Kashif Rahim

Geographic Distribution of Hepatitis C Virus Genotypes in Pakistan

Background: Distribution of Hepatitis C Virus (HCV) genotypes may be changed over time. Epidemiological Studies on distribution patterns of HCV genotypes in Pakistani population might assist for better treatment options and preventive strategies. Objectives: This study was conducted to determine distribution patterns of HCV genotypes in different geographical regions of Pakistan. Patients and Methods: In this cross-sectional study, 1818 randomly selected patients from different geographical regions of Pakistan, diagnosed with HCV infection by the third generation Enzyme Linked Immunosorbent Assay (ELISA), were included between April 2011 and December 2013. HCV RNA was detected in serum samples of patients by Reverse Transcription Polymerase Chain Reaction (RT- PCR) of the core region. Qualitative PCR was performed to determine viral load. HCV genotyping was performed by RT-nested PCR using type-specific primers of the core region. Frequency of different genotypes among patients was assessed according to gender, age and geographical region at the time of sampling. Results: Of 1818 HCV RNA positive samples, HCV genotypes PCR fragments were detected in 1552 (85.5%) samples. HCV genotype 3a was the predominant genotype (39.4%) followed by genotype 2a (24.93%). HCV genotype 3 was the predominant genotype in Punjab and Sindh regions, while genotype 2 was the most predominant genotype in Khyber Pakhtunkhwa region and the second predominant genotype after genotype 3 in Sindh region. The incidence of genotype 2a is increasing in our country with decrease in the incidence of genotype 3a. A higher incidence of HCV various genotypes were observed among male patients and those younger than 45 years. Conclusions: This study may facilitate treatment options and preventive strategies in Pakistan.

Thermophilic xylanases: from bench to bottle

Abstract Lignocellulosic biomass is a valuable raw material. As technology has evolved, industrial interest in new ways to take advantage of this raw material has grown. Biomass is treated with different microbial cells or enzymes under ideal industrial conditions to produce the desired products. Xylanases are the key enzymes that degrade the xylosidic linkages in the xylan backbone of the biomass, and commercial enzymes are categorized into different glycoside hydrolase families. Thermophilic microorganisms are excellent sources of industrially relevant thermostable enzymes that can withstand the harsh conditions of industrial processing. Thermostable xylanases display high-specific activity at elevated temperatures and distinguish themselves in biochemical properties, structures, and modes of action from their mesophilic counterparts. Natural xylanases can be further improved through genetic engineering. Rapid progress with genome editing, writing, and synthetic biological techniques have provided unlimited potential to produce thermophilic xylanases in their natural hosts or cell factories including bacteria, yeasts, and filamentous fungi. This review will discuss the biotechnological potential of xylanases from thermophilic microorganisms and the ways they are being optimized and produced for various industrial applications.

First report on molecular characterization of Leishmania species from cutaneous leishmaniasis patients in southern Khyber Pakhtunkhwa province of Pakistan

OBJECTIVE To report presence of Leishmania major in Khyber Pakhtunkhwa of Pakistan, where cutaneous leishmaniasis (CL) is endemic and was thought to be caused by Leishmania tropica only. METHODS Biopsy samples from 432 CL suspected patients were collected from 3 southern districts of Khyber Pakhtunkhwa during years 2011-2016. Microscopy on Giemsa stained slides were done followed by amplification of the ribosomal internal transcribed spacer 1 gene. RESULTS Leishmania amastigotes were detected by microscopy in 308 of 432 samples (71.3%) while 374 out of 432 samples (86.6%) were positive by ribosomal internal transcribed spacer 1 PCR. Subsequent restriction fragment length polymorphism confirmed L. tropica in 351 and L. major in 6 biopsy samples. CONCLUSIONS This study is the first molecular characterization of Leishmania species in southern Khyber Pakhtunkhwa. It confirmed the previous assumptions that anthroponotic CL is the major CL form present in Khyber Pakhtunkhwa province. Furthermore, this is the first report of L. major from a classical anthroponotic CL endemic focus identified in rural areas of Kohat district in southern Khyber Pakhtunkhwa.

Genome-Wide Identification of circRNAs in Pathogenic Basidiomycetous Yeast Cryptococcus neoformans Suggests Conserved circRNA Host Genes over Kingdoms

Circular RNAs (circRNAs), a novel class of ubiquitous and intriguing noncoding RNA, have been found in a number of eukaryotes but not yet basidiomycetes. In this study, we identified 73 circRNAs from 39.28 million filtered RNA reads from the basidiomycete Cryptococcus neoformans JEC21 using next-generation sequencing (NGS) and the bioinformatics tool circular RNA identification (CIRI). Furthermore, mapping of newly found circRNAs to the genome showed that 73.97% of the circRNAs originated from exonic regions, whereas 20.55% were from intergenic regions and 5.48% were from intronic regions. Enrichment analysis of circRNA host genes was conducted based on the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway databases. The results reveal that host genes are mainly responsible for primary metabolism and, interestingly, ribosomal protein production. Furthermore, we uncovered a high-level circRNA that was a transcript from the guanosine triphosphate (GTP)ase gene CNM01190 (gene ID: 3255052) in our yeast. Coincidentally, YPT5, CNM01190′s ortholog of the GTPase in Schizosaccharomyces pombe, protists, and humans, has already been proven to generate circRNAs. Additionally, overexpression of RNA debranching enzyme DBR1 had varied influence on the expression of circRNAs, indicating that multiple circRNA biosynthesis pathways exist in C. neoformans. Our study provides evidence for the existence of stable circRNAs in the opportunistic human pathogen C. neoformans and raises a question regarding their role related to pathogenesis in this yeast.

Identification of a basidiomycete-specific Vilse-like GTPase activating proteins (GAPs) and its roles in the production of virulence factors in Cryptococcus neoformans

ABSTRACT Cryptococcus neoformans is a basidiomycetous pathogenic yeast that causes fatal infections in both immunocompetent and immunocompromised patients. Regulation on the production of its virulence factors is not fully understood. Here we reported the characterization of a gene, named CVH1(CNA06260), encoding a Drosophila Vilse‐like RhoGAP homolog, which is hallmarked by three conserved functional domains: WW, MyTH4 and RhoGAP. Phylogenetic analysis suggests that CVH1 is highly conserved from protists to mammals and interestingly in basidiomycetes, but absent in plants or Ascomycota and other lower fungi. This phylogenetic distribution indicates an evolutionary link among these groups of organisms. Functional analyses demonstrated that CVH1 was involved in stress tolerance and virulence factor production. By disrupting CVH1, we created a second mutant cvh1&Dgr; with the CRISPR‐Cas9 editing tool. The mutant strain exhibited hypersensitivity to osmotic stress by 2 M sorbitol and NaCl, suggesting defects in the HOG signaling pathway and an interaction of Cvh1 with the HOG pathway. Hypersensitivity of cvh1&Dgr; to 1% Congo red and 0.01% SDS suggests that the cell wall integrity was impaired in the mutant. And cvh1&Dgr; hardly produced the pigment melanin and capsule. Our study for the first time demonstrates that the fungal Vilse‐like RhoGAP CVH1 is an important regulator of multiple biological processes in C. neoformans, and provides novel insights into the regulatory circuit of stress resistance/cell wall integrity, and laccase and capsule synthesis in C. neoformans. &NA; Graphical Abstract Figure. Identification of a basidiomycete‐specific Vilse‐like GTPase activating proteins (GAPs) and its roles in the production of virulence factors in Cryptococcus neoformans.

Characterization of dengue virus in Aedes aegypti and Aedes albopictus spp. of mosquitoes: A study in Khyber Pakhtunkhwa, Pakistan

Dengue is a vector-borne disease caused by dengue virus. According to the recent report of CDC that one-third population of the world are at high risk with Dengue fever. The prevalence of the dengue hemorrhagic fever was found more in tropical and sub-tropical regions of the world. Aedes mosquitoes was reported as the main cause of transmission of dengue virus. So the current study was planned to characterize the virus in Aedes mosquitoes collected from different area of Pakistan. In current investigation, Aedes mosquitoes and larvae were trapped under conducive conditions which are counted as 495 Aedes mosquitoes and 260 Aedes larvae. First of all, adult mosquitoes were identified morphologically under microscopy, counted as 73.3% Ae. aegypti and 26.7% Ae. albopictus. Finally, reverse transcriptase polymerase chain reaction analyses that only 4 adults of Aedes mosquitoes and 10 Aedes larvae as naturally infected with dengue virus with possible source Ae. aegypti. This study basically uncovered the presence of virus in different species of mosquitoes in southern regions of Pakistan. The present study will also give us an insight for vector control programs of dengue virus in the affected area.

Role of the fungus-specific flavin carrier Flc1 in antifungal resistance in the fungal pathogen Cryptococcus neoformans

FLC family, a conserved fungus-specific family of integral membrane proteins, has been demonstrated to play important roles in flavin transport, growth, and virulence in several fungi but not yet in Cryptococcus neoformans. In this study, we have identified the single homologue of flavin adenine dinucleotide transporter in the opportunistic pathogen C. neoformans. The computational and phylogenetic analysis confirmed the fungal specificity of cryptococcal Flc1 protein, thus providing a promising drug target for clinical treatment of cryptococcosis. Disruption of FLC1 conferred sensitivity to 1% Congo red and 0.02% SDS, as well as leading to impaired chitin distribution in cell wall as observed with Calcofluor White staining, which collectively indicated the roles of FLC1 in maintenance of cell wall integrity. Further investigations revealed the defects of flc1Δ mutant in resistance to poor nutrition and elevated temperatures, and the ability to undergo invasive growth under nutrient-depleted conditions was reduced as well in flc1Δ mutant, suggesting the roles of Flc1 in response to environmental stresses. More importantly, our results showed that flc1Δ mutant exhibited severe susceptibility to antifungal aminoglycosides (hygromycin B and geneticin) and amphotericin B, but developed multidrug resistance to flucytosine and rapamycin, which provided great hints for therapeutic failure of cryptococcosis in clinic with the standard combination therapy. Finally, typical virulence factors including melanin biosynthesis and capsule formation in flc1Δ mutant were reduced as well, indicating the possible involvement of Flc1 in virulence.

Characterization of Two Endo-β-1, 4-Xylanases from Myceliophthora thermophila and Their Saccharification Efficiencies, Synergistic with Commercial Cellulase

The xylanases with high specific activity and resistance to harsh conditions are of high practical value for biomass utilization. In the present study, two new GH11 xylanase genes, MYCTH_56237 and MYCTH_49824, have been cloned from thermophilic fungus Myceliophthora thermophila and expressed in Pichia pastoris. The specific activities of purified xylanases reach approximately 1,533.7 and 1,412.5 U/mg, respectively. Based on multiple template-based homology modeling, the structures of their catalytic domains are predicted. Enzyme activity was more effective in 7.5 L fermentor, yielding 2,010.4 and 2,004.2 U/mL, respectively. Both enzymes exhibit optimal activity at 60°C with pH of 6.0 and 7.0, respectively. Their activities are not affected by EDTA and an array of metal ions. The kinetic constants have been determined for MYCTH_56237 (Km = 8.80 mg/mL, Vmax = 2,380 U/mg) and MYCTH_49824 (Km = 5.67 mg/mL, Vmax = 1,750 U/mg). More importantly, both xylanases significantly cooperate with the commercial cellulase Celluclast 1.5 L in terms of the saccharification efficiency. All these biochemical properties of the xylanases offer practical potential for future applications.

Molecular detection of Leishmania species in human and animals from cutaneous leishmaniasis endemic areas of Waziristan, Khyber Pakhtunkhwa, Pakistan

Objectives: To detect Leishmania species in human patients, animal reservoirs and Phlebotomus sandflies in Waziristan, Pakistan. Methods: Tissue smears and aspirates from 448 cutaneous leishmaniasis (CL) suspected patients were analyzed. To sort out role of the reservoir hosts, skin scrapings, spleen and liver samples from 104 rodents were collected. Furthermore, buffy coat samples were obtained from 60 domestic animals. Sandflies were also trapped. All human, animals and sandfly samples were tested by microscopy, kinetoplastic PCR and internal transcribed spacer 1 (ITS1) PCR followed by restriction fragment length polymorphism for detection of Leishmania species. Results: An overall prevalence of 3.83% and 5.21% through microscopy and ITS1 PCR respectively was found. However, the statistically non-significant correlation was found between area, gender, and number of lesions. The presence of rodents, sandflies, domestic animals and internally displaced people increased the risk of CL. Using ITS1-PCR-RFLP, Leishmania tropica (L. tropica) was confirmed in 106 samples while 25 of the isolates were diagnosed as Leishmania major (L. major). Similarly, 3/104 rodents were positive for L. major and 14 pools of DNA samples containing Phlebotomus sergenti sandflies were positive for L. tropica. None of samples from domestic animals were positive for leishmaniasis. Conclusions: In the present study, L. tropica and L. major are found to be the main causative agents of CL in study area. Movement of internally displaced people from CL endemic areas presents a risk for nearby CL free areas. To the best of our knowledge, we report for the first time L. major infection in rodents (Rattus rattus) and L. tropica in Phlebotomus sergenti sandflies trapped in Waziristan, Pakistan.