Exploring clinical implications and role of non-coding RNAs in lung carcinogenesis.

Lung cancer is the utmost familiar category of cancer with greatest fatality rate worldwide and several regulatory mechanisms exercise cellular control on critical oncogenic trails implicated in lung associated carcinogenesis. The non-coding RNAs (ncRNAs) are shown to play a variety of regulatory roles, including stimulating cell proliferation, inhibiting programmed cell death, enhancing cancer cell metastatic ability and acquiring resistance to drugs. Furthermore, ncRNAs exhibit tissue-specific expression as well as great stability in bodily fluids. As a consequence, they are strong contenders for cancer based theragnostics. microRNA (miRNA) alters gene expression primarily by either degrading or interfering with the translation of targeted mRNA and long non-coding RNAs (lncRNAs) can influence gene expression by targeting transcriptional activators or repressors, RNA polymers and even DNA-duplex. lncRNAs are typically found to be dysregulated in lung cancer and hence targeting ncRNAs could be a viable strategy for developing potential therapies as well as for overcoming chemoresistance in lung cancer. The purpose of this review is to elucidate the role of ncRNAs, revisiting the recent studies in lung cancer.

Proviral ALV-LTR Sequence Is Essential for Continued Proliferation of the ALV-Transformed B Cell Line.

Avian leukosis virus (ALV) induces B-cell lymphomas and other malignancies in chickens through insertional activation of oncogenes, and c-myc activation has been commonly identified in ALV-induced tumors. Using ALV-transformed B-lymphoma-derived HP45 cell line, we applied in situ CRISPR-Cas9 editing of integrated proviral long terminal repeat (LTR) to examine the effects on gene expression and cell proliferation. Targeted deletion of LTR resulted in significant reduction in expression of a number of LTR-regulated genes including c-myc. LTR deletion also induced apoptosis of HP45 cells, affecting their proliferation, demonstrating the significance of LTR-mediated regulation of critical genes. Compared to the global effects on expression and functions of multiple genes in LTR-deleted cells, deletion of c-myc had a major effect on the HP45 cells proliferation with the phenotype similar to the LTR deletion, demonstrating the significance of c-myc expression in ALV-induced lymphomagenesis. Overall, our studies have not only shown the potential of targeted editing of the LTR for the global inhibition of retrovirus-induced transformation, but also have provided insights into the roles of LTR-regulated genes in ALV-induced neoplastic transformation.

Impact of spliceosome mutations on RNA splicing in myelodysplasia: dysregulated genes/pathways and clinical associations.

SF3B1, SRSF2, and U2AF1 are the most frequently mutated splicing factor genes in the myelodysplastic syndromes (MDS). We have performed a comprehensive and systematic analysis to determine the effect of these commonly mutated splicing factors on pre-mRNA splicing in the bone marrow stem/progenitor cells and in the erythroid and myeloid precursors in splicing factor mutant MDS. Using RNA-seq, we determined the aberrantly spliced genes and dysregulated pathways in CD34+ cells of 84 patients with MDS. Splicing factor mutations result in different alterations in splicing and largely affect different genes, but these converge in common dysregulated pathways and cellular processes, focused on RNA splicing, protein synthesis, and mitochondrial dysfunction, suggesting common mechanisms of action in MDS. Many of these dysregulated pathways and cellular processes can be linked to the known disease pathophysiology associated with splicing factor mutations in MDS, whereas several others have not been previously associated with MDS, such as sirtuin signaling. We identified aberrantly spliced events associated with clinical variables, and isoforms that independently predict survival in MDS and implicate dysregulation of focal adhesion and extracellular exosomes as drivers of poor survival. Aberrantly spliced genes and dysregulated pathways were identified in the MDS-affected lineages in splicing factor mutant MDS. Functional studies demonstrated that knockdown of the mitosis regulators SEPT2 and AKAP8, aberrantly spliced target genes of SF3B1 and SRSF2 mutations, respectively, led to impaired erythroid cell growth and differentiation. This study illuminates the effect of the common spliceosome mutations on the MDS phenotype and provides novel insights into disease pathophysiology.

Portable Self-Powered Piezoelectric Nanogenerator and Self-Charging Photo-Power Pack Using In Situ Formed Multifunctional Calcium Phosphate Nanorod-Doped PVDF Films.

Herein, biocompatible Ca3(PO4)2 nanorod-incorporated poly(vinylidene) difluoride films have been prepared via an in situ process. A good piezoelectricity (d33 ≈ 56.6 pC/N) along with a large dielectric constant of ∼3.48 × 105 at frequency 20 Hz has been achieved. Then, we have designed a biocompatible, highly durable, low-cost piezoelectric nanogenerator (CPNG) which shows the superiority in open-circuit voltage ∼47 V and current ∼1.8 µA generation with power density ∼47.4 mW cm-3 under the gentle touch of a finger. Excellent mechanical to electrical energy conversion efficiency (∼65.5%) of our developed CPNG leads to fast charging of a capacitor of 1 µF in 18 s and glowing of 26 light-emitting diodes (LEDs) under finger impartation. Further, a portable light-charging power pack (LCPP) has been developed using the high dielectric film as the storage function. Under light illumination, our LCPP generates open-circuit output voltage ∼1.29 V with short-circuit current 5.7 mA cm-2. Areal capacitance ∼1779 F m-2 and storage efficiency ∼88% are achieved. The device is able to lighten up 22 LEDs for 10 days after charging once.

Photo-Rechargeable Organic-Inorganic Dye-Integrated Polymeric Power Cell with Superior Performance and Durability.

In the present work, we propose a simple and unique approach to design a lightweight, low-cost, self-charging power cell with considerable capacity to generate and store photocharges named self-charged photo-power cell (SCPPC). Initially, highly electroactive sodium dodecyl sulfate (SDS)-incorporated poly(vinylidene fluoride) (PVDF) composite thin films with a large dielectric constant of ∼525 are synthesized via a simplistic solution casting process. Then, the as-prepared high-dielectric SDS/PVDF thin film is used as a charge-storage medium in combination with an inorganic-organic dye film, i.e., ZnO nanoparticles-eosin Y-poly(vinylpyrrolidone) film, as a photoelectron generator in our SCPPC. An open-circuit voltage of ∼1.2 V is attained after charging SCPPC under illumination light with intensity ∼110 mW/cm2 and then discharging fully with a constant current density of ∼4.5 mA/cm2. A specific areal capacitance of ∼450 F/m2 is obtained with large energy and power densities of ∼90 mWh/m2 and 54 W/m2, respectively. The improved overall efficiency, ∼3.78%, along with 89% storage efficiency leads to promising application possibilities of our rechargeable photo-power cell. The recyclability, i.e., rechargeability and storage durability, of the photo-power cell are also checked for 35 days without no such reduction in voltage generation and storage. Also, multicolored light-emitting diodes are lightened up using the photo-power cell as power source.

Learning a reactive potential for silica-water through uncertainty attribution.

The reactivity of silicates in aqueous solution is relevant to various chemistries ranging from silicate minerals in geology, to the C-S-H phase in cement, nanoporous zeolite catalysts, or highly porous precipitated silica. While simulations of chemical reactions can provide insight at the molecular level, balancing accuracy and scale in reactive simulations in the condensed phase is a challenge. Here, we demonstrate how a machine-learning reactive interatomic potential trained on PaiNN architecture can accurately capture silicate-water reactivity. The model was trained on a dataset comprising 400,000 energies and forces of molecular clusters at the ωB97X-D3/def2-TZVP level. To ensure the robustness of the model, we introduce a general active learning strategy based on the attribution of the model uncertainty, that automatically isolates uncertain regions of bulk simulations to be calculated as small-sized clusters. The potential reproduces static and dynamic properties of liquid water and solid crystalline silicates, despite having been trained exclusively on cluster data. Furthermore, we utilize enhanced sampling simulations to recover the self-ionization reactivity of water accurately, and the acidity of silicate oligomers, and lastly study the silicate dimerization reaction in a water solution at neutral conditions and find that the reaction occurs through a flanking mechanism.

BCR-ABL1 tyrosine kinase sustained MECOM expression in chronic myeloid leukaemia.

MECOM oncogene expression correlates with chronic myeloid leukaemia (CML) progression. Here we show that the knockdown of MECOM (E) and MECOM (ME) isoforms reduces cell division at low cell density, inhibits colony-forming cells by 34% and moderately reduces BCR-ABL1 mRNA and protein expression but not tyrosine kinase catalytic activity in K562 cells. We also show that both E and ME are expressed in CD34(+) selected cells of both CML chronic phase (CML-CP), and non-CML (normal) origin. Furthermore, MECOM mRNA and protein expression were repressed by imatinib mesylate treatment of CML-CP CD34(+) cells, K562 and KY01 cell lines whereas imatinib had no effect in non-CML BCR-ABL1 -ve CD34(+) cells. Together these results suggest that BCR-ABL1 tyrosine kinase catalytic activity regulates MECOM gene expression in CML-CP progenitor cells and that the BCR-ABL1 oncoprotein partially mediates its biological activity through MECOM. MECOM gene expression in CML-CP progenitor cells would provide an in vivo selective advantage, contributing to CML pathogenesis.

Enabling Fast Na+ Transfer Kinetics in the Whole-Voltage-Region of Hard-Carbon Anodes for Ultrahigh-Rate Sodium Storage.

Efficient electrode materials, that combine high power and high energy, are the crucial requisites of sodium-ion batteries (SIBs), which have unwrapped new possibilities in the areas of grid-scale energy storage. Hard carbons (HCs) are considered as the leading candidate anode materials for SIBs, however, the primary challenge of slow charge-transfer kinetics at the low potential region (<0.1 V) remains unresolved till date, and the underlying structure-performance correlation is under debate. Herein, ultrafast sodium storage in the whole-voltage-region (0.01-2 V), with the Na+ diffusion coefficient enhanced by 2 orders of magnitude (≈10-7 cm2 s-1 ) through rationally deploying the physical parameters of HCs using a ZnO-assisted bulk etching strategy is reported. It is unveiled that the Na+ adsorption energy (Ea ) and diffusion barrier (Eb ) are in a positive and negative linear relationship with the carbon p-band center, respectively, and balance of Ea and Eb is critical in enhancing the charge-storage kinetics. The charge-storage mechanism in HCs is evidenced through comprehensive in(ex) situ techniques. The as prepared HCs microspheres deliver a record high rate performance of 107 mAh g-1 @ 50 A g-1 and unprecedented electrochemical performance at extremely low temperature (426 mAh g-1 @ -40 °C).

Recent Progress in Amorphous Carbon-Based Materials for Anodes of Sodium-Ion Batteries: Synthesis Strategies, Mechanisms, and Performance.

Sodium-ion batteries (SIBs) are gaining renewed interest as a promising alternative to the already commercialized lithium-ion batteries. The large abundance, low cost, and similar electrochemistry of sodium (compared with lithium) is attracting the attention of the research community for their deployment in energy storage devices. Despite the fact that there are adequate cathode materials, the choice of suitable anodes for SIBs is limited. Graphite, the most versatile anode for LIBs, exhibits poor performance in case of SIBs. Amorphous or disordered carbons (hard and soft carbon) have been the most promising and cost-effective anode materials for SIBs. This Review discusses the recent advances of various forms of amorphous or disordered carbons used in SIBs with emphasis on their synthesis processes and relationship between microstructure, morphology, and performance. A profound understanding of the charge storage mechanisms of sodium in these carbon materials has been deliberated. The performance of these anode materials also depends upon electrolyte optimization, which has been aptly conferred. However, these anodes are often plagued with large voltage loss, low initial coulombic efficiency, and formation of solid electrolyte interphase. In order to overcome these challenges, several mitigation strategies have been put forward in a concise way to offer visions for the deployment of these amorphous carbon materials for the progress and commercial success of SIBs.

Senescence-associated miR-34a and miR-126 in middle-aged Indians with type 2 diabetes.

Rapid urbanization and unhealthy dietary patterns critically increase the risk of type 2 diabetes (T2D) in middle-aged Indians. However, despite recent evidence of senescence-associated microRNAs (SA-miRNAs) in regulating complex pathways of ageing, their expressions in middle-aged Indians with T2D remain unexplored. Hence we aimed to investigate the changes in expressions of SA-miRNAs miR-34a and miR-126 in middle-aged T2D patients. A total of 30 T2D patients and 30 controls were recruited of age 31-50 years. The expressions of plasma miR-34a and miR-126 were determined by quantitative PCR. Oxidized LDL (OxLDL) and malondialdehyde (MDA) levels were quantified using enzyme-linked immunosorbent assay (ELISA). The effect of different glucose concentrations on miR-34a, miR-126, senescence-associated, and oxidative stress-responsive genes were also studied in an in vitro model of mice pancreatic ß-cells. MiR-34a was significantly upregulated, whereas miR-126 was nonsignificantly reduced in T2D patients as compared to controls. T2D patients showed elevated levels of oxidative stress markers than controls. Analysis of cultured mice pancreatic ß-cells exposed to high glucose showed significant upregulation of miR-34a, miR-126, p53, and superoxide dismutase 2 (SOD2). We found that circulating miR-34a levels and oxidative stress markers levels were elevated in the middle-aged Indians with T2D as compared to controls. The presence of diabetes may aggravate the normal ageing process in the middle-aged Indians. These SA-miRNAs can also be used to check the cellular dysfunctions and ageing of pancreatic ß-cells.

Translational control of the interferon regulatory factor 2 mRNA by IRES element.

Translational control represents an important mode of regulation of gene expression under stress conditions. We have studied the translation of interferon regulatory factor 2 (IRF2) mRNA, a negative regulator of transcription of interferon-stimulated genes and demonstrated the presence of internal ribosome entry site (IRES) element in the 5'UTR of IRF2 RNA. Various control experiments ruled out the contribution of leaky scanning, cryptic promoter activity or RNA splicing in the internal initiation of IRF2 RNA. It seems IRF2-IRES function is not sensitive to eIF4G cleavage, since its activity was only marginally affected in presence of Coxsackievirus 2A protease. Interferon alpha treatment did not affect the IRF2-IRES activity or the protein level significantly. Also, in cells treated with tunicamycin [an agent causing endoplasmic reticulum (ER) stress], the IRF2-IRES activity and the protein levels were unaffected, although the cap-dependent translation was severely impaired. Analysis of the cellular protein binding with the IRF2-IRES suggests certain cellular factors, which might influence its function under stress conditions. Interestingly, partial knockdown of PTB protein significantly inhibited the IRF2-IRES function. Taken together, it appears that IRF2 gene expression during stress condition is controlled by the IRES element, which in turn influences the cellular response.

Electroactive and High Dielectric Folic Acid/PVDF Composite Film Rooted Simplistic Organic Photovoltaic Self-Charging Energy Storage Cell with Superior Energy Density and Storage Capability.

Herein we report a simplistic prototype approach to develop an organic photovoltaic self-charging energy storage cell (OPSESC) rooted with biopolymer folic acid (FA) modified high dielectric and electroactive ß crystal enriched poly(vinylidene fluoride) (PVDF) composite (PFA) thin film. Comprehensive and exhaustive characterizations of the synthesized PFA composite films validate the proper formation of ß-polymorphs in PVDF. Significant improvements of both ß-phase crystallization (F(ß) ≈ 71.4%) and dielectric constant (ε ≈ 218 at 20 Hz for PFA of 7.5 mass %) are the twosome realizations of our current study. Enhancement of ß-phase nucleation in the composites can be thought as a contribution of the strong interaction of the FA particles with the PVDF chains. Maxwell-Wagner-Sillars (MWS) interfacial polarization approves the establishment of thermally stable high dielectric values measured over a wide temperature spectrum. The optimized high dielectric and electroactive films are further employed as an active energy storage material in designing our device named as OPSESC. Self-charging under visible light irradiation without an external biasing electrical field and simultaneous remarkable self-storage of photogenerated electrical energy are the two foremost aptitudes and the spotlight of our present investigation. Our as fabricated device delivers an impressively high energy density of 7.84 mWh/g and an excellent specific capacitance of 61 F/g which is superior relative to the other photon induced two electrode organic self-charging energy storage devices reported so far. Our device also proves the realistic utility with good recycling capability by facilitating commercially available light emitting diode.

Er3+/Fe3+ Stimulated Electroactive, Visible Light Emitting, and High Dielectric Flexible PVDF Film Based Piezoelectric Nanogenerators: A Simple and Superior Self-Powered Energy Harvester with Remarkable Power Density.

The design of an energy-harvesting unit with superior output characteristics, i.e., high power density, is a great technological challenge in the present time. Here, simple, lightweight, flexible, and cost-effective piezoelectric nanogenerators (PENGs) have been fabricated by integrating the aluminum electrodes onto Er3+/Fe3+ stimulated electroactive, visible-light-emitting, and large dielectric PVDF films in which ErCl3·6H2O and Fe(NO3)3·9H2O act as the catalytic agents for electroactive ß polymorph nucleation and the enhancement of dielectric properties. The developed PENGs exhibit excellent energy-harvesting performance with very high power density and very fast charging ability compared with the previously reported PVDF-assisted prototype nanogenerators. The PENGs lead to very large power density (∼160 and ∼55.34 mW cm-3) under periodic finger imparting for Er3+- and Fe3+-stimulated PVDF-film-based energy-harvester units, respectively. The fabricated self-powered PENG is also able to light up 54 commercially available light-emitting diodes.

The U2AF1S34F mutation induces lineage-specific splicing alterations in myelodysplastic syndromes.

Mutations of the splicing factor-encoding gene U2AF1 are frequent in the myelodysplastic syndromes (MDS), a myeloid malignancy, and other cancers. Patients with MDS suffer from peripheral blood cytopenias, including anemia, and an increasing percentage of bone marrow myeloblasts. We studied the impact of the common U2AF1S34F mutation on cellular function and mRNA splicing in the main cell lineages affected in MDS. We demonstrated that U2AF1S34F expression in human hematopoietic progenitors impairs erythroid differentiation and skews granulomonocytic differentiation toward granulocytes. RNA sequencing of erythroid and granulomonocytic colonies revealed that U2AF1S34F induced a higher number of cassette exon splicing events in granulomonocytic cells than in erythroid cells. U2AF1S34F altered mRNA splicing of many transcripts that were expressed in both cell types in a lineage-specific manner. In hematopoietic progenitors, the introduction of isoform changes identified in the U2AF1S34F target genes H2AFY, encoding an H2A histone variant, and STRAP, encoding serine/threonine kinase receptor-associated protein, recapitulated phenotypes associated with U2AF1S34F expression in erythroid and granulomonocytic cells, suggesting a causal link. Furthermore, we showed that isoform modulation of H2AFY and STRAP rescues the erythroid differentiation defect in U2AF1S34F MDS cells, suggesting that splicing modulators could be used therapeutically. These data have critical implications for understanding MDS phenotypic heterogeneity and support the development of therapies targeting splicing abnormalities.

Impact of Splicing Factor Mutations on Pre-mRNA Splicing in the Myelodysplastic Syndromes.

Splicing is an essential cellular process which is carried out by the spliceosome in order to remove the introns and join the exons present in pre-mRNA transcripts. A variety of spliceosomal mutations have been recently identified in the myelodysplastic syndromes (MDS), a heterogeneous group of hematopoietic stem cell malignancies, revealing a new leukemogenic pathway involving spliceosomal dysfunction. Splicing factor genes are the most frequently mutated genes found in MDS, with mutations occurring in more than half of all patients. The high mutation frequency in different components of the spliceosome in MDS indicates that aberrant splicing may be a common consequence of these mutations in this disorder. RNA sequencing studies using MDS patient bone marrow cells and different mouse models have identified several downstream targets of the splicing factor mutations. Aberrant splicing of these target genes may contribute to MDS pathogenesis, however functional studies are required in order to fully determine the effects of the aberrant isoforms on disease phenotype. Splicing inhibitors are currently being developed and may be used as therapeutic agents to target aberrant pre-mRNA splicing in MDS and other cancers with splicing factor mutations. The mouse models expressing splicing factor mutations may prove particularly valuable for pre-clinical testing of these drugs.

ASXL1 mutation correction by CRISPR/Cas9 restores gene function in leukemia cells and increases survival in mouse xenografts.

Recurrent somatic mutations of the epigenetic modifier and tumor suppressor ASXL1 are common in myeloid malignancies, including chronic myeloid leukemia (CML), and are associated with poor clinical outcome. CRISPR/Cas9 has recently emerged as a powerful and versatile genome editing tool for genome engineering in various species. We have used the CRISPR/Cas9 system to correct the ASXL1 homozygous nonsense mutation present in the CML cell line KBM5, which lacks ASXL1 protein expression. CRISPR/Cas9-mediated ASXL1 homozygous correction resulted in protein re-expression with restored normal function, including down-regulation of Polycomb repressive complex 2 target genes. Significantly reduced cell growth and increased myeloid differentiation were observed in ASXL1 mutation-corrected cells, providing new insights into the role of ASXL1 in human myeloid cell differentiation. Mice xenografted with mutation-corrected KBM5 cells showed significantly longer survival than uncorrected xenografts. These results show that the sole correction of a driver mutation in leukemia cells increases survival in vivo in mice. This study provides proof-of-concept for driver gene mutation correction via CRISPR/Cas9 technology in human leukemia cells and presents a strategy to illuminate the impact of oncogenic mutations on cellular function and survival.

Targeted delivery of hepatitis C virus-specific short hairpin RNA in mouse liver using Sendai virosomes.

Internal ribosome entry site (IRES)-mediated translation of input viral RNA is the initial required step for the replication of the positive-stranded genome of hepatitis C virus (HCV). We have shown previously the importance of the GCAC sequence near the initiator AUG within the stem and loop IV (SLIV) region in mediating ribosome assembly on HCV RNA. Here, we demonstrate selective inhibition of HCV-IRES-mediated translation using short hairpin (sh)RNA targeting the same site within the HCV IRES. sh-SLIV showed significant inhibition of viral RNA replication in a human hepatocellular carcinoma (Huh7) cell line harbouring a HCV monocistronic replicon. More importantly, co-transfection of infectious HCV-H77s RNA and sh-SLIV in Huh7.5 cells successfully demonstrated a significant decrease in viral RNA in HCV cell culture. Additionally, we report, for the first time, the targeted delivery of sh-SLIV RNA into mice liver using Sendai virosomes and demonstrate selective inhibition of HCV-IRES-mediated translation. Results provide the proof of concept that Sendai virosomes could be used for the efficient delivery of shRNAs into liver tissue to block HCV replication.

Sequence-specific cleavage of hepatitis C virus RNA by DNAzymes: inhibition of viral RNA translation and replication.

DNAzyme (Dz) molecules have been shown to be highly efficient inhibitors of virus replication. Hepatitis C virus RNA translation is mediated by an internal ribosome entry site (IRES) element located mostly in the 5' untranslated region (UTR), the mechanism of which is fundamentally different from cap-dependent translation of cellular mRNAs, and thus an attractive target for designing antiviral drugs. Inhibition of HCV IRES-mediated translation has drastic consequences for the replication of viral RNA as well. We have designed several Dzs, targeting different regions of HCV IRES specific for 1b and also sequences conserved across genotypes. The RNA cleavage and translation inhibitory activities of these molecules were tested in a cell-free system and in cell culture using transient transfections. The majority of Dzs efficiently inhibited HCV IRES-mediated translation. However, these Dz molecules did not show significant inhibition of coxsackievirus B3 IRES-mediated translation or cap-dependent translation of reporter gene, showing high level of specificity towards target RNA. Also, Northern blot hybridization analysis showed significant cleavage of HCV IRES by the Dz molecules in Huh7 cells transiently transfected with the HCV-FLuc monocistronic construct. Interestingly, one of the Dzs was more effective against genotype1b, whereas the other showed significant inhibition of viral RNA replication in Huh7 cells harbouring a HCV 2a monocistronic replicon. As expected, mutant-Dz failed to cleave RNA and inhibit HCV RNA translation, showing the specificity of inhibition. Taken together, these findings suggest that the Dz molecule can be used as selective and effective inhibitor of HCV RNA replication, which can be explored further for development of a potent therapeutic agent against HCV infection.

Analysis of mutations within the 5' untranslated region, interferon sensitivity region, and PePHD region as a function of response to interferon therapy in hepatitis C virus-infected patients in India.

Mutations in several subgenomic regions have been implicated in influencing response to interferon therapy; however, a comprehensive picture of Indian patients was lacking. Based on the viral load and clinical factors, 10 out of 15 patients were found to be complete responders, whereas 5 patients were nonresponders. The pretreatment viral RNA load of the patients was found to be between 5.20 and 6.13 log10 IU/ml, which eventually fell to 2.77 log10 IU/ml after 24 weeks of treatment, whereas in the case of nonresponders, the average was 5.38 log10 IU/ml. In order to study the influence of the hepatitis C virus genotype on the response to interferon therapy, the 5' untranslated region sequences of the samples were analyzed, which showed that genotype 3 patients responded better than genotype 1 patients. Additionally, the mutations in the interferon sensitivity-determining region (ISDR) of the NS5A protein and the double-stranded RNA-activated protein kinase-eukaryotic initiation factor 2 alpha phosphorylation homology domain (PePHD) of the E2 envelope protein, before and after treatment, were compared with nonresponder prototype J. Although, no clear correlation was found in the case of the mutated ISDR, some significant changes in residues were observed in the PePHD region, which could be helpful in understanding the molecular basis of resistance to therapy. Interestingly, analysis of the quasispecies variations showed a change in genotype in one sample during treatment, which might have contributed to the resistance. The results suggest that the mutations in different regions of the viral genome might have a concerted effect on the response to interferon therapy.