miR-4716-3p and the target AKT2 Gene/rs2304186 SNP are associated with blood cancer pathogenesis in Pakistani population.

AKT2 is crucial for cancer cells' invasion, metastasis, and survival. It is a possible downstream gene target of cancer glycolysis-related microRNAs. The study investigated the role of miRNA-4716-3p, rs2304186, and the AKT2 gene in blood cancer pathogenesis. RT-qPCR was used to analyze AKT2 gene mRNA and miRNA-4716-3p expression in 200 blood cancer samples and 200 healthy controls. Furthermore, Tetra-ARMS PCR was used to examine the rs2304186 AKT2 SNP in 300 patients and 290 control samples. miRNA-4716-3p was shown to be significantly downregulated (p = 0.0294), whereas mRNA expression of the AKT2 gene was found to be significantly upregulated (p = 0.0034) in blood cancer patients compared to healthy individuals. miRNA-4716-3p downregulation (p = 0.0466) was more pronounced, while AKT2 upregulation was non-significant (p = 0.1661) in untreated patients compared to chemotherapy-treated patients. Blood cancer risk was significantly associated with the rs2304186 GT genotype (p = 0.0432), TT genotype (p = 0.0502), and mutant allele (T) frequency (p = 0.0008). Polymorphism rs2304186 was associated with an increased risk of blood cancer in dominant (p = 0.0011), recessive (p = 0.0502), and additive (p = 0.0008) genetic models. The results suggested that the rs2304186 and the deregulated expression of miRNA-4716-3p and AKT2 gene at the mRNA level may significantly increase the incidence of blood cancer, particularly in the Pakistani population. Therefore, these may function as suitable biomarkers for blood cancer diagnosis and prognosis. Additional, larger-scale investigations may be required to affirm these results.

Autophagy related genes mediated mitophagy in yeast, mammals and higher plants.

Autophagy is classified into macro-autophagy and micro-autophagy. Two major types of autophagy in the complex eukaryotic organism are microautophagy and macroautophagy. During microautophagy, cytoplasmic components that need to be degraded are taken up by lysosomes in animals and by vacuole in yeast and plants via the invagination of tonoplast. While macroautophagy is initiated after the formation of a cup-shaped membrane structure, a phagophore develops at cargo that grows in size and is sealed by double-membrane vesicles to form autophagosome; a generalized mechanism for degradation of the organelle. Autophagic removal of damaged mitochondria is a conserved cellular process to maintain a healthy mitochondrion called Mitophagy. In plants and animals, mitophagy has crucial roles in stress responses, senescence, development, and programmed cell death. Mitophagy appears in mammals, fungi, and plants but many genes that controlled mitophagy are absent from plants. Numerous studies have been conducted by using ATG mutants for the identification of functional roles of Autophagy Related Genes (ATG) required during the autophagy process at various steps like; auto phagosome formation, ATG protein recruitment, etc. The role of more than 25 ATG genes in mitophagy has been discussed in this review paper. The main parameters, reviewed and summarized in this review paper, are the name of species, common name, function, domain, deletion, induction, and localization of these autophagy-related genes in the cell. This review will facilitate the students, researchers, and academics for their further research insights.

miRNA-767 and its binding site polymorphism in the mTOR gene act as potential biomarkers for female reproductive cancers.

Aims: The present study aimed to understand the relationship between the mTOR gene SNP (rs2536) and reproductive cancer risk. The expression level of miRNA-767 was also assessed. Methods: 700 tumor samples (300 breast, 200 ovarian and 200 cervical cancers), along with adjacent uninvolved control tissue, were used. rs2536 was screened using Tetra-ARMS PCR and expression level of miRNA-767 was assessed using quantitative PCR. Results: The frequency of the homozygous mutant genotype of rs2536 was observed significantly higher in breast (p < 0.04), ovarian (p < 0.005) and cervical (p < 0.003) cancers. Significant downregulation of miRNA-767 was observed in tumors compared with controls. Conclusion: The present study demonstrates that increased mutant frequency of rs2536 and deregulation of miRNA-767 are associated with increased reproductive cancer risk.

Role of exosomal miRNA-19a/19b and PTEN in brain tumor diagnosis.

Aim: The current study was designed to evaluate the diagnostic significance of the exosomal miRNAs miR-19a and miR-19b and the PTEN gene in brain tumor patients versus controls. Methods: Exosomes were extracted from the serum samples of 400 brain tumor patients and 400 healthy controls. The exosomes were characterized by scanning electron microscopy, dynamic light scattering and ELISA. Quantitative PCR was used to analyze selected exosome miRNAs and gene expression levels. Results: Analysis showed significant deregulated expression of miR-19a (p < 0.0001), miR-19b (p < 0.0001) and PTEN (p < 0.001) in patients versus controls. Spearman correlation showed a significant correlation among the selected exosomal miRNAs and the PTEN gene. Conclusion: Receiver operating characteristic curve analysis showed the good diagnostic value of exosomal miRNAs and the PTEN gene in brain tumor patients.

Expression variations of DNA damage response genes ATM and ATR in blood cancer patients.

Hematological malignancies (HM) constitute a variety of cancers originating in blood, bone marrow (BM), and lymphatic systems. During the last two decades, the incidence of HM has dramatically increased worldwide. The etiology of HM is still debatable. Genetic instability is a major risk factor for HM. DDR network is a complex signal transduction cellular machinery that detects DNA damage and activates cellular repair factors, thus maintaining genomic integrity. DDR network detects a variety of DNA damage and triggers the activation of cell cycle control, DNA repair, senescence, and apoptosis. Among the DNA repairing pathways, the DNA damage response (DDR) pathway includes DNA damage signaling apparatus such as ATM and ATR genes. ATM tends to detect double-strand breaks (DSBs) while ATR detects single-strand DNA (ssDNA). The study was conducted to observe the expression deregulations of DNA damage response (DDR) pathway genes (ATM, ATR) at mRNA level in 200 blood cancer patients and 200 controls. The real-time PCR was used to analyze the expression of the target genes. The expression results showed statistically significant downregulation of ATM (p < 0.0001) and ATR (p < 0.0001) genes in blood cancer patients vs. controls. Moreover, a significant downregulation of ATM (p < 0.0001) and ATR (p < 0.0001) was obtained in chemotherapy-treated patients vs. healthy controls. The results suggest that dysregulation in ATM and ATR genes may be associated with increased blood cancer risk.

Fyn and Lyn gene polymorphisms impact the risk of thyroid cancer.

Thyroid cancer is the most common malignancy of the endocrine glands, and during last couple of decades, its incidence has risen alarmingly, across the globe. Etiology of thyroid cancer is still debatable. There are a few worth mentioning risk factors which contribute to initiation of abnormalities in thyroid gland leading to cancer. Genetic instability is major risk factors in thyroid carcinogenesis. Among the genetic factors, the Src family of genes (Src, Yes1, Fyn and Lyn) have been implicated in many cancers but there is little data regarding the association of these (Src, Yes1, Fyn and Lyn) genes with thyroid carcinogenesis. Fyn and Lyn genes of Src family found engaged in proliferation, migration, invasion, angiogenesis, and metastasis in different cancers. This study was planned to examine the effect of Fyn and Lyn SNPs on thyroid cancer risk in Pakistani population in 500 patients and 500 controls. Three polymorphisms of Fyn gene (rs6916861, rs2182644 and rs12910) and three polymorphisms of Lyn gene (rs2668011, rs45587541 and rs45489500) were analyzed using Tetra-primer ARMS-PCR followed by DNA sequencing. SNP rs6916861 of Fyn gene mutant genotype (CC) showed statistically significant threefold increased risk of thyroid cancer (P < 0.0001). In case of rs2182644 of Fyn gene, mutant genotype (AA) indicated statistically significant 17-fold increased risk of thyroid cancer (P < 0.0001). Statistically significant threefold increased risk of thyroid cancer was observed in genotype AC (P < 0.0001) of Fyn gene polymorphism rs12910. In SNP rs2668011 of Lyn gene, TT genotype showed statistically significant threefold increased risk of thyroid cancer (P < 0.0001). In case of rs45587541 of Lyn gene, GA genotypes showed statistically significant 11-fold increased risk in thyroid cancer (P < 0.0001). Haplotype analysis revealed that AAATAG*, AGACAG*, AGCCAA*, AGCCAG*, CAATAG*, CGCCAG* and CGCCGA* haplotypes of Fyn and Lyn polymorphisms are associated with increased thyroid cancer risk. These results showed that genotypes and allele distribution of Fyn and Lyn are significantly linked with increased thyroid cancer risk and could be genetic adjuster for said disease.

Role of Chk1 gene in molecular classification and prognosis of gastric cancer using immunohistochemistry and LORD-Q assay.

Purpose: The aim of the current study was to assess the prognostic value of the Chk1 gene in the DNA damage response pathway in gastric cancer (GC). Methods: Expression levels of the Chk1 were measured in 220 GC tumor tissues and adjacent healthy/noncancerous tissues using real-time PCR and immunohistochemical staining. Genomic instability in GC patients was measured using the long-run real-time PCR technique for DNA-damage quantification assay and comet assay. Results: Significantly downregulated expression of Chk1 was observed at the mRNA level (p < 0.0001) and protein level (p < 0.0001). Significantly increased frequency of lesions/10 kb and comets was observed in tumor tissues compared with control tissues. Conclusion: The data suggest that downregulated expression of Chk1 and positive Heliobacter pylori infection status may have prognostic significance in GC.

In silico analysis of quranic and prophetic medicinals plants for the treatment of infectious viral diseases including corona virus.

Coronavirus disease (COVID-19) is an infection of the respiratory system caused by single standard RNA viruses named as Severe Acute Respiratory Syndrome 2 (SARS-CoV-2). The disease appeared as a serious problem and the leading cause of death in human beings throughout the world. The main source of different phytochemicals are plants, which helps in the development of new drugs against various ailments. Islam is comprehensive religion and a complete code of life for Muslims. The teaching of Islam, according to the Holy Quran and Hadith are universal for the benefit of humanity. Islam believes that every ailment is from God and who made the disease definitely made its medication. There is a complete guideline with regard to taking measures against infectious diseases such as quarantine and seeking medicinal treatment. The research objective is to gather the knowledge of medicinal plants described in the Holy Quran or utilized by the Prophet (SAW) for the treatment of different ailments or advised to use them to boost immunity and strengthen the body. Scientists across the globe have found these plants beneficial for many diseases and have antiviral potential. In present study, the six plant species including Olea europaea, Nigella sativa, Allium Sativum, Allium cepa, Zingiber officinale and Cassia senna were selected which contain phytochemicals like Calcium Elenolate, Thymoquinone, S-Allylcysteine, Dipropyl Disulfide, Sesquiterpene, Monoterpene, Pelargonidin 3-Galactoside ion and Kaempferol. The phytochemicals monoterpene (from Zingiber officinale) shows best interaction with target proteins RdRP, 3CLPro, ACE2. Calcium Elonate (from olive) bonds with 3CLPro, ACE2 and Kemoferol and Pelargomidine (from Senna Makki) bonds with RdRP, ACE2. The ligands show a unique set of intersections i.e. hydrogen bonding, and alkyl interaction. These medicinal plants can be utilized immediately for the treatment of COVID-19 as their safety is already established. This treatment can enhance recovery when combined with other treatments. Furthermore, the screening of bioactive compounds or phytochemicals found in these plants can be utilized to design new therapeutic drug to treat COVID-19 pandemic.

In-silico network-based analysis of drugs used against COVID-19: Human well-being study.

INTRODUCTION: Researchers worldwide with great endeavor searching and repurpose drugs might be potentially useful in fighting newly emerged coronavirus. These drugs show inhibition but also show side effects and complications too. On December 27, 2020, 80,926,235 cases have been reported worldwide. Specifically, in Pakistan, 471,335 has been reported with inconsiderable deaths. PROBLEM STATEMENT: Identification of COVID-19 drugs pathway through drug-gene and gene-gene interaction to find out the most important genes involved in the pathway to deal with the actual cause of side effects beyond the beneficent effects of the drugs. METHODOLOGY: The medicines used to treat COVID-19 are retrieved from the Drug Bank. The drug-gene interaction was performed using the Drug Gene Interaction Database to check the relation between the genes and the drugs. The networks of genes are developed by Gene MANIA, while Cytoscape is used to check the active functional association of the targeted gene. The developed systems cross-validated using the EnrichNet tool and identify drug genes' concerned pathways using Reactome and STRING. RESULTS: Five drugs Azithromycin, Bevacizumab, CQ, HCQ, and Lopinavir, are retrieved. The drug-gene interaction shows several genes that are targeted by the drug. Gene MANIA interaction network shows the functional association of the genes like co-expression, physical interaction, predicted, genetic interaction, co-localization, and shared protein domains. CONCLUSION: Our study suggests the pathways for each drug in which targeted genes and medicines play a crucial role, which will help experts in-vitro overcome and deal with the side effects of these drugs, as we find out the in-silico gene analysis for the COVID-19 drugs.

Proteomic Analysis of Medicinal Plant Calotropis Gigantea by In Silico Peptide Mass Fingerprinting.

Medicinal plants are the basic source of medicinal compounds traditionally used for the treatment of human diseases. Calotropis gigantea is a medicinal plant belonging to the family of Apocynaceae in the plant kingdom and subfamily Asclepiadaceae usually bearing multiple medicinal properties to cure a variety of diseases. BACKGROUND: The Peptide Mass Fingerprinting (PMF) identifies the proteins from a reference protein database by comparing the amino acid sequence that is previously stored in the database and identified. OBJECTIVE: The purpose of the study is to identify the peptides having anti-cancerous properties by in silico peptide mass fingerprinting. METHODS: The calculation of in silico peptide masses is done through the ExPASy PeptideMass and these masses are used to identify the peptides from the MASCOT online server. Anticancer probability is calculated by iACP server, docking of active peptides is done by CABS-dock the server. RESULTS: The anti-cancer peptides are identified with the MASCOT peptide mass fingerprinting server, the identified peptides are screened and only the anti-cancer are selected. De-novo peptide structure prediction is used for 3D structure prediction by PEP-FOLD 3 server. The docking results confirm strong bonding with the interacting amino acids of the receptor protein of breast cancer BRCA1 which shows the best peptide binding to the active chain, the human leukemia protein docking with peptides shows the accurate binding. CONCLUSION: These peptides are stable and functional and are the best way for the treatment of cancer and many other deadly diseases.

nCOV-19 peptides mass fingerprinting identification, binding, and blocking of inhibitors flavonoids and anthraquinone of Moringa oleifera and hydroxychloroquine.

An rare pandemic of viral pneumonia occurs in December 2019 in Wuhan, China, which is now recognized internationally as Corona Virus Disease 2019 (COVID-19), the etiological agent classified as Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2). According to the World Health Organization (WHO), it has so far expanded to more than 213 countries/territories worldwide. Our study aims to find the viral peptides of SARS-COV-2 by peptide mass fingerprinting (PMF) in order to predict its novel structure and find an inhibitor for each viral peptide. For this reason, we calculated the mass of amino acid sequences translated from the SARS-CoV2 whole genome and identify the peptides that may be a target for inhibition. Molecular peptide docking with Moringa oleifera, phytochemicals (aqueous and ethanolic) leaf extracts of flavonoids (3.56 ± 0.03), (3.83 ± 0.02), anthraquinone (11.68 ± 0.04), (10.86 ± 0.06) and hydroxychloroquine present therapy of COVID-19 in Pakistan for comparative study. Results indicate that 15 peptides of SARS-CoV2 have been identified from PMF, which is then used as a selective inhibitor. The maximum energy obtained from AutoDock Vina for hydroxychloroquine is -5.1 kcal/mol, kaempferol (flavonoid) is -6.2 kcal/mol, and for anthraquinone -6 kcal/mol. Visualization of docking complex, important effects are observed regarding the binding of peptides to drug compounds. In conclusion, it is proposed that these compounds are effective antiviral agents against COVID-19 and can be used in clinical trials.Communicated by Ramaswamy H. Sarma.

Association between single nucleotide polymorphisms of DNA damage response pathway genes and increased risk in breast cancer.

Aim: We aimed to evaluate the role of selected single nucleotide polymorphisms of DNA damage response pathway genes in breast cancer (BC). Materials & methods: In present study, 500 BC patients and 500 controls was used to estimate the frequency of single nucleotide polymorphisms of DNA damage response pathway genes. Tetra-amplification refractory mutation system-PCR technique was used for screening of the six selected polymorphisms. Results: Logistic regression analysis showed that heterozygous mutant genotype of rs1800057 (p < 0.0001) and homozygous mutant genotype of rs1801516 (p < 0.0001) was associated with significant increased risk of BC. In the ATR gene, heterozygous mutant genotype of rs2227931 (p < 0.0001) was associated with significant increased risk of BC. However, significant decreased risk of BC was found associated with heterozygous mutant genotype of rs2227928 (p < 0.0002) and homozygous mutant genotype of rs2229032 (p < 0.0001) in patients compared with controls. Conclusion: The present results showed that alteration in DNA damage response pathway gene (ATM & ATR) results in increased BC risk.

Linkage disequilibrium and haplotype analysis of Src and Yes1 genes in thyroid cancer.

Purpose: This study was planned to examine the effects of Src and Yes1 single nucleotide polymorphism (SNPs) on the risk of thyroid cancer in 499 patients and 500 controls. Materials & methods: Three SNPs of Src gene and three SNPs of Yes1 gene were analyzed using Tetra-primer ARMS-PCR followed by sequencing. Results: rs121913314 of Src gene genotype TT showed 32-fold increased risk of thyroid cancer and rs2305994 of Yes1 genotypes TT and CT showed 2.7-fold and 16-fold increased risk in thyroid cancer (p < 0.0001). Haplotype analysis revealed that CATGCC, CATGCT, CATGTC, CATGTT, TATGCC and TATGTTA haplotypes are associated with thyroid cancer risk. Conclusion: Results showed that genotypes and allele distribution of Src and Yes1 genes are significantly linked with increased risk of thyroid cancer.

In silico T-cell and B-cell Epitope Based Vaccine Design Against Alphavirus Strain of Chikungunya.

BACKGROUND: Chikungunya an arbovirus, is transmitted to humans by the bite of Aedes mosquito. The virus occurrences have been reported in Southeast Asian countries including Pakistan. Its symptoms include typical febrile illness and arthralgic syndrome. The virus has not decisively proved to be life-threatening. METHODS: The attempt was to design T-cell and B-cell epitope-based vaccine for Chikungunya. The proteome of chikungunya was retrieved, antigenic proteins were identified and T-cell epitopes and B-cell epitopes were predicted. Interacting HLA alleles were also identified. The final analysis was done to confirm that predicted T-cell epitopes and B-cell epitopes can be used as a vaccine. RESULTS: About 32 T-cell epitopes and a 10mer B-cell epitope were identified. Both T-cell and Bcell epitopes demonstrated strong interactions with HLA alleles. The predicted T-cell and B-cell epitopes were docked with respective HLA alleles. The docking analysis showed that the predicted respective epitopes best fit into the binding pockets of the alleles. CONCLUSION: On the basis of this computational analysis, it is suggested that these predicted epitopes can be used as a remedy against Alphavirus strain of chikungunya. Further laboratory experiments can be conducted to determine the efficacy and stability of this work.

Subtractive Proteome Mining Approach towards Unique Putative Drug Targets Identification for Salmonella typhimurium.

BACKGROUND: Salmonella typhimurium is a rod-shaped bacteria with a Gram-negative genus, belonging to the Enterobacteriaceae family of microbes, which invades the intestinal lumen of Human. Salmonella typhimurium is a root source, accounting for gastroenteritis in humans as well as in other mammals. Gastroenteritisis associated with Salmonella Typhimurium interacts with the contaminated food and water and spreads to nearby people in the area. Small intestine is attacked by Salmonella, which then enter into the bloodstream momentarily, and are responsible for millions of mortalities and morbidities around the globe. Salmonella typhimurium toxins cause gastrointestiritis due to inflammation in the stomach and intestine in infants and young children. It accounts for millions of deaths with a higher incidence rate in developing countries. METHODS: In the current research, subtractive proteome mining has been done to recognize putative drug targets. The proteome was analyzed through blast in order to exclude homologous proteins. Bacterial essential proteins were predicted and the participation of the essential genes in the metabolic pathways has been analyzed. RESULTS: 36 essential genes and 15 unique pathways have been identified as potential drug targets among the total of 1934 proteins. The location of proteins is determined as an outer membrane. 3 proteins out of 36 essential proteins are recognized as putative drug targets. CONCLUSION: In the future, virtual screening for the evaluation of novel clinical compounds for the identified proteins will be effective and valuable for Salmonella Typhimurium infection in Homo sapiens.

Relationship of single nucleotide polymorphisms and haplotype interaction of mitochondrial unfolded protein response pathway genes with head and neck cancer.

Aim: In this study, we evaluated the effect of selected polymorphisms of mitochondrial unfolded protein response (UPRmt) pathway in 500 head and neck cancer (HNC) patients and 500 healthy controls from Pakistan. Materials & methods: The experiments were conducted using tetra-ARMS PCR followed by DNA sequencing. Results: Multivariate analysis showed that AA genotype of rs3782116 showed fivefold, GG genotype of rs6598072 approximately twofold and CC genotype of rs4946936 and TT genotype of rs12212067 showed twofold increased risk of HNC. Furthermore, haplotype analysis showed that certain haplotypes of UPRmt pathway single nucleotide polymorphisms have significant association with increased HNC risk. Conclusion: These results show that genetic aberrations in UPRmt pathway genes have association with increased HNC risk and can be an indicator of advance clinical outcome especially invasion and metastasis.

Author Correction: Effect of Plant Density, Boron Nutrition and Growth Regulation on Seed Mass, Emergence and Offspring Growth Plasticity in Cotton.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Effect of Plant Density, Boron Nutrition and Growth Regulation on Seed Mass, Emergence and Offspring Growth Plasticity in Cotton.

Seed nutrients reserves have direct relationship with seed functional traits and influence offspring performance. Effects of plant density, foliage boron (B) nutrition and mepiquat chloride (MC) growth regulation on seed nutrients reserves, seed mass and production, and emergence and offspring growth traits of cotton were studied in two years field experiment. Seed nutrients reserves and seed mass were decreased at higher maternal plant density relative to lower plant density with concomitant decrease in emergence and offspring seedling growth. However, maternal foliage B nutrition and MC growth regulation enhanced seed nutrients reserves, seed mass, emergence and offspring seedling growth performance. There was a significant positive relationship between seed mass and seed nutrients reserves indicating that changes in nutrient availability/uptake in response to maternal ecological factors determine variation in seed functional traits. Nonetheless, seed mass was positively correlated with emergence percentage and negatively with emergence timing. Furthermore, variation in offspring seedling growth traits with seed mass indicated the significance of initial seed nutrients reserves for early seedling vigour and establishment. In conclusion, lower maternal plant density, B nutrition and MC growth regulation ensued in higher emergence and offspring seedling growth of cotton because of higher seed nutrient reserves and seed mass.

Identification of phytotoxins in different plant parts of Brassica napus and their influence on mung bean.

Plants in Brassica genus have been found to possess strong allelopathic potential. They may inhibit seed germination and emergence of subsequent crops following them in a rotation system. Series of laboratory and greenhouse experiments were conducted to determine the allelopathic impacts of Brassica napus L. against mung bean. We studied (1) the effects of aqueous extract (5%) of different plant parts (root, stem, leaf, flower, and whole plant) of B. napus, (2) the effects of leaf and flower extracts of B. napus at 0, 1, 2, 3, and 4% concentrations, and (3) the effect of residues of different B. napus plant parts and decomposition periods (0, 7, 14, and 21 days) on germination and seedling growth of mung bean. Various types of phenolics including quercitin, chlorogenic acid, p-coumeric acid, m-coumaric acid, benzoic acid, caffeic acid, syringic acid, vanillic acid, ferulic acid, cinamic acid, and gallic acid were identified in plant parts of B. napus. Among aqueous extracts of various plant parts, leaf and flower were found to have stronger inhibitory effects on germination and seedling growth traits of mung bean, higher concentrations were more toxic. The decomposition period changed the phtotoxic effect of residues, more inhibitory effect was shown at 14 days decomposition while decomposition for 21 days reduced inhibitory effect. The more total water-soluble phenolic was found in 5% (w/v) aqueous extract and 5% (w/w) residues of B. napus flowers at 14 days of decomposition (89.80 and 10.47 mg L-1), respectively. The strong inhibitory effects of B. napus should be managed when followed in rotation.

The Absence of HCV RNA and NS5A Protein in Peripheral Blood Mononuclear Cells Is a Prognostic Tool for Sustained Virological Response.

Hepatitis C Virus (HCV) infection is a major health concern worldwide. The presence of both HCV viral RNA and NS5A proteins in peripheral blood mononuclear cells (PBMCs) indicate the efficacy of the treatment during sustained virological response (SVR) and end of treatment response (ETR). The main objective of this study was to detect the absence or presence of HCV RNA and NS5A proteins in PBMCs. Blood samples were taken from selected patients (Islamabad, Pakistan) before treatment, at ETR, and during SVR. Two hundred HCV responders to pegylated IFN-α-2a plus ribavirin were selected. HCV RNA was extracted from the patients to determine the viral load by reverse transcription (RT)-polymerase chain reaction before treatment. Out of 200 patients, 152 (76%) and 48 (24%) achieved positive and negative ETR, respectively. Among ETR patients, 134 (88.2%) showed SVR, whereas 18 (11.8%) displayed relapse. The male to female ratio was 92:108 with mean age of 37.4 years. Among 152 ETR-positive patients, 29 (19%) patients' PBMCs were positive for HCV RNA and 27 (17.8%) were positive for NS55A proteins. Patients having HCV RNA in PBMCs showed higher relapse frequency compared with patients lacking it. Similarly, patients having NS5A protein showed significantly higher relapse frequency compared with patients lacking it. All PBMC-positive samples were of genotype 3a. In addition, patients with positive NS5A in their PBMCs showed greater risk of relapse compared with patients having HCV RNA. We conclude that the absence of both viral HCV and proteins can be used as an indicator for diagnosis of SVR in the future.