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Objective: The primary objective of this study was to quantify changes in performance of task-specific motor activities over 12 motor rehabilitation sessions with lumbosacral spinal cord stimulation (SCS) via either transcutaneous stimulation or epidural stimulation. Both stimulation modalities have been used in recent years to restore functions lost to spinal cord injury (SCI). Secondary outcomes examine participants' perspectives captured via the User Experience Questionnaire (UEQ) upon study completion to further understand their perception of SCS., Methods: Six individuals with SCI completed 12 sessions with one modality of SCS during supine and/or side-lying, seated forward reaching, and standing activities. Changes in volitional lower extremity movement, the number of points of contact needed at hips and/or knees to facilitate standing, and changes in seated forward reaching distance were used to quantify performance. The UEQ was administered to gauge participants' perspectives following use of SCS to enable functions impaired due to SCI., Results: For all participants, performance of motor activities improved with SCS compared to without stimulation. Responses for the UEQ showed an overall positive perception of trialing SCS with rehabilitation to enhance motor functions impaired by SCI., Conclusions: Regardless of injury severity, location of injury, time since SCI, or SCS modality, all participants experienced gains in motor function in the presence of SCS combined with a condensed rehabilitation program. However, no evidence of sustained motor functions was found in the absence of SCS. UEQ results highlight the positive perception of SCS with rehabilitation as well as the importance of consulting persons with lived experience of SCS during clinical trial design and protocol development. Trial registration: ClinicalTrials.gov identifier: NCT05095454.

The use of artificial intelligence and, more specifically, deep learning methods in chemistry is becoming increasingly common. Applications in informatics fields, such as cheminformatics and proteomics, structural biology, and spectroscopy, including NMR, are on the rise. Recent developments in model architectures, such as graph convolutional neural networks and transformers, have been enabled by advancements in computational hardware and software. However, model architectures with more predictive power often require larger amounts of training data, which can be challenging to acquire, but this requirement can be mitigated through techniques like pretraining and fine-tuning. In spite of these successes, challenges remain, such as normalization and scaling of data, availability of experimentally acquired data, and model explainability., (© 2022 John Wiley & Sons Ltd.)

Background: Neuromodulation using epidural electrical stimulation (EES) has shown functional restoration in humans with chronic spinal cord injury (SCI). EES during body weight supported treadmill training (BWSTT) enhanced stepping performance in clinical trial participants with paraplegia. Unfortunately, tools are lacking in availability to quantify clinician assistance during BWSTT with and without EES. Force sensitive resistors (FSRs) have previously quantified clinician assistance during static standing; however, dynamic tasks have not been addressed. Objective: To determine the validity of FSRs in measurements of force and duration to quantify clinician assistance and participant progression during BWSTT with EES in participants with SCI. Design: A feasibility study to determine the effectiveness of EES to restore function in individuals with SCI. Methods: Two male participants with chronic SCI were enrolled in a pilot phase clinical trial. Following implantation of an EES system in the lumbosacral spinal cord, both participants underwent 12 months of BWSTT with EES. At monthly intervals, FSRs were positioned on participants' knees to quantity forces applied by clinicians to achieve appropriate mechanics of stepping during BWSTT. The FSRs were validated on the benchtop using a leg model instrumented with a multiaxial load cell as the gold standard. The outcomes included clinician-applied force duration measured by FSR sensors and changes in applied forces indicating progression over the course of rehabilitation. Results: The force sensitive resistors validation revealed a proportional bias in their output. Loading required for maximal assist training exceeded the active range of the FSRs but were capable of capturing changes in clinician assist levels. The FSRs were also temporally responsive which increased utility for accurately assessing training contact time. The FSRs readings were able to capture independent stance for both participants by study end. There was minimal to no applied force bilaterally for participant 1 and unilaterally for participant 2. Conclusions: Clinician assistance applied at the knees as measured through FSRs during dynamic rehabilitation and EES (both on and off) effectively detected point of contact and duration of forces; however, it lacks accuracy of magnitude assessment. The reduced contact time measured through FSRs related to increased stance duration, which objectively identified independence in stepping during EES-enabled BWSTT following SCI., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Linde, Thoreson, Lopez, Gill, Veith, Hale, Calvert, Grahn, Fautsch, Sayenko and Zhao.)

Transcutaneous (TSS) and epidural spinal stimulation (ESS) are electrophysiological techniques that have been used to investigate the interactions between exogenous electrical stimuli and spinal sensorimotor networks that integrate descending motor signals with afferent inputs from the periphery during motor tasks such as standing and stepping. Recently, pilot-phase clinical trials using ESS and TSS have demonstrated restoration of motor functions that were previously lost due to spinal cord injury (SCI). However, the spinal network interactions that occur in response to TSS or ESS pulses with spared descending connections across the site of SCI have yet to be characterized. Therefore, we examined the effects of delivering TSS or ESS pulses to the lumbosacral spinal cord in nine individuals with chronic SCI. During low-frequency stimulation, participants were instructed to relax or attempt maximum voluntary contraction to perform full leg flexion while supine. We observed similar lower-extremity neuromusculature activation during TSS and ESS when performed in the same participants while instructed to relax. Interestingly, when participants were instructed to attempt lower-extremity muscle contractions, both TSS- and ESS-evoked motor responses were significantly inhibited across all muscles. Participants with clinically complete SCI tested with ESS and participants with clinically incomplete SCI tested with TSS demonstrated greater ability to modulate evoked responses than participants with motor complete SCI tested with TSS, although this was not statistically significant due to a low number of subjects in each subgroup. These results suggest that descending commands combined with spinal stimulation may increase activity of inhibitory interneuronal circuitry within spinal sensorimotor networks in individuals with SCI, which may be relevant in the context of regaining functional motor outcomes.

Background: Regaining control of movement following a spinal cord injury (SCI) requires utilization and/or functional reorganization of residual descending, and likely ascending, supraspinal sensorimotor pathways, which may be facilitated via task-specific training through body weight supported treadmill (BWST) training. Recently, epidural electrical stimulation (ES) combined with task-specific training demonstrated independence of standing and stepping functions in individuals with clinically complete SCI. The restoration of these functions may be dependent upon variables such as manipulation of proprioceptive input, ES parameter adjustments, and participant intent during step training. However, the impact of each variable on the degree of independence achieved during BWST stepping remains unknown. Objective: To describe the effects of descending intentional commands and proprioceptive inputs, specifically body weight support (BWS), on lower extremity motor activity and vertical ground reaction forces (vGRF) during ES-enabled BWST stepping in humans with chronic sensorimotor complete SCI. Furthermore, we describe perceived changes in the level of assistance provided by clinicians when intent and BWS are modified. Methods: Two individuals with chronic, mid thoracic, clinically complete SCI, enrolled in an IRB and FDA (IDE G150167) approved clinical trial. A 16-contact electrode array was implanted in the epidural space between the T11-L1 vertebral regions. Lower extremity motor output and vertical ground reaction forces were obtained during clinician-assisted ES-enabled treadmill stepping with BWS. Consecutive steps were achieved during various experimentally-controlled conditions, including intentional participation and varied BWS (60% and 20%) while ES parameters remain unchanged. Results: During ES-enabled BWST stepping, the knee extensors exhibited an increase in motor activation during trials in which stepping was passive compared to active or during trials in which 60% BWS was provided compared to 20% BWS. As a result of this increased motor activation, perceived clinician assistance increased during the transition from stance to swing. Intentional participation and 20% BWS resulted in timely and purposeful activation of the lower extremities muscles, which improved independence and decreased clinician assistance. Conclusion: Maximizing participant intention and optimizing proprioceptive inputs through BWS during ES-enabled BWST stepping may facilitate greater independence during BWST stepping for individuals with clinically complete SCI. Clinical Trial Registration: ClinicalTrials.gov identifier: NCT02592668., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Gill, Linde, Hale, Lopez, Fautsch, Calvert, Veith, Beck, Garlanger, Sayenko, Lavrov, Thoreson, Grahn and Zhao.)

The zero- and double-quantum methyl TROSY Hahn-echo and the methyl 1 H- 1 H dipole-dipole cross-correlation nuclear magnetic resonance experiments enable estimation of multiple quantum chemical exchange broadening in methyl groups in proteins. The two relaxation rate constants are established to be linearly dependent using molecular dynamics simulations and empirical analysis of experimental data. This relationship allows chemical exchange broadening to be recognized as an increase in the Hahn-echo relaxation rate constant. The approach is illustrated by analyzing relaxation data collected at three temperatures for E. coli ribonuclease HI and by analyzing relaxation data collected for different cofactor and substrate complexes of E. coli AlkB.

Epidural electrical stimulation (EES) of the spinal cord has been shown to restore function after spinal cord injury (SCI). Characterization of EES-evoked motor responses has provided a basic understanding of spinal sensorimotor network activity related to EES-enabled motor activity of the lower extremities. However, the use of EES-evoked motor responses to guide EES system implantation over the spinal cord and their relation to post-operative EES-enabled function in humans with chronic paralysis attributed to SCI has yet to be described. Herein, we describe the surgical and intraoperative electrophysiological approach used, followed by initial EES-enabled results observed in 2 human subjects with motor complete paralysis who were enrolled in a clinical trial investigating the use of EES to enable motor functions after SCI. The 16-contact electrode array was initially positioned under fluoroscopic guidance. Then, EES-evoked motor responses were recorded from select leg muscles and displayed in real time to determine electrode array proximity to spinal cord regions associated with motor activity of the lower extremities. Acceptable array positioning was determined based on achievement of selective proximal or distal leg muscle activity, as well as bilateral muscle activation. Motor response latencies were not significantly different between intraoperative recordings and post-operative recordings, indicating that array positioning remained stable. Additionally, EES enabled intentional control of step-like activity in both subjects within the first 5 days of testing. These results suggest that the use of EES-evoked motor responses may guide intraoperative positioning of epidural electrodes to target spinal cord circuitry to enable motor functions after SCI.

Spinal sensorimotor networks that are functionally disconnected from the brain because of spinal cord injury (SCI) can be facilitated via epidural electrical stimulation (EES) to restore robust, coordinated motor activity in humans with paralysis 1-3 . Previously, we reported a clinical case of complete sensorimotor paralysis of the lower extremities in which EES restored the ability to stand and the ability to control step-like activity while side-lying or suspended vertically in a body-weight support system (BWS) 4 . Since then, dynamic task-specific training in the presence of EES, termed multimodal rehabilitation (MMR), was performed for 43 weeks and resulted in bilateral stepping on a treadmill, independent from trainer assistance or BWS. Additionally, MMR enabled independent stepping over ground while using a front-wheeled walker with trainer assistance at the hips to maintain balance. Furthermore, MMR engaged sensorimotor networks to achieve dynamic performance of standing and stepping. To our knowledge, this is the first report of independent stepping enabled by task-specific training in the presence of EES by a human with complete loss of lower extremity sensorimotor function due to SCI.

Hepatitis B is a clinically important public health issue. Infection leads to hepatocellular carcinoma. Therefore, patients need antiviral therapy for prolonged period to prevent the complication of the disease. Data concerning chronic hepatitis B (CHB) patients with high hepatitis B virus (HBV) DNA are limited. The aim of the study was to check the efficacy of the nucleoside reverse transcriptase inhibitors (tenofovir) in terms of suppression of HBV DNA. The secondary end point in the study is to evaluate trends of predictive variables that predict outcome of treatment. In this specific study, we evaluated 140 CHB male and female patients, of these 110 completed 48 weeks of treatment. On the basis of hepatitis B e antigen (HBeAg), patients were stratified; HBV DNA and hepatitis B surface antigen (HBsAg) levels were measured along with liver function tests. All enrolled patients were given tenofovir disoproxil fumarate 300 mg daily before breakfast. Overall, 69.1% of patients showed virologic response. HBeAg-negative patient group showed 68% viral suppression and HBeAg-positive patient group showed 45.9% over 24 months of treatment, while at 48 months it was shown to be 76.7% and 54.1%, respectively. None of the patients suffered HBsAg loss during the 48 months. Baseline high HBV DNA level was found as a significant predictor of response (OR, 1.9; 95% CI = 1.23-3.9, p = 0.005). None of the patients observed had serious adverse events. Mutations in the RT region of polymerase gene are shown to be associated with resistance to antiviral drugs. Among patients suffering with chronic HBV infection, HBeAg-negative patient group have better virologic response as compared with HBeAg-positive group. Higher concentration of HBV DNA at baseline has negative prediction for sustained viral suppression. The A-B motif interdomain rtL122F mutation was found in nonresponder patients in our study. Another mutation rtN248H observed in E motif considered to have effect on DNA primer grip, which forms part of binding pocket.

Hepatitis C virus (HCV) signifies an important health issue as it is a globally prevalent pathogen and poses a great threat to human health. Direct antiviral therapy became a landmark in treatment against chronic HCV infection as they have proven to increase sustained virological response (SVR) rate, provide shortened and simplified regimens. This study aimed to evaluate efficacy of Sofosbuvir and classify factors of treatment success and their function in therapy continuation decision. We studied host and viral factors in 310 patients who received Sofosbuvir (DAA) at a dose of 400 mg daily along with ribavirin 10 mg/kg body weight for 24 weeks and followed by 12 weeks after completion of treatment. A total of 302 patients (97.42%) showed end-of-treatment response. However, 8 patients (2.58%) were virological nonresponders and 286 patients (94.70%) achieved SVR. Among 310 HCV positive, the percentage of viral genotype 3-infected patients was 89.6%, whereas only 10.32% were affected by HCV genotype 1. Multivariable analysis showed that baseline HCV RNA (≤8 × 10 6 IU/mL vs. >8 × 10 6 IU/mL) [95% confidence interval (CI), odds ratio (OR) 3.0, 2.0 to 8.4; p = 0.004], age (<50 vs. ≥50 years) (95% CI, OR 1.9, 3.0 to 10.1; p = 0.03), liver texture (normal liver vs. coarse) are related to virological response. None of the patients included in the study has S282T substitution in NS5b of HCV. No significant difference in response was observed in patients with different genotypes of rs12979860. Liver texture, age, and viral load are predictors of SVR. In this real-life data no substitution of S282T in NS5b was observed related to failure of therapy. Oral Sofosbuvir generally has a high tolerance and can effortlessly beat the safety concerns of past regimens.

The catalytic domain of protein tyrosine kinases can interconvert between active and inactive conformations in response to regulatory inputs. We recently demonstrated that Src kinase features an allosteric network that couples substrate-binding sites. However, the extent of conformational and dynamic changes that are propagated throughout the kinase domain remains poorly understood. Here, we monitor by NMR the effect of conformationally selective inhibitors on kinase backbone dynamics. We find that inhibitor binding and activation loop autophosphorylation induces dynamic changes across the entire kinase. We identify a highly conserved amino acid, Gly449, that is necessary for Src activation. Finally, we show for the first time how the SH3-SH2 domains perturb the dynamics of the kinase domain in the context of the full length protein. We provide experimental support for long-range communication in Src kinase that leads to the relative stabilization of active or inactive conformations and modulation of substrate affinity.

We report a case of chronic traumatic paraplegia in which epidural electrical stimulation (EES) of the lumbosacral spinal cord enabled (1) volitional control of task-specific muscle activity, (2) volitional control of rhythmic muscle activity to produce steplike movements while side-lying, (3) independent standing, and (4) while in a vertical position with body weight partially supported, voluntary control of steplike movements and rhythmic muscle activity. This is the first time that the application of EES enabled all of these tasks in the same patient within the first 2 weeks (8 stimulation sessions total) of EES therapy., (Copyright © 2017 Mayo Foundation for Medical Education and Research. Published by Elsevier Inc. All rights reserved.)

Intrinsically disordered proteins (IDPs) and proteins with intrinsically disordered regions (IDRs) are known to play important roles in regulatory and signaling pathways. A critical aspect of these functions is the ability of IDP/IDRs to form highly specific complexes with target molecules. However, elucidation of the contributions of conformational dynamics to function has been limited by challenges associated with structural heterogeneity of IDP/IDRs. Using NMR spin relaxation parameters ((15)N R1, (15)N R2, and {(1)H}-(15)N heteronuclear NOE) collected at four static magnetic fields ranging from 14.1 to 21.1 T, we have analyzed the backbone dynamics of the basic leucine-zipper (bZip) domain of the Saccharomyces cerevisiae transcription factor GCN4, whose DNA binding domain is intrinsically disordered in the absence of DNA substrate. We demonstrate that the extended model-free analysis can be applied to proteins with IDRs such as apo GCN4 and that these results significantly extend previous NMR studies of GCN4 dynamics performed using a single static magnetic field of 11.74 T [Bracken, et al., J. Mol. Biol., 1999, 285, 2133-2146] and correlate well with molecular dynamics simulations [Robustelli, et al., J. Chem. Theory Comput., 2013, 9, 5190-5200]. In contrast to the earlier work, data at multiple static fields allows the time scales of internal dynamics of GCN4 to be reliably quantified. Large amplitude dynamic fluctuations in the DNA-binding region have correlation times (τs ≈ 1.4-2.5 ns) consistent with a two-step mechanism in which partially ordered bZip conformations of GCN4 form initial encounter complexes with DNA and then rapidly rearrange to the high affinity state with fully formed basic region recognition helices.

Background: The gold standard treatment for chronic hepatitis C virus (HCV) infection is pegylated interferon (PEG-IFN) in combination with ribavirin. Most patients treated with PEG-IFN achieve a sustained virological response (SVR). However host genetic factors play a vital role in the spontaneous and treatment-induced clearance of HCV infection from these infected patients. In the current study, polymorphisms of IL28B (rs8099917 and rs12979860) were analyzed and their association with the virological response to PEG-IFN alpha treatment was determined., Methods: One hundred and fifty HCV genotype 3 patients were assessed to study the correlation of IL28B with a therapeutic regimen of PEG-IFN alpha plus ribavirin. Twenty patients were excluded due to a refusal to participate in the study and 25 patients failed to meet the inclusion criteria. Of the 105 patients recruited, 49 (46.7%) were male and 56 (53.3%) were female. In order to determine single nucleotide polymorphisms of rs8099917 and rs12979860, the sample was amplified by PCR and then IL28B typing was carried out by restriction fragment length polymorphism (RFLP) followed by standard sequencing., Results: We found three types of genotype in rs8099917 of IL28B: wild-type TT in 60.0% of patients, heterozygous GT minor genotype in 36.2%, and GG in 3.8%. The frequency of the CC genotype of rs12979860 was 54.3%, CT was 37.1%, and TT was 8.6%. Overall, SVR was achieved in 68.6% of patients. A higher SVR was achieved for patients with the favorable genotype CC of rs12979860, with 84.2% as compared to 56.4% and 22.2% for minor genotype CT and TT, respectively (p=0.0001). We did not find a significant association for SVR to antiviral treatment in patients with genotype TT (rs8099917) (71.9%, p=0.36). The rapid virological response (RVR) rate was significantly higher in patients with major genotype TT (88.9%, p=0.04). These results show that IL28B polymorphism is highly associated with SVR to therapy in the Pakistani population infected with HCV genotype 3., Conclusions: HCV-infected patients carrying homozygous C/C have a higher chance of SVR. In addition, patients who carry T/T (rs8099917) have a higher chance of RVR., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

A central goal of enzymology is to understand the physicochemical mechanisms that enable proteins to catalyze complex chemical reactions with high efficiency. Recent methodological advances enable the contribution of protein dynamics to enzyme efficiency to be explored more deeply. Here, we utilize enzymological and biophysical studies, including NMR measurements of conformational dynamics, to develop a quantitative mechanistic scheme for the DNA repair enzyme AlkB. Like other iron/2-oxoglutarate-dependent dioxygenases, AlkB employs a two-step mechanism in which oxidation of 2-oxoglutarate generates a highly reactive enzyme-bound oxyferryl intermediate that, in the case of AlkB, slowly hydroxylates an alkylated nucleobase. Our results demonstrate that a microsecond-to-millisecond time scale conformational transition facilitates the proper sequential order of substrate binding to AlkB. Mutations altering the dynamics of this transition allow generation of the oxyferryl intermediate but promote its premature quenching by solvent, which uncouples 2-oxoglutarate turnover from nucleobase oxidation. Therefore, efficient catalysis by AlkB depends upon the dynamics of a specific conformational transition, establishing another paradigm for the control of enzyme function by protein dynamics., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

The present work demonstrates that NMR spin relaxation rate constants for molecules interconverting between states with different diffusion tensors can be modeled theoretically by combining orientational correlation functions for exchanging spherical molecules with locally isotropic approximations for the diffusion anisotropic tensors. The resulting expressions are validated by comparison with correlation functions obtained by Monte Carlo simulations and are accurate for moderate degrees of diffusion anisotropy typically encountered in investigations of globular proteins. The results are complementary to an elegant, but more complex, formalism that is accurate for all degrees of diffusion anisotropy [Ryabov, Y.; Clore, G. M.; Schwieters, C. D. J. Chem. Phys. 2012, 136, 034108].

Introduction: Hepatitis C viral (HCV) infection is caused by an RNA virus. HCV infection is considered to induce systemic disease that causes steatosis, alters lipid metabolism, and results in metabolic syndrome. This study aimed to investigate the therapeutic outcome in HCV genotype 3 patients with metabolic syndrome., Materials and Methods: A total of 621 HCV-positive patients who visited the hospital for treatment were screened. Among these, 441 patients were enrolled for antiviral therapy. These enrolled patients were assessed for metabolic syndrome according to the International Diabetes Federation criteria. Group A included patients with metabolic syndrome and group B included patients without metabolic syndrome. All patients received peginterferon-α2a (180 μg/week) and ribavirin (10 mg/kg/day) for 6 months., Results: The prevalence of metabolic syndrome in chronic HCV patients was 37.9%. We observed that metabolic syndrome was more common among female compared with male participants (43.9 vs. 28.8%, P=0.005). It was found that sustained virologic response (SVR) rates were significantly higher in the patients in group B (without metabolic syndrome) compared with the patients in group A who had metabolic syndrome (72.2 vs. 43.7%, P<0.05). Older patients were at a higher risk for metabolic syndrome and a correlation of metabolic syndrome with nonresponse to antiviral therapy was observed. An interesting correlation among metabolic syndrome, age, and SVR was found: with age, SVR decreases, while metabolic syndrome increases., Conclusion: Metabolic syndrome has an influence on therapeutic outcomes in terms of SVR. Moreover, this information can identify patients who might have a low chance of attaining an SVR and a timely decision may protect the patients from the adverse effects of therapy.

Background: Rapid virological response (RVR) is now thought to be the strongest predictor of sustained virological response (SVR) in hepatitis C virus (HCV) patients undergoing antiviral therapy. It can be used as a guide to individualize treatment duration. The aim of this study was to assess the role of RVR in tailoring the duration of treatment., Methods: Patients with HCV genotype 3 infections were enrolled and treated with pegylated interferon alfa-2a (PEG IFN alfa-2a) 180 μg/week and ribavirin. HCV RNA was analyzed at weeks 4, 12, 16, and 24. Treatment duration was individualized on the basis of RVR. Patients who achieved RVR and who were aged ≤ 40 years with a body mass index (BMI) ≤ 27 kg/m(2) received 16 weeks of treatment (group A). Patients who achieved RVR and were aged >40 years with a BMI >27 kg/m(2), aged >40 years with a BMI ≤ 27 kg/m(2), and aged ≤ 40 years with a BMI >27 kg/m(2) received 24 weeks of treatment (group B). Patients who did not achieve RVR but who achieved an early virological response (EVR; HCV PCR-negative or ≥ 2 log drop in HCV RNA at week 12) were treated with 24 weeks of therapy (group C)., Results: SVR was observed in 86% in group A, 82.2% in group B, and 46.8% in group C. A difference was observed in SVR for patients with and without RVR and receiving the standard duration of treatment (82.2% vs. 46.8%, p<0 .001). The results show that the rate of SVR is not inferior in those with RVR treated with 16 weeks of therapy compared to 24 weeks (86% vs. 82.2%, p=0.004)., Conclusions: RVR is useful to individualize the duration of treatment and to predict the treatment outcome. A short treatment of 16 weeks is as effective as 24 weeks in HCV genotype 3 patients who achieve RVR, who have a low BMI, and are younger in age., (Copyright © 2013 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Background: The recommended standard therapeutic regimen for chronic hepatitis patients with hepatitis C virus (HCV) genotype 3 is pegylated interferon plus ribavirin for 24 weeks. The aim of the present study was to evaluate treatment efficacy and variables predictive of treatment success, interactions among variables contributing to a response to therapy, and the utility of the rapid virological response (RVR; week 4 virological response) to predict treatment outcomes in HCV genotype 3-infected patients in routine clinical practice., Methods: We prospectively studied baseline and during-treatment factors associated with a sustained virological response (SVR) in HCV genotype 3-infected patients who received pegylated interferon alfa-2a (PEG-IFN α2a) 180 μg/week plus ribavirin 800 mg daily for 24 weeks and who were followed for 24 weeks after the completion of treatment., Results: Four hundred and twenty-six treated patients were included in the analysis; 320 (75.1%) showed an SVR. The following factors were assessed for their ability to predict SVR by means of univariable and multivariable logistic regression analysis: patient age, sex, pre-treatment viral load, pre-treatment alanine aminotransferase (ALT), body mass index (BMI), and RVR. Four factors - age, pre-treatment viral load, pre-treatment ALT, and RVR - were statistically significant predictors of SVR (p<0.05) in the univariable analysis. Factors showing a significant association with SVR were assessed by multivariable logistic regression analysis. In the multivariable analysis, independent factors associated with SVR were the attainment of RVR (odds ratio (OR) 11, 95% confidence interval (CI) 6.15-20.69; p<0.0001), patient age ≤40 years (OR 4.2, 95% CI 2.30-7.96, p<0.0001), and a low pre-treatment viral load (≤8 × 10(5) IU/ml; OR 3.4, 95% CI 1.87-6.25; p<0.0001). The effect of RVR in patients aged >40 years was more pronounced than in those aged ≤40 years: 81.1% of patients aged >40 years who achieved an RVR had an SVR, whereas only 7.5% of patients aged >40 years who did not achieve an RVR had an SVR (p<0.05)., Conclusions: RVR is an independent variable that is predictive of SVR. Moreover older patients (>40 years) who achieve an RVR are likely to have an SVR, while patients who do not achieve an RVR and who have a high pre-treatment viral load (>8 × 10(5) IU/ml) are unlikely to have an SVR., (Copyright © 2012 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Multiplet-filtered and gradient-selected heteronuclear zero-quantum coherence (gsHZQC) TROSY experiments are described for measuring (1)H-(13)C correlations for (13)CH(3) methyl groups in proteins. These experiments provide improved suppression of undesirable, broad outer components of the heteronuclear zero-quantum multiplet in medium-sized proteins, or in flexible sites of larger proteins, compared to previously described HZQC sequences (Tugarinov et al. in J Am Chem Soc 126:4921-4925, 2004; Ollerenshaw et al. in J Biomol NMR 33:25-41, 2005). Hahn-echo versions of the gsHZQC experiment also are described for measuring zero- and double-quantum transverse relaxation rate constants for identification of chemical exchange broadening. Application of the proposed pulse sequences to Escherichia coli ribonuclease HI, with a molecular mass of 18 kD, indicates that improved multiplet suppression is obtained without substantial loss of sensitivity., (© Springer Science+Business Media B.V. 2011)

Understanding the relationship between protein dynamics and stability is of paramount importance to the fields of biology and pharmaceutics. Clarifying this relationship is complicated by the large amount of experimental data that must be generated and analyzed if motions that exist over the wide range of timescales are to be included. To address this issue, we propose an approach that utilizes a multidimensional vector-based empirical phase diagram (EPD) to analyze a set of dynamic results acquired across a temperature-pH perturbation plane. This approach is applied to a humanized immunoglobulin G1 (IgG1), a protein of major biological and pharmaceutical importance whose dynamic nature is linked to its multiple biological roles. Static and dynamic measurements are used to characterize the IgG and to construct both static and dynamic EPDs. Between pH 5 and 8, a single, pH-dependent transition is observed that corresponds to thermal unfolding of the IgG. Under more acidic conditions, evidence exists for the formation of a more compact, aggregation resistant state of the immunoglobulin, known as A-form. The dynamics-based EPD presents a considerably more detailed pattern of apparent phase transitions over the temperature-pH plane. The utility and potential applications of this approach are discussed.

U.S. chemical plants currently have potentially catastrophic vulnerabilities as terrorist targets. The possible consequences of these vulnerabilities echo from the tragedies of the Bhopal incident in 1984 to the terrorist attacks on 11 September 2001 and, most recently, Hurricanes Katrina and Rita. Findings from a 2004 nationwide participatory research study of 125 local union leaders at sites with very large volumes of highly hazardous chemicals suggest that voluntary efforts to achieve chemical plant security are not succeeding. Study respondents reported that companies had only infrequently taken actions that are most effective in preventing or in preparing to respond to a terrorist threat. In addition, companies reportedly often failed to involve key stakeholders, including workers, local unions, and the surrounding communities, in these efforts. The environmental health community thus has an opportunity to play a key role in advocating for and supporting improvements in prevention of and preparation for terrorist attacks. Policy-level recommendations to redress chemical site vulnerabilities and the related ongoing threats to the nation's security are as follows: a) specify detailed requirements for chemical site assessment and security ; b) mandate audit inspections supported by significant penalties for cases of noncompliance ; c) require progress toward achieving inherently safer processes, including the minimizing of storage of highly hazardous chemicals ; d) examine and require additional effective actions in prevention, emergency preparedness, and response and remediation ; e) mandate and fund the upgrading of emergency communication systems ; and f) involve workers and community members in plan creation and equip and prepare them to prevent and respond effectively to an incident.

The crystal structure of the Tl+ form of the G-quadruplex formed from the Oxytricha nova telomere sequence, d(G4T4G4), has been solved to 1.55 A. This G-quadruplex contains five Tl+ ions, three of which are interspersed between adjacent G-quartet planes and one in each of the two thymine loops. The structure displays a high degree of similarity to the K+ crystal structure [Haider et al. (2002), J. Mol. Biol., 320, 189-200], including the number and location of the monovalent cation binding sites. The highly isomorphic nature of the two structures, which contain such a large number of monovalent binding sites (relative to nucleic acid content), verifies the ability of Tl+ to mimic K+ in nucleic acids. Information from this report confirms and extends the assignment of 205Tl resonances from a previous report [Gill et al. (2005), J. Am. Chem. Soc., 127, 16 723-16 732] where 205Tl NMR was used to study monovalent cation binding to this G-quadruplex. The assignment of these resonances provides evidence for the occurrence of conformational dynamics in the thymine loop region that is in slow exchange on the 205Tl timescale.

Monovalent cations play an important role in many biological functions. The guanine rich sequence, d(G4T4G4), requires monovalent cations for formation of the G-quadruplex, d(G4T4G4)2. This requirement can be satisfied by thallium (Tl+), a potassium (K+) surrogate. To verify that the structure of d(G4T4G4)2 in the presence of Tl+ is similar to the K+-form of the G-quadruplex, the solution structure of the Tl+-form of d(G4T4G4)2 was determined. The 10 lowest energy structures have an all atom RMSD of 0.76 +/- 0.16 A. Comparison of this structure to the identical G-quadruplex formed in the presence of K+ validates the isomorphous nature of Tl+ and K+. Using a 1H-205Tl spin-echo difference experiment we show that, in the Tl+-form of d(G4T4G4)2, small scalar couplings (<1 Hz) exist between 205Tl and protons in the G-quadruplex. These data comprise the first 1H-205Tl scalar couplings observed in a biological system and have the potential to provide important constraints for structure determination. These experiments can be applied to any system in which the substituted Tl+ cations are in slow exchange with the bulk ions in solution.

Background and Aims: Hepatitis C virus (HCV) diagnosis causes significant psychological stress and anxiety. We thought it would be important to illustrate the anxiety caused by HCV diagnosis in patients from the developing world., Methods: This study was conducted at the Shifa International Hospital, Islamabad, between February 2004 and April 2004. All patients who were recently diagnosed with HCV (those who tested positive to anti-HCV and HCV polymerase chain reaction) were given a questionnaire that compared stress due to HCV infection with four other variables, including death of a close family member, divorce, loss of source of income and move to another city. We also analyzed the anxiety level among these patients using the Beck Anxiety Inventory (BAI)., Results: We studied 98 patients and 100 healthy controls. Thirty-one (31.63%) patients who were diagnosed with HCV presented only with fatigue. Sixty-three (64.28%) patients admitted that diagnosis of HCV interfered with their daily life in some way. Among the newly diagnosed cases, 48 (48.97%) patients had moderate to severe anxiety (BAI Class B and C). Multi-logistic regression analysis showed that anxiety was related to HCV-related stress (P < 0.002) and self-perceived severity of disease (P < 0.001). HCV diagnosis was significantly more stressful than divorce (59.87 vs 70.95; P < 0.013), loss of source of income (50.52 vs 70.80; P < 0.001), and a move to another city (28.32 vs 70.80; P < 0.001)., Conclusions: Diagnosis with HCV is reported to be more stressful than divorce, loss of source of income and a move to another city.

Objective: To estimate the sero-prevalence of anti-HCV among the spouses of HCV positive patients., Design: A cross-sectional survey., Place and Duration of Study: Shifa International Hospital, Islamabad, from January 2001 to January 2004., Patients and Methods: Spouses of patients of hepatitis C were studied. Spouses of patients who had hepatitis C by the presence of anti-HCV and HCV RNA (PCR) were tested for anti-HCV by ELISA method., Results: A total of 347 consecutive patients were included in the study. Fifty-three patients were single, 48 were widows or divorced and 19 individuals refused to be tested. Thus, 227 patients were available for anti-HCV testing. Out of these 227 spouses tested, 10 (4.4%) were found to be positive for anti-HCV. The mean age of HCV-positive spouses was 44.2 +/- 8.31 years, while that of HCV-negative spouses was 46.37 +/- 11.31 years (p = 0.55). The mean years of marriage of HCV-positive spouses was 20.1 +/- 9.79 years as compared to 21.1 +/- 9.96 years for HCV-negative spouses (p = 0.75). The male proportion of HCV-positive spouses was 60% as compared to 57.6% for HCV-negative spouses (p = 0.9). The patients and spouses had common risk factor of history of injections., Conclusion: A positive anti-HCV was found in 4.4% spouses and common risk factors of history of injection and transfusion were noted in the patients and spouses.

Solution NMR spin-relaxation experiments were used to compare mus-ms dynamics in RNase A in the apo form and as complexed to the substrate-mimic, pTppAp. The crystal structure of the RNase A/pTppAp complex was determined and demonstrates that this ligand binds at the active site and utilizes established substrate binding sites in its interaction with RNase A. Relaxation-compensated CPMG experiments identify flexible residues in and around the active site in both the apo and pTppAp-bound enzyme. Quantitative analysis of the NMR spin-relaxation dispersion curves show that the time scale of motion in RNase A is unchanged when pTppAp binds and is similar to the time scale for the rate-determining step of the catalytic reaction. Temperature-dependent measurements provide an activation barrier for motion of 5.2 +/- 1.0 kcal/mol and 4.5 +/- 1.2 kcal/mol for the apo and pTppAp forms of RNase A, respectively. These data indicate very similar motion exists in the free and bound enzyme. Additionally, chemical shift data suggests that the magnitude of motion is also similar for these two forms and that it is likely that apo enzyme interconverts to a structure that resembles a ligand-bound form. Likewise, it appears that the bound conformation samples the apo enzyme form even when ligand is present. Taken together the data imply that RNase A is in a preexisting dynamic equilibrium between two conformations that represent the open and closed enzyme forms. These data suggest that ligand binding stabilizes the bound conformer but does not induce it.

Objective: To determine the efficacy of long acting octreotide (LAR) in the treatment of inoperable hepatocellular carcinoma; as well as to estimate the improvement in the quality of life., Methods: This study was carried out at the Shifa International Hospital, Islamabad between February and September 2003. Patients were recruited after an informed consent. There were 22 patients who decided to take the medication whereas 20 patients refused due to socio-economic issues. They served as controls. The patients agreeable for treatment were administered octreotide 100 mcg subcutaneously thrice daily for two weeks. This was followed by monthly administration of 20 mg intramuscular octreotide. The patients were followed up for 6 months. Tumor size, alpha-fetoprotein levels, and improvement in quality of life (QOL) were monitored during therapy., Results: Out of 22 patients, 19 patients completed the treatment. All were males. Mean age at presentation was 55 years. Tumor size regression was seen in 10 out of 22 patients (45.5%). Mean alpha-fetoprotein levels decreased in 11 out of 22 (50%) patients. An improvement in the quality of life was seen in 10 out of 22 (45.5%) patients after treatment with long acting octreotide. In the treatment arm, 14 out of 22 (64%) patients were alive at the end of six months as compared to 10 out of 20 (50%) in the control group., Conclusion: LAR causes tumor size regression, decreases AFP levels and improves quality of life in patients with inoperable hepatocellular carcinoma.

Objective: To review the experience of percutaneous endoscopic gastrostomy (PEG) tube placement and its management in the last nine years, at a tertiary care center in Pakistan., Methods: All patients who underwent percutaneous endoscopic gastrostomy from January 1995 to January 2004 at Shifa International Hospital, Islamabad, were included in this study. The indications, technique, complications and follow up were reviewed., Results: A total of 182 persons underwent this procedure. There were 118 (65.0%) males and 64 (35.0%) females. Age ranged from 55-86 years. One hundred seventy five (96.0%) patients had cerebro-vascular accident, five (2.75%) had Parkinson's disease and two (1.25%) malignancy. More than 99% procedures were successful and no procedure related mortality was noted. Mild PEG site infections were encountered in eighteen (9.8%) patients which were manageable with local treatment and oral antibiotics. Four (2.2%) patients had severe tube site infection and needed parenteral antibiotics. Tube dislodgement took place in five (2.75%) patients and had to be removed and reinserted. Thirty days follow up was uneventful with regard to the tube. Longest follow up was 736 days., Conclusion: Percutaneous endoscopic gastrostomy had proved a viable means of enteral nutrition in patients with neurological impairment. Complications were insignificant. However, patient and care giver's education could be improved for more effective tube management, and prevention of PEG insertion site infection.

Objective: To study the efficacy of antiviral drug lamivudine for treatment of chronic hepatitis B in Pakistani population., Design: Experimental study., Place and Duration of Study: A tertiary Care Center, from January 2000 to January 2004., Patients and Methods: Patients who had positive hepatitis B surface antigen (HBsAg) and elevated ALT were recruited. Both hepatitis Be antigen (HBeAg) positive and HBeAg negative patients were included. Treatment was given with lamivudine 100mg once a day for 12 months and they were followed every 2-3 months with ALT, HBeAg, and in certain cases with, HBV DNA (PCR). Those patients who were HBeAg positive were considered to have achieved end of treatment response (ETR) when ALT was normal and HBeAg was negative and this was documented at two consecutive occasions, at least one month apart. Those patients who were HBeAg negative were considered to have achieved ETR when their ALT was normal and HBV DNA was undetectable. Patients were followed for further 6 months to evaluate for any relapse., Results: A total of 105 patients were included. Eighty-two (78.1%) were males and 23 (21.9%) were females. Mean ALT of all patients was 87.15 +/- 46.01 u/dl. Seventy-two HBeAg positive and 15 HBeAg negative patients completed treatment with 23.6 % and 80.0 % response rate respectively (p <0.001)., Conclusion: The response of lamivudine in chronic hepatitis B in Pakistani population is encouraging in HBeAg negative patients but is rather low in those who are HBeAg positive.

We present the case of a 49 years old male, who was HCV RNA positive with an ALT of 260, showing no response to standard interferon (INF) plus ribavirin treatment for 48 weeks and to a further therapy with high dose standard IFN plus ribavirin for 24 weeks. Pegylated (PEG) IFN was then started at a dose of 80 mcg/week with ribavirin, but proved to be of no avail. Finally, a dose of 120 mcg/week of PEG INF and ribavirin was administered for 48 weeks. This resulted in negative HCV RNA PCR after two months and remained undetectable with normal ALT. A six-month follow-up showed a sustained viral response.

A recently reported crystal structure of an intact bacterial group I self-splicing intron in complex with both its exons provided the first molecular view into the mechanism of RNA splicing. This intron structure, which was trapped in the state prior to the exon ligation reaction, also reveals the architecture of a complex RNA fold. The majority of the intron is contained within three internally stacked, but sequence discontinuous, helical domains. Here the tertiary hydrogen bonding and stacking interactions between the domains, and the single-stranded joiner segments that bridge between them, are fully described. Features of the structure include: (1) A pseudoknot belt that circumscribes the molecule at its longitudinal midpoint; (2) two tetraloop-tetraloop receptor motifs at the peripheral edges of the structure; (3) an extensive minor groove triplex between the paired and joiner segments, P6-J6/6a and P3-J3/4, which provides the major interaction interface between the intron's two primary domains (P4-P6 and P3-P9.0); (4) a six-nucleotide J8/7 single stranded element that adopts a mu-shaped structure and twists through the active site, making critical contacts to all three helical domains; and (5) an extensive base stacking architecture that realizes 90% of all possible stacking interactions. The intron structure was validated by hydroxyl radical footprinting, where strong correlation was observed between experimental and predicted solvent accessibility. Models of the pre-first and pre-second steps of intron splicing are proposed with full-sized tRNA exons. They suggest that the tRNA undergoes substantial angular motion relative to the intron between the two steps of splicing.

Objective: To develop non-invasive laboratory variables for the identification of esophageal varices in patients with cirrhosis at Digestive Disease Center, Shifa International Hospital, Islamabad., Patients and Methods: All patients with chronic viral hepatitis who presented to the Gastroenterology Division between July 2002 to July 2003 were enrolled in the study. All patients with a diagnosis of Chronic Hepatitis who had platelet counts of 140,000, I.N.R of greater than 1.5 and portal vein (PV) diameter of 13 mm or greater were included in the study. All patients underwent endoscopy to see the presence of varices which were graded following the standard criteria, Results: Of 140 patients, 100 (71%) were males and 40 (29%) females. Esophageal varices were present in 70% of the patients while 30% had no varices., Conclusion: It was seen that using the standard criteria of a PV diameter 13 mm, I.N.R 1.5 and platelet counts 100,000 for the diagnosis of portal hypertension about 70% patients had endoscopic evidence of esophageal varices. As prophylactic beta blockers are recommended to reduce the chances of a bleed from esophageal varices therefore it is suggested that endoscopy should be done only in patients who fulfill the standard criteria of portal hypertension and those found to have varices should be put on beta blockers.

Objective: To determine the prevalence of hepatitis C virus (HCV) and hepatitis B virus (HBV) infection by detection of anti-HCV and hepatitis B surface antigen (HbsAg) in general population of Pakistan., Design: Observational., Place and Duration of Study: Community clinic of Shifa International Hospital, Islamabad, from January 1998 to June 2004., Materials and Methods: Sera of healthy adult individuals who presented for medical evaluation as a pre-employment criteria in the Gulf region were examined for presence of hepatitis B surface antigen and anti-HCV antibody. Alanine aminotransferase levels were also determined., Results: A total of 47,538 individuals were examined. Out of these, 2528 (5.31%) were positive for anti-HCV and 1221 (2.56%) individuals had positive HBsAg. Hepatitis B surface antigen and anti-HCV both were found in 92 (0.19%) individuals. Mean age of subjects, positive for HCV antibody was 44 years and 40.5 years for HBV. Ninety-four percent individuals were males and 6% were females. Alanine aminotransferase (ALT) was normal in 56 % of subjects with positive HCV and 84% of individuals with HBV., Conclusion: This study which evaluated predominantly a healthy male population, showed a high seroprevalence of anti-HCV and average seroprevalence of hepatitis B virus infection. A large majority of these patients was young and had normal ALT.

Objective: To evaluate the serological profile of patients with incidentally detected positive hepatitis-B surface antigen (HBsAg) and to asses the risk factors., Design: An observational study., Place and Duration of Study: This study was conducted at Shifa International Hospital, Islamabad from 1999 to 2003., Patients and Methods: All patients who presented to gastroenterology clinic of Shifa International Hospital, Islamabad with positive HbsAg, detected incidentally, were tested for alamine transaminase (ALT), hepatitis Beantigen (HbeAg) and in certain cases hepatitis-B virus DNA (HBV DNA) by polymerase chain reaction (PCR). Their risk factors for acquisition of infection were assessed with specific questions., Results: A total of 224 patients were examined. One hundred sixty-four (73.2%) were male and 60 (26.8%) female. Mean age fo all the subjects was 32.45 -/+ 11.85 years. Out of 224 patients, 48 (21.4%) were positive for HBeAg and 176 (78.6%) were negative. Out of 48 subjects who were positive for HBeAg, 36 underwent HBV DNA determination and 32 (88.8%) were positive for HBV DNA. Out of 176 subjects who had negative HBeAg, 46 had elevated ALT and in those HBV DNA was performed and 14 had positive HBV DNA. Most common risk factors detected in these patients were intramuscular injections and surgery, however, in a large number, risk factors were unknown., Conclusion: Twenty-one percent asymptomatic subjects with positive HBsAg were found to be HBeAg positive. A large number of subjects with negative HBeAg had HBV DNA positive suggesting presence of precore mutants. Intramuscular injections and surgery were noted to be frequent risk factors in these subjects.

Objective: To find out if advanced liver disease causes decrement in health related quality of life in Pakistani patients., Methods: An observational study was conducted at the Shifa International Hospital Islamabad. Chronic Liver Disease Questionnaire (CLDQ) was administered to patients with liver disease who were either admitted or seen as outpatients. Patients were divided into two groups. Those with no cirrhosis or Child's Class A were placed into group A. Those with advanced liver disease i.e. Child's Class B and C into group B., Results: A total of 56 patients (mean age 50.6 years) were included in this study. Hepatitis C was the cause of chronic liver disease (CLD) in 52 (92.9%) patients. Twenty-one (37.5%) patients were in group A and 35 (62.5%) in group B. Patients with advanced liver disease had significantly lower CLDQ scores as well as decrement in five out of the six categories for assessing quality of life., Conclusion: Chronic liver disease causes significant impairment in the Quality of Life Index (QLI) in Pakistani patients.

Objective: To review the experience of patients presenting with clinical manifestations of tropical sprue and assess their diagnosis and management, response to treatment and follow up., Methods: This single center retrospective descriptive study was done at Shifa International Hospital, Islamabad from January 1994 to January 2003. All patients who presented with diarrhea, anorexia, weight loss and anemia and who proved to have partial villous atrophy on intestinal biopsy and had responded to treatment with antibiotic and folic acid,were included in this review., Results: A total of 42 patients were encountered during these years. There were 31 (74.0%) males and 11 (26%) females. The age ranged from 17-66 years. All patients presented with diarrhea, weight loss, anorexia and had megaloblastic anemia. In all patients, a distal duodenal biopsy showed partial villous atrophy. All were treated with tetracycline 1 g per day and folic acid 5 mg per day and responded to treatment within 4 weeks. Total treatment lasted 3 months and resulted in complete resolution of symptoms and gain of weight. The follow up lasted for a mean of 5 years and no relapses were noted., Conclusion: Tropical sprue presents with diarrhea, anorexia, weight loss, and megaloblastic anemia. The partial villous atrophy has been a constant finding. The response to treatment to tetracycline and folic acid has been uniformly successful.

Objective: To review the experience of endoscopic dilation of esophageal strictures at a tertiary care hospital., Design: Descriptive study., Place and Duration of Study: Shifa International Hospital, Islamabad January 1985 to January 2003., Materials and Methods: Patients presenting with dysphagia who presented at our institution during the past 8 years were reviewed. Their etiology and the number of dilations required for improvement in dysphagia and follow up with any complications were reviewed. All procedures were performed with savary dilators using guide wire without fluoroscopy., Results: A total of 369 patients were seen during this period and 583 procedures were performed. Two hundred thirty-five (63.7%) patients had peptic stricture, 123 (33.3%) had malignant strictures, 08 (2.2%) had non obstructive dysphagia and 6 (1.6%) had corrosive ingestions. The age ranged from 28-68 years. There were 254 (88.5%) males and 115 (31.2%) females. Sixty-seven (11.5%) procedures were performed as inpatient and 516 (88.5%) procedures were done as outpatient. Two to three attempts at dilatation were required to achieve satisfactory swallowing. There were no serious complication. Follow-up period was 6-8 weeks on an average., Conclusion: Most common esophageal stricture found in our practice were peptic strictures. Dilation with savary system was safe and effective and was able to relieve dysphagia in large number of patients. No fluoroscopic guidance was needed.

Background: The aim of this study was to review etiological and clinical features of patients with hepatocellular carcinoma (HCC) at a tertiary care centre in past nine years. Relevant data on HCC in other parts of country and world were reviewed., Methods: Patients who had biopsy proven HCC were reviewed retrospectively. Demographic features were noted and positivity for serology. presence of cirrhosis. level of alpha-fetoprotein, tumour size and distribution of liver lesions were noted., Results: A total of 67 patients were found to have biopsy proven HCC. Mean age was 58.64 +/- 12.77 years. Males were 79%. Hepatitis B surface antigen was noted to be positive in 23% of the patients, who were tested and hepatitis C antibody was found to be positive in 67% of the patients who were tested. Alpha fetoprotein level was 632.09 +/- 1332.31. Cirrhosis was noted in 69% patients. Tumour size in patients with single lesion was 6.6 +/- 1.14 cm. Patients with single lesion had 70% time involvement of the left lobe and 30% times had involvement of the right lobe. Fifty one percent of the patients in this series had multilocular distribution., Conclusion: Hepatocellular carcinoma has become a common tumour in Pakistan and studies are showing that this cancer is related to hepatitis C virus infection in majority of the patients. A large number of them have underlying cirrhosis and are multifocal in origin and are presented in an advanced condition.

The cationic lipids 1,2-dioleoyl-3-trimethylammonium-propane and dimethyldioctadecylammonium bromide, with or without the helper lipids 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine or cholesterol, and the cationic polymer polyethyleneimine, were compared for their ability to displace fluorescent dyes from DNA. Differences in displacement of the intercalating dyes ethidium bromide and ethidium homodimer correlate with their relative affinities with DNA, with the extent of ethidium homodimer displacement significantly less. Differences in ethidium homodimer and ethidium bromide displacement as a function of the ratio of polycation to DNA and the charge density of the polycation suggest a greater sensitivity of the former to topological changes in condensed DNA. Marked differences in the ability of these cationic delivery systems to displace the minor groove binding dyes 4',6-diamidino-2-phenylindole and Hoechst 33258 upon interaction with DNA are also apparent, with the majority of Hoechst 33258 remaining bound to DNA. Changes in the spectral properties of Hoechst 33258 were further used to characterize polycation-induced changes in solvent accessibility of the DNA minor groove. Taken together, these studies demonstrate differences in the interaction of various cationic lipids and polyethyleneimine in terms of regional displacement of dyes, polycation-induced structural changes in DNA, as well as polycation-mediated changes in solvent accessibility of the minor groove. The relevance of these studies to current models of the structure and assembly of polycation/DNA complexes are discussed., (Copyright 2003 Wiley-Liss, Inc.)

The interaction of cationic liposomes with supercoiled plasmid DNA results in a major rearrangement of each component to form compact multilamellar structures comprised of alternating layers of two-dimensional arrays of DNA sandwiched between lipid bilayers. Fluorescence resonance energy transfer was used to estimate the distance of closest approach of DNA to the lipid bilayers in these complexes. The effect of several compositional variables on this distance, including the ratio of cationic lipid to DNA, and the charge density, intrinsic curvature, and fluidity of the lipid bilayer were examined. Additionally, the effect of ionic strength was studied. For complexes prepared at or above a 3:1 charge ratio (+/-), the observed distance of closest approach was found to be in agreement with the intercalation of DNA between lipid bilayers. As the charge ratio was decreased, a monotonic increase in the distance was observed with a maximum observed at 0.5:1. Correlations between differences in the proximity of DNA to the lipid bilayer and the hydrodynamic size of the complexes were also found. A model based on these observations and previous reports suggests the formation of discrete populations of complexes below a charge ratio of 0.5:1 and above 3:1. The structure of the negatively charged complexes is consistent with DNA extending from the surface of the particles, whereas those possessing excess positive charge were multilamellar aggregates with the DNA effectively condensed between lipid bilayers. Complexes between these two states consist of weighted fractions of these two species.