde condensation reactions. These experiments also revealed a 2.5-fold increase in acetaldehyde transformation to 1,3BDO (in combination with AKR) in the BH1352 F160Y and F160Y/M173I variants. The replacement of the WT BH1352 by the F160Y or F160Y/M173I variants in E. coli cells expressing the DERA + AKR pathway increased the production of 1,3BDO from glucose five and six times, respectively. Thus, our work provides detailed insights into the molecular mechanisms of substrate selectivity and activity of DERAs and identifies two DERA variants with enhanced activity for in vitro and in vivo 1,3BDO biosynthesis.

Current state of and need for enzyme engineering of 2-deoxy-D-ribose 5-phosphate aldolases and its impact.

Deoxyribose-5-phosphate aldolases (DERAs, EC 4.1.2.4) are acetaldehyde-dependent, Class I aldolases catalyzing in nature a reversible aldol reaction between an acetaldehyde donor (C2 compound) and glyceraldehyde-3-phosphate acceptor (C3 compound, C3P) to generate deoxyribose-5-phosphate (C5 compound, DR5P). DERA enzymes have been found to accept also other types of aldehydes as their donor, and in particular as acceptor molecules. Consequently, DERA enzymes can be applied in C-C bond formation reactions to produce novel compounds, thus offering a versatile biocatalytic alternative for synthesis. DERA enzymes, found in all kingdoms of life, share a common TIM barrel fold despite the low overall sequence identity. The catalytic mechanism is well-studied and involves formation of a covalent enzyme-substrate intermediate. A number of protein engineering studies to optimize substrate specificity, enzyme efficiency, and stability of DERA aldolases have been published. These have employed various engineering strategies including structure-based design, directed evolution, and recently also machine learning-guided protein engineering. For application purposes, enzyme immobilization and usage of whole cell catalysis are preferred methods as they improve the overall performance of the biocatalytic processes, including often also the stability of the enzyme. Besides single-step enzymatic reactions, DERA aldolases have also been applied in multi-enzyme cascade reactions both in vitro and in vivo. The DERA-based applications range from synthesis of commodity chemicals and flavours to more complicated and high-value pharmaceutical compounds. KEY POINTS: • DERA aldolases are versatile biocatalysts able to make new C-C bonds. • Synthetic utility of DERAs has been improved by protein engineering approaches. • Computational methods are expected to speed up the future DERA engineering efforts.

Substrate specificity of 2-deoxy-D-ribose 5-phosphate aldolase (DERA) assessed by different protein engineering and machine learning methods.

In this work, deoxyribose-5-phosphate aldolase (Ec DERA, EC 4.1.2.4) from Escherichia coli was chosen as the protein engineering target for improving the substrate preference towards smaller, non-phosphorylated aldehyde donor substrates, in particular towards acetaldehyde. The initial broad set of mutations was directed to 24 amino acid positions in the active site or in the close vicinity, based on the 3D complex structure of the E. coli DERA wild-type aldolase. The specific activity of the DERA variants containing one to three amino acid mutations was characterised using three different substrates. A novel machine learning (ML) model utilising Gaussian processes and feature learning was applied for the 3rd mutagenesis round to predict new beneficial mutant combinations. This led to the most clear-cut (two- to threefold) improvement in acetaldehyde (C2) addition capability with the concomitant abolishment of the activity towards the natural donor molecule glyceraldehyde-3-phosphate (C3P) as well as the non-phosphorylated equivalent (C3). The Ec DERA variants were also tested on aldol reaction utilising formaldehyde (C1) as the donor. Ec DERA wild-type was shown to be able to carry out this reaction, and furthermore, some of the improved variants on acetaldehyde addition reaction turned out to have also improved activity on formaldehyde. KEY POINTS: • DERA aldolases are promiscuous enzymes. • Synthetic utility of DERA aldolase was improved by protein engineering approaches. • Machine learning methods aid the protein engineering of DERA.

Haematological outcomes in progression of malaria: A cohort study from district Dera Ismail Khan, Pakistan.

Malaria is the second highest reported disease from public health sector which affects about 4 million people each year in Pakistan. The study sought to evaluate the haematological changes in malarial patients in order to find any relation of these changes with malarial pathogenesis. This cross sectional descriptive study was designed at the pathology department of Gomal Medical College, Dera Ismail Khan during March 2015 to February 2017. Blood samples were collected from 400 malaria microscopy positive in EDTA tubes for the analysis of haematological parameters like Total leucocyte count, Haemoglobin, Platelets, Mean Cell Volume, Mean Cell Haemoglobin and Mean Cell Haemoglobin Concentration. Out of the 400 cases of malaria, 366 (91.5%) were Plasmodium vivax, 24 (6.0%) were Plasmodium falciparum and 10 (2.5%) were co-infection of Plasmodium vivax and Plasmodium falciparum infection. Anaemia was seen in 148 (37%) of cases; thrombocytopenia was 316(79%) and 60 (15%) cases had leucopenia. This study shows that malarial patients exhibited important changes in haematological parameters like thrombocytopenia; anaemia and leucopenia are significant predictors of malaria infection. When used in combination with other clinical and microscopy methods, these parameters could improve the diagnosis and treatment of malaria.

2-Deoxy-D-ribose-5-phosphate aldolase (DERA): applications and modifications.

2-Deoxy-D-ribose-5-phosphate aldolase (DERA) is a class I aldolase that offers access to several building blocks for organic synthesis. It catalyzes the stereoselective C-C bond formation between acetaldehyde and numerous other aldehydes. However, the practical application of DERA as a biocatalyst is limited by its poor tolerance towards industrially relevant concentrations of aldehydes, in particular acetaldehyde. Therefore, the development of proper experimental conditions, including protein engineering and/or immobilization on appropriate supports, is required. The present review is aimed to provide a brief overview of DERA, its history, and progress made in understanding the functioning of the enzyme. Furthermore, the current understanding regarding aldehyde resistance of DERA and the various optimizations carried out to modify this property are discussed.

Engineered, highly productive biosynthesis of artificial, lactonized statin side-chain building blocks: The hidden potential of Escherichia coli unleashed.

We have recently reported the development of an efficient, whole-cell process for chemoenzymatic production of key chiral intermediates in statin synthesis by employing high-density Escherichia coli culture with the overexpressed deoxyribose-5-phosphate aldolase (DERA). The optically pure, 6-substituted cyclic hemiacetals can be used for the synthesis of atorvastatin, rosuvastatin and pitavastatin using further chemical steps. All of the synthetic routes established to date begin with a regiospecific oxidation of these lactol intermediates into the corresponding lactones, followed by several steps yielding 6-substituted, open-chain or lactonized derivatives which can be coupled by various approaches with the heterocyclic part of the statin molecule. Here we report for the first time the use of PQQ-dependent glucose dehydrogenases for a highly efficient, regioselective oxidation of artificial, derivatized aldohexoses, more specifically, the statin lactol intermediates. First, PQQ-dependent dehydrogenases of both soluble and membrane-bound type were characterized for their activity toward various DERA-derived lactols. Further, we describe a highly productive whole-cell system for oxidation of these 2,4-dideoxyaldopyranoses using a PQQ-dependent glucose dehydrogenase (Gcd) overexpressed in E. coli while taking advantage of the respiratory chain as the mediator of the electron transfer to oxygen. Finally, a two-step artificial biosynthetic pathway was developed by unleashing the intrinsic genetic potential of E. coli. The combined overexpression of the endogenous DERA and the membrane-bound, PQQ-dependent glucose dehydrogenase, the latter being coupled to the respiratory chain, allows direct biosynthesis of 6-substituted lactones in a highly productive, high-yield, cost-effective and industrially scalable process.

Gender, sexuality and cis-heteropatriarchal parental control: navigating the politics of neo-panopticon and the resilience of young Khawaja Sara and Hijra in Peshawar, Pakistan.

Background: Scholarly works have extensively explored the marginalized positions of transgender individuals in Pakistan. However, there is a noticeable gap in literature concerning the profound impact of cis-heteropatriarchal parental control on young Khawaja Sara and Hijra individuals-members of the transgender community-particularly when they reside in their parental homes in Peshawar, Pakistan. Aim: Grounded in Foucault's panopticon concept, this study seeks to illuminate the pervasive surveillance experienced by young Khawaja Sara and Hijra within the confines of their familial environments. Method: The study employed a qualitative methodology, which involved conducting face-to-face interviews with 10 members of the Khawaja Sara and Hijra communities in Peshawar. Findings: The findings demonstrate that trans identities are perceived as a breach of honor in the local Pashtun culture, leading to disrespect and disgrace for the family name. In this way, a meticulous monitoring through regular surveillance creates a neo-panoptic environment within their parental households. This pervasive surveillance not only obstructs their access to quality health services, educational facilities, and employment opportunities but also compels many transgender individuals to abandon their parental homes and migrate to urban areas in Peshawar for their security and protection. Despite grappling with societal pressures, encompassing corporal punishment, sexual abuse, and homelessness, the findings underscore the remarkable resilience and resistance displayed by young Khawaja Sara and Hijra members. Their resilience and resistance serve as a potent challenge against the entrenched cis-heteropatriarchal parental control in Peshawar, Pakistan. Conclusion: The study concludes on highlighting the harsh circumstances confronted by transgender individuals within their parental households in Peshawar, where their trans subjectivities label them as dishonorable. Despite enduring societal pressures, the resilience exhibited by young Khawaja Sara and Hijra emerges as a formidable challenge to the deeply entrenched cis-heteropatriarchal control. This describes the indomitable strength of the transgender community in overcoming systemic adversities in Peshawar.

Prevalence of under-nutrition and its associated factors among 6-23 months old children of employed and unemployed mothers in town kebeles of Dera district, northwest Ethiopia: a comparative cross-sectional study.

BACKGROUND: Child under-nutrition remains a widespread problem around the globe. Improving child nutrition and empowering women are two important and closely connected development goals. These two interconnected goals will affect one another through different mechanisms, and the net effect may not necessarily be positive. Yet, the impact of maternal employment, one method of empowering mothers, on children's nutritional is not well studied in Ethiopia. Hence, this study is to compare the prevalence of under-nutrition and its associated factors among 6-23 months old children of employed and unemployed mothers in town kebeles of Dera district, Northwest Ethiopia, 2022. METHODS: A community-based comparative cross-sectional study design was conducted among 356 employed and 356 unemployed mothers having 6-23 months old children. A systematic random sampling technique was used to select study participants. Epi-data version 3.1 and SPSS version 25.0 statistical software were used for data entry and analysis, respectively. Both bi-variable and multivariable binary logistic regression was done to assess the association between independent and dependent variables. A p-value of less than 0.05 in a multivariable binary logistic regression was declared as the level of statistical significance. RESULT: The prevalence of under-nutrition was 69.8% (95% CI: 65.0, 74.7) among children of unemployed mothers, compared to 27.4% (95% CI: 22.7, 32.2) among children of employed mothers. Under-nutrition among children of unemployed mothers was significantly associated with being a male child, age increase by one month, household food insecurity, lack of ANC follow-up, and not exclusively breastfed. Whereas, among children of employed mothers, being a male child, age increase by one month, being sick during the last two weeks prior to data collection, not immunized to their age, and low meal frequency were significantly associated with their under-nutrition. CONCLUSION: The prevalence of under-nutrition among children of unemployed women is significantly higher than those children of employed women which consolidate the evidence that women's employment status have a positive association with child nutrition. Different factors were also identified as significant predictors of child under-nutrition among these two groups (employed and unemployed women). Thus, multi-sectoral intervention approach together with agriculture and education offices should be strengthened.

Small Ruminant Farming in Tribal Areas of Dera Ghazi Khan, Punjab, Pakistan.

Provincially Administered Tribal Areas (PATA) of Punjab-Pakistan are comprised of hilly mountains with small ruminants as a sole source of income. In this study, farming practices, productivity, health and the economic value of sheep were evaluated in PATA through a survey of farmers (n = 138) holding 11,558 heads of sheep. Out of a total population, 87% were non-descriptive flocks, and 9% and 4% were purebred flocks belonging to the Kajli and Thali populations, respectively. Sheep flocks were mainly (86%) reared under the traditional production system and had a delayed onset of puberty. There was low influence of season on the reproduction, and the majority of flocks (78%) were bred throughout the year. The lack of proper vaccination and poor management exposed the flocks to bacterial, viral and parasitic infections, which lead to high mortality in lambs (~22%) and adults (~32%). The share of sheep in farmers livelihood was 42%, and only 20% of producers' living standard was improved with sheep farming, but the rise in rearing more sheep was quite low (20%). Although the livestock department arranged farmers' training, the majority of farmers (83%) never participated in training and had no knowledge of modern technologies. Collectively, the traditional sheep production systems, poor management, lack of vaccination, marketing channels and farmers training hampered the sheep rearing and producers' livelihood in the PATA of Punjab-Pakistan. However, developing model livestock farms, conducting farmer training, establishing a viable market for dairy products, and introducing subsidy policy interventions can improve the sheep farming in these areas.

Analysis of RHOA mutations and their significance in the proliferation and transcriptome of digestive tract cancer cells.

The ras homolog family member A (RHOA) gene encodes a member of the Rho family of small GTPases and is known to function in reorganization of the actin cytoskeleton, which is associated with regulation of cell shape, attachment and motility. RHOA has been found to be recurrently mutated in gastrointestinal cancer; however, the functional significance of the mutated RHOA protein in digestive tract cancers remains to be uncovered. The aim of the present study was to understand the role of mutant RHOA in the proliferation and transcriptome of digestive tract cancer cells. Mutations of RHOA in one esophageal cancer cell line, OE19, eight gastric cancer cell lines, namely, AGS, GCIY, HGC-27, KATO III, MKN1, MKN45, SNU16 and SNU719, as well as two colon cancer cell lines, CCK-81 and SW948, were determined using Sanger sequencing. The results uncovered several mutations, including p.Arg5Gln and p.Tyr42Cys in CCK-81, p.Arg5Trp and p.Phe39Leu in SNU16, p.Gly17Glu in SW948, p.Tyr42Ser in OE19, p.Ala61Val in SNU719, p.Glu64del in AGS. Wild-type RHOA was identified in GCIY, HGC-27, KATO III, MKN1 and MKN45. Knockdown of RHOA using small interfering RNA attenuated the in vitro proliferation in the three-dimensional culture systems of GCIY, MKN1, OE19 and SW948, whereas no apparent changes were seen in CCK-81, HGC-27 and SNU719. Transcriptome analysis revealed that downregulation of the long non-coding RNA (lnc)-DERA-1 was observed in all tested cell lines following RHOA knockdown in the RHOA-mutated cell lines. Gene Ontology analysis showed that the genes associated with small molecule metabolic process, oxidation-reduction processes, protein kinase activity, transport, and cell junction were commonly downregulated in cells whose proliferation was attenuated by the knockdown of RHOA. These results suggested that certain RHOA mutations may result in upregulation of lnc-DERA-1 and genes associated with cellular metabolism and proliferation in digestive tract cancers.

Community-Based Health Insurance Membership Dropout Rate and Associated Factors in Dera District, Northwest Ethiopia.

BACKGROUND: Community-based health insurance is an emerging strategy for providing financial protection against health-related poverty. They have developed into alternative health financing mechanisms for out-of-pocket expenses in low- and middle-income countries. Hence, the aim of this study was to assess the drop-out rate of community-based health insurance membership and associated factors in Dera district, Northwest Ethiopia, 2020. METHODS: Through systematic random sampling techniques, a community-based cross-sectional study was conducted on 584 participants. A structured interviewer-based administered questionnaire was used for data collection. EpI data is used for data entry, while SPSS 20 version is used for analysis. P-value <0.2 binary logistic regression was entered into multivariable logistic regression. Variables with a P-value of <0.05 and a 95% confidence level were considered to be significantly associated with the outcome variable. RESULTS: The CBHI dropout rate in the district is calculated to be 37.3% (95% CI: 34, 41%) in the district. Length of enrollment, ≥4 years in the CBHI program (AOR=0.39, 95% CI: 0.26, 0.59), households visit the health facilities 4-6 times a year (AOR=1.92, 95% CI: 1.10, 3.32), have no access to the hospital (AOR=1.68, 95% CI: 1.02, 2.77), knowledge of CBHI (AOR=1.93, 95% CI: 1.32, 2.82) and official position holder for decision-making in the households (AOR=2.07, 95% CI: 1.33, 3.23) were factors associated with CBHI dropout rate in the scheme. CONCLUSION: This finding confirmed that the CBHI dropout rate in the district was high. Length of enrollment, health facility visit, hospital accessibility, knowledge of CBHI, and official position holders used for decision-making are significantly associated with the CBHI dropout rate. Therefore, emphasis should be given on improving members' understanding of the CBHI package of benefits; increasing access to hospitals and empowering women will increase the utilization of CBHI. In addition, the quality of care for CBHI patients can be improved when they had access to health services.

Wild birds trade in Dera Ismael Khan and Bannu divisions of Khyber PakhtunKhwa (KPK) Province, Pakistan / Comércio de aves selvagens nas divisões Dera Ismail Khan e Bannu da província de Khyber Pakhtunkhwa (KPK), Paquistão

Wildlife trade is a profitable economic activity. Birds are among the most heavily traded animals worldwide, with numerous species threatened by pet trade. The present case study was stretched for period of one year to find out the status of wild birds’ trade in Bannu and Dera Ismael Khan Divisions. The study was aimed to find out the types of species, commercial prices of bird, trade routes, overall income generated from birds' trade and the status of dealers whether licensed or not. Bimonthly visits were conducted to interview 52 bird dealers/pet shops owners/venders (23 in Dera Ismael Khan and 29 in Bannu). A sum of 16 conceivable sites were visited during the study period. Out of these, 8 sites were located in Dera Ismael Khan and 8 sites in Bannu Division. The highest priced species was Demoiselle crane (Grus virgo) and the lowest priced species was house sparrow (Passer domesticus). Total number of species traded in both regions were 8 and 14 while the income generated by bird trade was amounting to PkR 1,322,200 and 7,128,950 in Dera Ismael Khan and Bannu respectively. Majority of the birds were trapped locally with the exception of few from adjacent areas. Commercial activity led by illegal bird traders was found 75% and 100% species in Dera Ismael Khan and Bannu respectively. There was no significant difference between prices of birds, number of species sold and income generated by bird trade in two divisions.

O comércio de animais selvagens é uma atividade econômica lucrativa. Os pássaros estão entre os animais mais comercializados em todo o mundo, com inúmeras espécies ameaçadas pelo comércio de animais de estimação. O presente estudo de caso foi estendido por um período de um ano para descobrir a situação do comércio de aves selvagens nas Divisões Bannu e Dera Ismael Khan. O estudo teve como objetivo descobrir os tipos de espécies, os preços comerciais das aves, as rotas comerciais, a receita geral gerada pelo comércio de aves e o status dos negociantes, licenciados ou não. Visitas bimestrais foram realizadas para entrevistar 52 comerciantes de pássaros / proprietários / vendedores de pet shops (23 em Dera Ismael Khan e 29 em Bannu). Uma soma de 16 locais concebíveis foram visitados durante o período de estudo. Destes, 8 sites estavam localizados em Dera Ismael Khan e 8 sites na Divisão Bannu. A espécie com preço mais alto foi o guindaste Demoiselle (Grus virgo) e a espécie com preço mais baixo foi o pardal (Passer domesticus). O número total de espécies comercializadas em ambas as regiões foi de 8 e 14, enquanto a receita gerada pelo comércio de aves foi de PkR 1.322.200 e 7.128.950 em Dera Ismael Khan e Bannu, respectivamente. A maioria das aves foi capturada localmente, com exceção de algumas das áreas adjacentes. A atividade comercial liderada por comerciantes ilegais de aves foi encontrada com 75% e 100% de espécies em Dera Ismael Khan e Bannu, respectivamente. Não houve diferença significativa entre os preços das aves, o número de espécies vendidas e a receita gerada pelo comércio de aves nas duas divisões.

Wild birds trade in Dera Ismael Khan and Bannu divisions of Khyber PakhtunKhwa (KPK) Province, Pakistan

Abstract Wildlife trade is a profitable economic activity. Birds are among the most heavily traded animals worldwide, with numerous species threatened by pet trade. The present case study was stretched for period of one year to find out the status of wild birds trade in Bannu and Dera Ismael Khan Divisions. The study was aimed to find out the types of species, commercial prices of bird, trade routes, overall income generated from birds trade and the status of dealers whether licensed or not. Bimonthly visits were conducted to interview 52 bird dealers/pet shops owners/venders (23 in Dera Ismael Khan and 29 in Bannu). A sum of 16 conceivable sites were visited during the study period. Out of these, 8 sites were located in Dera Ismael Khan and 8 sites in Bannu Division. The highest priced species was Demoiselle crane (Grus virgo) and the lowest priced species was house sparrow (Passer domesticus). Total number of species traded in both regions were 8 and 14 while the income generated by bird trade was amounting to PkR 1,322,200 and 7,128,950 in Dera Ismael Khan and Bannu respectively. Majority of the birds were trapped locally with the exception of few from adjacent areas. Commercial activity led by illegal bird traders was found 75% and 100% species in Dera Ismael Khan and Bannu respectively. There was no significant difference between prices of birds, number of species sold and income generated by bird trade in two divisions.

Resumo O comércio de animais selvagens é uma atividade econômica lucrativa. Os pássaros estão entre os animais mais comercializados em todo o mundo, com inúmeras espécies ameaçadas pelo comércio de animais de estimação. O presente estudo de caso foi estendido por um período de um ano para descobrir a situação do comércio de aves selvagens nas Divisões Bannu e Dera Ismael Khan. O estudo teve como objetivo descobrir os tipos de espécies, os preços comerciais das aves, as rotas comerciais, a receita geral gerada pelo comércio de aves e o status dos negociantes, licenciados ou não. Visitas bimestrais foram realizadas para entrevistar 52 comerciantes de pássaros / proprietários / vendedores de pet shops (23 em Dera Ismael Khan e 29 em Bannu). Uma soma de 16 locais concebíveis foram visitados durante o período de estudo. Destes, 8 sites estavam localizados em Dera Ismael Khan e 8 sites na Divisão Bannu. A espécie com preço mais alto foi o guindaste Demoiselle (Grus virgo) e a espécie com preço mais baixo foi o pardal (Passer domesticus). O número total de espécies comercializadas em ambas as regiões foi de 8 e 14, enquanto a receita gerada pelo comércio de aves foi de PkR 1.322.200 e 7.128.950 em Dera Ismael Khan e Bannu, respectivamente. A maioria das aves foi capturada localmente, com exceção de algumas das áreas adjacentes. A atividade comercial liderada por comerciantes ilegais de aves foi encontrada com 75% e 100% de espécies em Dera Ismael Khan e Bannu, respectivamente. Não houve diferença significativa entre os preços das aves, o número de espécies vendidas e a receita gerada pelo comércio de aves nas duas divisões.

Wild birds trade in Dera Ismael Khan and Bannu divisions of Khyber PakhtunKhwa (KPK) Province, Pakistan / Comércio de aves selvagens nas divisões Dera Ismail Khan e Bannu da província de Khyber Pakhtunkhwa (KPK), Paquistão

Abstract Wildlife trade is a profitable economic activity. Birds are among the most heavily traded animals worldwide, with numerous species threatened by pet trade. The present case study was stretched for period of one year to find out the status of wild birds' trade in Bannu and Dera Ismael Khan Divisions. The study was aimed to find out the types of species, commercial prices of bird, trade routes, overall income generated from birds' trade and the status of dealers whether licensed or not. Bimonthly visits were conducted to interview 52 bird dealers/pet shops owners/venders (23 in Dera Ismael Khan and 29 in Bannu). A sum of 16 conceivable sites were visited during the study period. Out of these, 8 sites were located in Dera Ismael Khan and 8 sites in Bannu Division. The highest priced species was Demoiselle crane (Grus virgo) and the lowest priced species was house sparrow (Passer domesticus). Total number of species traded in both regions were 8 and 14 while the income generated by bird trade was amounting to PkR 1,322,200 and 7,128,950 in Dera Ismael Khan and Bannu respectively. Majority of the birds were trapped locally with the exception of few from adjacent areas. Commercial activity led by illegal bird traders was found 75% and 100% species in Dera Ismael Khan and Bannu respectively. There was no significant difference between prices of birds, number of species sold and income generated by bird trade in two divisions.

Resumo O comércio de animais selvagens é uma atividade econômica lucrativa. Os pássaros estão entre os animais mais comercializados em todo o mundo, com inúmeras espécies ameaçadas pelo comércio de animais de estimação. O presente estudo de caso foi estendido por um período de um ano para descobrir a situação do comércio de aves selvagens nas Divisões Bannu e Dera Ismael Khan. O estudo teve como objetivo descobrir os tipos de espécies, os preços comerciais das aves, as rotas comerciais, a receita geral gerada pelo comércio de aves e o status dos negociantes, licenciados ou não. Visitas bimestrais foram realizadas para entrevistar 52 comerciantes de pássaros / proprietários / vendedores de pet shops (23 em Dera Ismael Khan e 29 em Bannu). Uma soma de 16 locais concebíveis foram visitados durante o período de estudo. Destes, 8 sites estavam localizados em Dera Ismael Khan e 8 sites na Divisão Bannu. A espécie com preço mais alto foi o guindaste Demoiselle (Grus virgo) e a espécie com preço mais baixo foi o pardal (Passer domesticus). O número total de espécies comercializadas em ambas as regiões foi de 8 e 14, enquanto a receita gerada pelo comércio de aves foi de PkR 1.322.200 e 7.128.950 em Dera Ismael Khan e Bannu, respectivamente. A maioria das aves foi capturada localmente, com exceção de algumas das áreas adjacentes. A atividade comercial liderada por comerciantes ilegais de aves foi encontrada com 75% e 100% de espécies em Dera Ismael Khan e Bannu, respectivamente. Não houve diferença significativa entre os preços das aves, o número de espécies vendidas e a receita gerada pelo comércio de aves nas duas divisões.

1H, 13C, and 15N backbone and sidechain resonance assignments of a monomeric variant of E. coli deoxyribose-5-phosphate aldolase.

Deoxyribose-5-phosphate aldolase (DERA) catalyses the reversible conversion of 2-deoxyribose-5-phosphate (dR5P) into glyceraldehyde-3-phosphate (G3P) and acetaldehyde. For industrial applications, this enzyme is used in organic synthesis for aldol reactions between acetaldehyde as a donor and a wide range of aldehydes as acceptors. Here, we present a near complete set of sequence-specific 1H, 13C and 15N resonance assignments of a 28 kDa monomeric variant of the Escherichia coli DERA. These assignments provide the basis for ongoing structural and dynamic analysis of DERA substrate specificity.

Structural insight for substrate tolerance to 2-deoxyribose-5-phosphate aldolase from the pathogen Streptococcus suis.

2-deoxyribose-5-phosphate aldolase (DERA) is a class I aldolase that catalyzes aldol condensation of two aldehydes in the active site, which is particularly germane in drug manufacture. Structural and biochemical studies have shown that the active site of DERA is typically loosely packed and displays broader substrate specificity despite sharing conserved folding architecture with other aldolases. The most distinctive structural feature of DERA compared to other aldolases is short and flexible C-terminal region. This region is also responsible for substrate recognition. Therefore, substrate tolerance may be related to the C-terminal structural features of DERA. Here, we determined the crystal structures of full length and C-terminal truncated DERA from Streptococcus suis (SsDERA). In common, both contained the typical (α/ß)8 TIM-barrel fold of class I aldolases. Surprisingly, C-terminal truncation resulting in missing the last α9 and ß8 secondary elements, allowed DERA to maintain activity comparable to the fulllength enzyme. Specifically, Arg186 and Ser205 residues at the C-terminus appeared mutually supplemental or less indispensible for substrate phosphate moiety recognition. Our results suggest that DERA might adopt a shorter C-terminal region than conventional aldolases during evolution pathway, resulting in a broader range of substrate tolerance through active site flexibility.

Genomic Characterization of the Genus Nairovirus (Family Bunyaviridae).

Nairovirus, one of five bunyaviral genera, includes seven species. Genomic sequence information is limited for members of the Dera Ghazi Khan, Hughes, Qalyub, Sakhalin, and Thiafora nairovirus species. We used next-generation sequencing and historical virus-culture samples to determine 14 complete and nine coding-complete nairoviral genome sequences to further characterize these species. Previously unsequenced viruses include Abu Mina, Clo Mor, Great Saltee, Hughes, Raza, Sakhalin, Soldado, and Tillamook viruses. In addition, we present genomic sequence information on additional isolates of previously sequenced Avalon, Dugbe, Sapphire II, and Zirqa viruses. Finally, we identify Tunis virus, previously thought to be a phlebovirus, as an isolate of Abu Hammad virus. Phylogenetic analyses indicate the need for reassignment of Sapphire II virus to Dera Ghazi Khan nairovirus and reassignment of Hazara, Tofla, and Nairobi sheep disease viruses to novel species. We also propose new species for the Kasokero group (Kasokero, Leopards Hill, Yogue viruses), the Ketarah group (Gossas, Issyk-kul, Keterah/soft tick viruses) and the Burana group (Wenzhou tick virus, Huángpí tick virus 1, Tǎchéng tick virus 1). Our analyses emphasize the sister relationship of nairoviruses and arenaviruses, and indicate that several nairo-like viruses (Shayáng spider virus 1, Xinzhou spider virus, Sanxiá water strider virus 1, South Bay virus, Wǔhàn millipede virus 2) require establishment of novel genera in a larger nairovirus-arenavirus supergroup.