Bioenergy production in Pakistan: Potential, progress, and prospect.

Pakistan is a developing country with a rapidly growing population. It is currently facing serious economic and energy challenges. Pakistan's energy demand is increasing by the day, and it now stands at 84 MTOE. Currently, the use of fossil fuels dominates Pakistan's energy sector. Conversely, indigenous fossil fuel resources are rapidly depleting and will be unable to meet rising energy demands in the future. Therefore, to withstand its energy needs, the country will need to explore alternative energy production methods. Biomass is one of the alternatives that has enormous potential to help Pakistan combat its growing energy crisis. In this review, we first present an overview of bioenergy, biomass resources, and biomass conversion technologies. We then discuss in detail the current state of the energy mix of Pakistan. Subsequently, we show that annual production of about 121 MT of agricultural residues, 427 MT of animal manure, and 7.5 MT of MSW in Pakistan offer a variety of bioenergy options ranging from biofuels to bio-electricity production. Overall, these biomass resources in Pakistan have the potential to generate 20,709 MW of bio-electricity and 12,615 million m3 of biogas annually in Pakistan. Though these resources hold promising potential for bioenergy production in the country, however, there are some critical challenges that need to be considered, and some of which are extremely difficult to overcome for a developing country like Pakistan. This work is expected to provide a useful basis for biomass management and utilization in Pakistan to harvest eco-friendly and sustainable green energy locally.

Genome-Wide Analyses of Proteome and Acetylome in Zymomonas mobilis Under N2-Fixing Condition.

Zymomonas mobilis, a promising candidate for industrial biofuel production, is capable of nitrogen fixation naturally without hindering ethanol production. However, little is known about the regulation of nitrogen fixation in Z. mobilis. We herein conducted a high throughput analysis of proteome and protein acetylation in Z. mobilis under N2-fixing conditions and established its first acetylome. The upregulated proteins mainly belong to processes of nitrogen fixation, motility, chemotaxis, flagellar assembly, energy production, transportation, and oxidation-reduction. Whereas, downregulated proteins are mainly related to energy-consuming and biosynthetic processes. Our acetylome analyses revealed 197 uniquely acetylated proteins, belonging to major pathways such as nitrogen fixation, central carbon metabolism, ammonia assimilation pathway, protein biosynthesis, and amino acid metabolism. Further, we observed acetylation in glycolytic enzymes of central carbon metabolism, the nitrogenase complex, the master regulator NifA, and the enzyme in GS/GOGAT cycle. These findings suggest that protein acetylation may play an important role in regulating various aspects of N2-metabolism in Z. mobilis. This study provides new knowledge of specific proteins and their associated cellular processes and pathways that may be regulated by protein acetylation in Z. mobilis.

Bio-conversion of CO2 into biofuels and other value-added chemicals via metabolic engineering.

Carbon dioxide (CO2) occurs naturally in the atmosphere as a trace gas, which is produced naturally as well as by anthropogenic activities. CO2 is a readily available source of carbon that in principle can be used as a raw material for the synthesis of valuable products. The autotrophic organisms are naturally equipped to convert CO2 into biomass by obtaining energy from sunlight or inorganic electron donors. This autotrophic CO2 fixation has been exploited in biotechnology, and microbial cell factories have been metabolically engineered to convert CO2 into biofuels and other value-added bio-based chemicals. A variety of metabolic engineering efforts for CO2 fixation ranging from basic copy, paste, and fine-tuning approaches to engineering and testing of novel synthetic CO2 fixing pathways have been demonstrated. In this paper, we review the current advances and innovations in metabolic engineering for bio-conversion of CO2 into bio biofuels and other value-added bio-based chemicals.

Analyses of mitogenomic markers shed light on the divergence, population dynamics, and demographic history of Pakistani chickens.

Pakistan is one of a few sites, associated with the earliest known independent domestication event in the evolutionary history of chicken, which is socio-economically and historically the most important poultry bird in the country. However, the divergence, past population dynamics, and demographic history of Pakistani chickens have not been addressed so far. Therefore, we herein investigated the indigenous Pakistani chickens using mitogenomic markers. We first prepared individual DNA samples from the chicken feathers, and generated nucleotide sequence data, which was then subjected to various population genetics analyses. In molecular phylogenetic analysis, the Pakistani chickens were clustered under nine different clades. Among the wild fowls, the Indian red jungle fowl (IRJF) shared very close affinities to Pakistani chickens. The Bayesian skyline plot showed an increase in the effective population size of Pakistani chickens during the last 50 years. Finally, a time-calibrated phylogeny inferred molecular divergence of the Pakistani chickens. A molecular rate of 3.6 × 10-6 mutations/site/year (95% HPD interval: 2.28 × 10-8 to 9.32 × 10-6) was estimated for the data set. In a rooted tree with root-age of 12058 years (95% HPD interval: 1161-38411), the Pakistani chicken haplotypes showed divergence from IRJF haplotypes around 6987 years (95% HPD interval: 1132-20746) ago, and they shared their most recent common ancestor with Gallus gallus spadiceus, and G. g. jabouillei at the root of the tree. Overall, these results suggest that Pakistani chicken haplotypes share their ancestral gene pool with the IRJF as compared to other red jungle fowl subspecies.

Apparent nutrient digestibility of carbohydrate (corn) in Cirrhinus mrigala under the influence of chromium chloride hexahydrate / Digestibilidade aparente de nutrientes oriundos de carboidratos (milho) em Cirrhinus mrigala sob a influência do cloreto de cromo hexa-hidratado

Abstract A feeding trial was carried out to assess the effect of dietary chromium supplementation on apparent nutrient digestibility coefficient (%) of gelatinized and non-gelatinized corn in Cirrhinus mrigala fingerlings for 90 days. Using various levels of chromium chloride hexahydrate six test diets designated as T1 (G/0.0 Cr2Cl3.6H2O mg Kg-1), T2 (NG/0.0 Cr2Cl3.6H2O mg Kg-1), T3 (G/0.2 Cr2Cl3.6H2O mg Kg-1), T4 (NG/0.2 Cr2Cl3.6H2O mg Kg-1), T5 (G/0.4 Cr2Cl3.6H2O mg Kg-1) and T6 (NG/0.4 Cr2Cl3.6H2O mg Kg-1) were prepared. Results showed highest apparent digestibility coefficient (ADC) of nutrients dry matter, crude lipid and gross energy in test diet T5 that was gelatinized and supplemented with chromium 0.4 mg/Kg while, for crude protein higher value of nutrient digestibility was recorded in T3 test diet (G/0.2 Cr2Cl3.6H2O mg/Kg). It was concluded that chromium supplementation with gelatinized corn in fish (Cirrhinus mrigala) diet can improve the nutrients digestibility more efficiently.

Resumo Um experimento referente à alimentação foi realizado para avaliar o efeito da suplementação dietética de cromo, no coeficiente de digestibilidade aparente do nutriente (%) do milho gelatinizado e não gelatinizado em alevinos de Cirrhinus mrigala por 90 dias. Usando vários níveis de cloreto de cromo hexa-hidratado, seis dietas testes foram designadas e preparadas como: T1 (G/0,0 Cr2Cl3.6H2O mg Kg-1); T2 (NG/0,0 Cr2Cl3.6H2O mg Kg-1); T3 (G/0,2 Cr2Cl3.6H2O mg Kg-1); T4 (NG/0,2 Cr2Cl3.6H2O mg Kg-1); T5 (G/0,4 Cr2Cl3.6H2O mg Kg-1) e T6 (NG/0,4 Cr2Cl3.6H2O mg Kg-1). Os resultados mostraram maior coeficiente de digestibilidade aparente (CDA) em matéria seca de nutrientes, lipídios brutos e energia bruta na dieta teste T5, que foi gelatinizada e suplementada com cromo 0,4 mg / Kg; enquanto que para a proteína bruta, foi registrada maior digestibilidade dos nutrientes na dieta teste T3 (G/0,2 Cr2Cl3.6H2O mg/Kg). Concluiu-se que a suplementação de cromo com milho gelatinizado na dieta de peixes (Cirrhinus mrigala) pôde melhorar a digestibilidade dos nutrientes de forma mais eficiente.

Suppression of hyaluronidase reduces invasion and establishment of Haemonchus contortus larvae in sheep.

Haemonchus contortus is a hematophagous endoparasite of small ruminants, which is responsible for huge economic losses in livestock sector. Hyaluronidase produced by infective larvae of H. contortus can degrade hyaluronic acid present in the host's abomasal tissue. Thus, it facilitates larval tissue invasion and early establishment. We herein explored this ability of hyaluronidase in H. contortus, and tested whether hyaluronidase is utilized as a virulence factor by H. contortus while establishing the infection. We first successfully blocked the hyaluronidase gene in L3 larvae by RNA interference (RNAi), which was subsequently confirmed by qPCR, enzymatic activity, and immunohistochemistry assays. Using these larvae we then conducted in vivo and in vitro assays on sheep to assess the effects of hyaluronidase suppression on larval invasion and establishment of infection. The in vivo assay showed a significant drop in worm burden in siRNA treated group in comparison to control group. During in vitro assay we applied an ovine ex vivo model where siRNA treated group of larvae showed significantly reduced invasion of the abomasal tissue explants as compared to control group. These findings indicate that hyaluronidase plays a key role in host's tissue invasion and larval establishment, and it is used as a virulence factor by H. contortus while establishing the infection. As an invasive virulence molecule, its functional research is thus conducive to the prevention of haemonchosis.

A Whole Genome Re-Sequencing Based GWA Analysis Reveals Candidate Genes Associated with Ivermectin Resistance in Haemonchus contortus.

The most important and broad-spectrum drug used to control the parasitic worms to date is ivermectin (IVM). Resistance against IVM has emerged in parasites, and preserving its efficacy is now becoming a serious issue. The parasitic nematode Haemonchus contortus (Rudolphi, 1803) is economically an important parasite of small ruminants across the globe, which has a successful track record in IVM resistance. There are growing evidences regarding the multigenic nature of IVM resistance, and although some genes have been proposed as candidates of IVM resistance using lower magnification of genome, the genetic basis of IVM resistance still remains poorly resolved. Using the full magnification of genome, we herein applied a population genomics approach to characterize genome-wide signatures of selection among pooled worms from two susceptible and six ivermectin-resistant isolates of H. contortus, and revealed candidate genes under selection in relation to IVM resistance. These candidates also included a previously known IVM-resistance-associated candidate gene HCON_00148840, glc-3. Finally, an RNA-interference-based functional validation assay revealed the HCON_00143950 as IVM-tolerance-associated gene in H. contortus. The possible role of this gene in IVM resistance could be detoxification of xenobiotic in phase I of xenobiotic metabolism. The results of this study further enhance our understanding on the IVM resistance and continue to provide further evidence in favor of multigenic nature of IVM resistance.

Apparent nutrient digestibility of carbohydrate (corn) in Cirrhinus mrigala under the influence of chromium chloride hexahydrate.

A feeding trial was carried out to assess the effect of dietary chromium supplementation on apparent nutrient digestibility coefficient (%) of gelatinized and non-gelatinized corn in Cirrhinus mrigala fingerlings for 90 days. Using various levels of chromium chloride hexahydrate six test diets designated as T1 (G/0.0 Cr2Cl3.6H2O mg Kg-1), T2 (NG/0.0 Cr2Cl3.6H2O mg Kg-1), T3 (G/0.2 Cr2Cl3.6H2O mg Kg-1), T4 (NG/0.2 Cr2Cl3.6H2O mg Kg-1), T5 (G/0.4 Cr2Cl3.6H2O mg Kg-1) and T6 (NG/0.4 Cr2Cl3.6H2O mg Kg-1) were prepared. Results showed highest apparent digestibility coefficient (ADC) of nutrients dry matter, crude lipid and gross energy in test diet T5 that was gelatinized and supplemented with chromium 0.4 mg/Kg while, for crude protein higher value of nutrient digestibility was recorded in T3 test diet (G/0.2 Cr2Cl3.6H2O mg/Kg). It was concluded that chromium supplementation with gelatinized corn in fish (Cirrhinus mrigala) diet can improve the nutrients digestibility more efficiently.

Microsatellite analysis reveals extensive gene flow, and lack of population structure in the farm populations of Haemonchus contortus in northern China.

The parasitic nematode Haemonchus contortus is economically an important parasite of small ruminants across the globe. China is the world's largest producer, consumer, and importer of mutton. With ubiquitous distribution across the country H. contortus is one of the potential candidates to cause huge economic losses to small ruminant farming industry in China. We herein investigated genetic diversity and population structure of six farm populations of H. contortus in northern China, and also compared them to H. contortus isolates from UK and Australia. We first prepared individual DNA samples from 240 adult worms, and generated genotyping data using eight microsatellite markers. Obtained data was then subjected to allelic frequency and population genetic analyses. The overall allelic richness (mean/locus/pop = 7.375 ±â€¯0.844-10.125 ±â€¯1.109), and expected heterozygosity (mean/locus/pop = 0.646 ±â€¯0.040-0.735 ±â€¯0.025) indicated high degree of population genetic variation across the Chinese isolates. Low level of genetic differentiation (Fst = 0.010-0.066) was observed across all the populations. AMOVA results showed high level of variation (93%) within the populations. PCA analysis revealed mixed clustering of all the populations with no visible geographical sub-structuring. Finally the population admixture analysis resulted in extensive admixing of genotypes across all the populations. With these findings we conclude that there is no obvious population genetic structure with extensive gene flow across all the farm populations of H. contortus in northern China.

Molecular characterization and phylogenetic analysis of tomato leaf curl Palampur virus, a bipartite begomovirus, associated with Cucumis sativus L. in Pakistan.

Leaf samples of Cucumis Sativus L. (C. sativus) (Family; Cucurbitaceae) showing vein thickening, mild leaf curling and leaf enations were collected from the farmer's field. Amplification of the full-length viral molecules was performed through rolling circle amplification (RCA). Cloning of the full-length viral molecules was done through standard cloning procedure followed by sequencing. Sequence similarity analysis and phylogenetic studies showed that the virus associated with leaf curling and enations in C. sativus was a bipartite begomovirus, where DNA-A and DNA-B showed highest nucleotide sequence homology of 98% and 97% to tomato leaf curl Palampur virus (ToLCPMV) from India. Attempts to isolate betasatellites and alphasatellites through PCR using RCA product as template, did not result in any amplification. A maximum likelihood phylogenetic tree grouped DNA-A and B components with other isolates from India. SDT was used to find the pairwise identity scores of different sequences of ToLCPMV present in the database. Phylogenetic analysis showed that sequences of ToLCPMV DNA-A and B components in this study share high degree of homology with existing viruses and are isolates of ToLCPMV-India. Infectious molecules of both components (Accessions, MG252783 and MG252784, respectively) were constructed for infectivity analysis to fulfill the Koch's postulate. Infectivity analysis revealed that ToLCPMV DNA-A is infectious to model host plant Nicotiana benthamiana and viral accumulation was confirmed through Southern blot analysis. Accumulation of DNA-B was confirmed through PCR. Infectivity analysis was also conducted using the original host, C. sativus, but plants were unable to survive the agroinoculation. To our knowledge this is the first report of ToLCPMV associated with C. sativus L. in Pakistan.

Ethnogenetic analysis reveals that Kohistanis of Pakistan were genetically linked to west Eurasians by a probable ancestral genepool from Eurasian steppe in the bronze age.

Despite the unique geographic, ethnic, social and cultural features of Kohistan in Pakistan, the origin and descent of Kohistanis remain still obscure. In an effort to address questions concerning the genetic structure, origin and genetic affinities of Kohistanis, we herein applied an ethnogenetic approach consisting on mitochondrial DNA (mtDNA) analysis and dental morphology analysis. We sequenced HVS1 of mtDNA, observed 14 haplotypes and assigned a total of 9 haplogroups belonging to macrolineages M (17%) and N (83%). Genetic diversity estimates in Kohistanis (Hd = 0.910 ±â€¯0.014; Pi = 0.019 ±â€¯0.001; θw = 0.019 ±â€¯0.006) were similar to that of previous studies in other Pakistani populations. Overall, the analyses of dental morphology and mtDNA profile of Kohistanis resulted in similar findings. All the analyses indicate that Kohistanis share affinities to populations from Europe, Near East, Central Asia and South Asia. The Kohistani HVS1 haplotype 2 shares 100% identity to HVS1 haplotypes across the Europe. These results in light of recent insights into ancient genomics lead us to conclude that ancestry from Eurasian Steppe genetically linked Kohistanis to all these populations in the Bronze Age. This is consistent with linguistic evidence and also with the Indo-Aryan migration model for the peopling of South Asia.

Population structure, genetic diversity and phylogenetic analysis of different rural and commercial chickens of Pakistan using complete sequence of mtDNA D-loop.

Chicken is the most important poultry bird in Pakistan that not only provides nutrition but also contributes to country's economy. The Pakistani chicken and its germplasm resources are not genetically characterized and explored. Here, we focused at rural and commercial chickens of Pakistan and explored genetic diversity, population structure and phylogeny. We first collected feather samples from Rural and Broiler populations of Pakistani chickens, isolated DNA and sequenced complete D-loop of mtDNA. The length of complete D-loop ranged from 1231 to 1234 bp in Pakistani chickens. The GC content was 39%. Hotspots of mutations were three hypervariable sites (HVS). Most of the variations (77%) were in HVS1. In a total, 26 polymorphic sites defined 12 haplotypes and all major haplogroups (A-I) in genetic structure of Pakistani chickens. Genetic diversity remained relatively very low in Broiler (Pi = 0.00212 ± 0.00136). There was a low sharing of matrilineages between the two populations (Fst = 0.170). With high Hd value (0.825 ± 0.051) and presence of all nine major haplogroups the rural chicken population showed relatively rich genepool. Finally we did molecular phylogenetic analysis and inferred phylogeny. Presence of subcontinent specific haplogroups E3 and I and clustering of Indian red junglefowl closely with Pakistani chickens in Bayesian inference tree, provide further evidence for an independent domestication event of chicken in subcontinent.

The Understanding of Ebola Virus Disease (EVD) Among Medical Practitioners of Karachi, Pakistan.

The World Health Organization (WHO) has acknowledged the large West African Ebola virus disease (EVD) outbreak to be a community health disaster of global concern, and the spread of disease demands a synchronized response. Medical practitioners have an increased risk of contracting the disease as compared to others as they are directly exposed to patients' blood or fluids. This study evaluated the knowledge of medical practitioners in Karachi regarding EVD. It was descriptive and exploratory in nature and took place over a period of 4 months, i.e., August 2016 to November 2016. The respondents were randomly selected by convenience sampling and surveyed with a 20-item questionnaire. Overall, 403 questionnaires were included in the study and a response rate of 80.6% was achieved. The majority (56.3%) considered themselves to be somewhat knowledgeable; females had more knowledge as compared to male (p < 0.003). More than 80% knew about the 2014 Ebola outbreak in West Africa. Interestingly, the findings revealed that respondents' knowledge about diagnosis and identification of EVD is good. Respondents considered EVD a severe disease and emphasized on the need for protective measures when contacting affected patients. Interventions should be tailored to focus on areas where respondents showed a lack of knowledge about the disease.

Apparent nutrient digestibility of carbohydrate (corn) in Cirrhinus mrigala under the influence of chromium chloride hexahydrate

Abstract A feeding trial was carried out to assess the effect of dietary chromium supplementation on apparent nutrient digestibility coefficient (%) of gelatinized and non-gelatinized corn in Cirrhinus mrigala fingerlings for 90 days. Using various levels of chromium chloride hexahydrate six test diets designated as T1 (G/0.0 Cr2Cl3.6H2O mg Kg-1), T2 (NG/0.0 Cr2Cl3.6H2O mg Kg-1), T3 (G/0.2 Cr2Cl3.6H2O mg Kg-1), T4 (NG/0.2 Cr2Cl3.6H2O mg Kg-1), T5 (G/0.4 Cr2Cl3.6H2O mg Kg-1) and T6 (NG/0.4 Cr2Cl3.6H2O mg Kg-1) were prepared. Results showed highest apparent digestibility coefficient (ADC) of nutrients dry matter, crude lipid and gross energy in test diet T5 that was gelatinized and supplemented with chromium 0.4 mg/Kg while, for crude protein higher value of nutrient digestibility was recorded in T3 test diet (G/0.2 Cr2Cl3.6H2O mg/Kg). It was concluded that chromium supplementation with gelatinized corn in fish (Cirrhinus mrigala) diet can improve the nutrients digestibility more efficiently.

Resumo Um experimento referente à alimentação foi realizado para avaliar o efeito da suplementação dietética de cromo, no coeficiente de digestibilidade aparente do nutriente (%) do milho gelatinizado e não gelatinizado em alevinos de Cirrhinus mrigala por 90 dias. Usando vários níveis de cloreto de cromo hexa-hidratado, seis dietas testes foram designadas e preparadas como: T1 (G/0,0 Cr2Cl3.6H2O mg Kg-1); T2 (NG/0,0 Cr2Cl3.6H2O mg Kg-1); T3 (G/0,2 Cr2Cl3.6H2O mg Kg-1); T4 (NG/0,2 Cr2Cl3.6H2O mg Kg-1); T5 (G/0,4 Cr2Cl3.6H2O mg Kg-1) e T6 (NG/0,4 Cr2Cl3.6H2O mg Kg-1). Os resultados mostraram maior coeficiente de digestibilidade aparente (CDA) em matéria seca de nutrientes, lipídios brutos e energia bruta na dieta teste T5, que foi gelatinizada e suplementada com cromo 0,4 mg / Kg; enquanto que para a proteína bruta, foi registrada maior digestibilidade dos nutrientes na dieta teste T3 (G/0,2 Cr2Cl3.6H2O mg/Kg). Concluiu-se que a suplementação de cromo com milho gelatinizado na dieta de peixes (Cirrhinus mrigala) pôde melhorar a digestibilidade dos nutrientes de forma mais eficiente.