Nonlinear measures of respiration: Respiratory irregularity and increased chaos of respiration in patients with panic disorder

Background. Respiratory irregularity has been previously reported in patients with panic disorder using time domain measures. However, the respiratory signal is not entirely linear and a few previous studies used approximate entropy (APEN), a measure of regularity of time series. We have been studying APEN and other nonlinear measures including a measure of chaos, the largest Lyapunov exponent (LLE) of heart rate time series, in some detail. In this study, we used these measures of respiration to compare normal controls (n = 18) and patients with panic disorder (n = 22) in addition to the traditional time domain measures of respiratory rate and tidal volume. Methods: Respiratory signal was obtained by the Respitrace system using a thoracic and an abdominal belt, which was digitized at 500 Hz. Later, the time series were constructed at 4 Hz, as the highest frequency in this signal is limited to 0.5 Hz. We used 256 s of data (1,024 points) during supine and standing postures under normal breathing and controlled breathing at 12 breaths/min. Results: APEN was significantly higher in patients in standing posture during normal as well as controlled breathing (p = 0.002 and 0.02, respectively). LLE was also significantly higher in standing posture during normal breathing (p = 0.009). Similarly, the time domain measures of standard deviations and the coefficient of variation (COV) of tidal volume (TV) were significantly higher in the patient group (p = 0.02 and 0.004, respectively). The frequency of sighs was also higher in the patient group in standing posture (p = 0.02). In standing posture, LLE (p < 0.05) as well as APEN (p < 0.01) contributed significantly toward the separation of the two groups over and beyond the linear measure, i.e. the COV of TV. Conclusion: These findings support the previously described respiratory irregularity in patients with panic disorder and also illustrate the utility of nonlinear measures such as APEN and LLE as additional measures toward a better understanding of the abnormalities of respiratory physiology in similar patient populations as the correlation between LLE, APEN and some of the time domain measures only explained up to 50-60% of the variation. Copyright (C) 2002 S. Karger AG, Basel.

Development of recombinant coat protein antibody based IC-RT-PCR for detection and discrimination of sugarcane streak mosaic virus isolates from Southern India

Sugarcane streak mosaic virus (SCSMV), causes mosaic disease of sugarcane and is thought to belong to a new undescribed genus in the family Potyviridae. The coat protein (CP) gene from the Andhra Pradesh (AP) isolate of SCSMV (SCSMV AP) was cloned and expressed in Escherichia coli. The recombinant coat protein was used to raise high quality antiserum. The CP antiserum was used to develop an immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) based assay for the detection and discrimination of SCSMV isolates in South India. The sequence of the cloned PCR products encoding 3'untranslated region (UTR) and CP regions of the virus isolates from three different locations in South India viz. Tanuku (Coastal Andhra Pradesh), Coimbatore (Tamil Nadu) and Hospet (Karnataka) was compared with that of SCSMV AP The analysis showed that they share 89.4, 89.5 and 90% identity respectively at the nucleotide level. This suggests that the isolates causing mosaic disease of sugarcane in South India are indeed strains of SCSMV In addition, the sensitivity of the IC-RT-PCR was compared with direct antigen coating-enzyme linked immunosorbent assay (DAC-ELISA) and dot-blot immunobinding assays and was found to be more sensitive and hence could be used to detect the presence of virus in sugarcane breeding, germplasm centres and in quarantine programs.

Interplay between NS3 protease and human La protein regulates translation-replication switch of Hepatitis C virus

HCV NS3 protein plays a central role in viral polyprotein processing and RNA replication. We demonstrate that the NS3 protease (NS3(pro)) domain alone can specifically bind to HCV-IRES RNA, predominantly in the SLIV region. The cleavage activity of the NS3 protease domain is reduced upon HCV-RNA binding. More importantly, NS3(pro) binding to the SLIV hinders the interaction of La protein, a cellular IRES-trans acting factor required for HCV IRES-mediated translation, resulting in inhibition of HCV-IRES activity. Although overexpression of both NS3(pro) as well as the full length NS3 protein decreased the level of HCV IRES mediated translation, replication of HCV replicon RNA was enhanced significantly. These observations suggest that the NS3(pro) binding to HCV IRES reduces translation in favor of RNA replication. The competition between the host factor (La) and the viral protein (NS3) for binding to HCV IRES might regulate the molecular switch from translation to replication of HCV.

Modelling and classification of respiratory volume wavefourms

A technique is proposed for classifying respiratory volume waveforms(RVW) into normal and abnormal categories of respiratory pathways. The proposed method transforms the temporal sequence into frequency domain by using an orthogonal transform, namely discrete cosine transform (DCT) and the transformed signal is pole-zero modelled. A Bayes classifier using model pole angles as the feature vector performed satisfactorily when a limited number of RVWs recorded under deep and rapid (DR) manoeuvre are classified.

Investigations on the RNA binding and phosphorylation of groundnut bud necrosis virus nucleocapsid protein

Groundnut bud necrosis virus belongs to the genus Tospovirus, infects a wide range of crop plants and causes severe losses. To understand the role of the nucleocapsid protein in the viral life cycle, the protein was overexpressed in E. coli and purified by Ni-NTA chromatography. The purified N protein was well folded and was predominantly alpha-helical. Deletion analysis revealed that the C-terminal unfolded region of the N protein was involved in RNA binding. Furthermore, the N protein could be phosphorylated in vitro by Nicotiana benthamiana plant sap and by purified recombinant kinases such as protein kinase CK2 and calcium-dependent protein kinase. This is the first report of phoshphorylation of a nucleocapsid protein in the family Bunyaviridae. The possible implications of the present findings for the viral life cycle are discussed.

Advances in the Mathematical Modelling of Hepatitis C Virus Dynamics

Mathematical models have provided key insights into the pathogenesis of hepatitis C virus (HCV) in vivo, suggested predominant mechanism(s) of drug action, explained confounding patterns of viral load changes in HCV infected patients undergoing therapy, and presented a framework for therapy optimization. In this article, I present an overview of the major advances in the mathematical modeling of HCV dynamics.

Mathematical Model of Viral Kinetics In Vitro Estimates the Number of E2-CD81 Complexes Necessary for Hepatitis C Virus Entry

Interaction between the hepatitis C virus (HCV) envelope protein E2 and the host receptor CD81 is essential for HCV entry into target cells. The number of E2-CD81 complexes necessary for HCV entry has remained difficult to estimate experimentally. Using the recently developed cell culture systems that allow persistent HCV infection in vitro, the dependence of HCV entry and kinetics on CD81 expression has been measured. We reasoned that analysis of the latter experiments using a mathematical model of viral kinetics may yield estimates of the number of E2-CD81 complexes necessary for HCV entry. Here, we constructed a mathematical model of HCV viral kinetics in vitro, in which we accounted explicitly for the dependence of HCV entry on CD81 expression. Model predictions of viral kinetics are in quantitative agreement with experimental observations. Specifically, our model predicts triphasic viral kinetics in vitro, where the first phase is characterized by cell proliferation, the second by the infection of susceptible cells and the third by the growth of cells refractory to infection. By fitting model predictions to the above data, we were able to estimate the threshold number of E2-CD81 complexes necessary for HCV entry into human hepatoma-derived cells. We found that depending on the E2-CD81 binding affinity, between 1 and 13 E2-CD81 complexes are necessary for HCV entry. With this estimate, our model captured data from independent experiments that employed different HCV clones and cells with distinct CD81 expression levels, indicating that the estimate is robust. Our study thus quantifies the molecular requirements of HCV entry and suggests guidelines for intervention strategies that target the E2-CD81 interaction. Further, our model presents a framework for quantitative analyses of cell culture studies now extensively employed to investigate HCV infection.

Ribosome-RNA interaction: a potential target for developing antiviral against hepatitis C virus

Hepatitis C virus (HCV), a member of Flaviviridae, encoding a positive-sense single-stranded RNA translates by cap-independent mechanism using the internal ribosome entry site (IRES) present in the 5' UTR of the virus. The IRES has complex stem loop structures and is capable of recruiting the 40S ribosomal subunit in a factor-independent fashion. As the IRES sequence is highly conserved throughout the HCV genotypes and the translation is the first obligatory step of the HCV life cycle, the IRE'S-mediated translation, or more specifically, the ribosome HCV RNA interaction is an attractive target to design effective antivirals. This article will focus on the mechanism of the HCV IRES translation and the various ways in which the interaction of ribosome and IRES has been targeted.

Hypoxia-Mediated Impairment of the Mitochondrial Respiratory Chain Inhibits the Bactericidal Activity of Macrophages

In infected tissues oxygen tensions are low. As innate immune cells have to operate under these conditions, we analyzed the ability of macrophages (M phi) to kill Escherichia coli or Staphylococcus aureus in a hypoxic microenvironment. Oxygen restriction did not promote intracellular bacterial growth but did impair the bactericidal activity of the host cells against both pathogens. This correlated with a decreased production of reactive oxygen intermediates (ROI) and reactive nitrogen intermediates. Experiments with phagocyte NADPH oxidase (PHOX) and inducible NO synthase (NOS2) double-deficient M phi revealed that in E. coli- or S. aureus-infected cells the reduced antibacterial activity during hypoxia was either entirely or partially independent of the diminished PHOX and NOS2 activity. Hypoxia impaired the mitochondrial activity of infected M phi. Inhibition of the mitochondrial respiratory chain activity during normoxia (using rotenone or antimycin A) completely or partially mimicked the defective antibacterial activity observed in hypoxic E. coli-or S. aureus-infected wild-type M phi, respectively. Accordingly, inhibition of the respiratory chain of S. aureus-infected, normoxic PHOX-/- NOS2(-/-) M phi further raised the bacterial burden of the cells, which reached the level measured in hypoxic PHOX-/- NOS2(-/-) M phi cultures. Our data demonstrate that the reduced killing of S. aureus or E. coli during hypoxia is not simply due to a lack of PHOX and NOS2 activity but partially or completely results from an impaired mitochondrial antibacterial effector function. Since pharmacological inhibition of the respiratory chain raised the generation of ROI but nevertheless phenocopied the effect of hypoxia, ROI can be excluded as the mechanism underlying the antimicrobial activity of mitochondria.

Sesbania Mosaic Virus (SeMV) Infectious Clone: Possible Mechanism of 3 ` and 5 ` End Repair and Role of Polyprotein Processing in Viral Replication

Sesbania mosaic virus (SeMV) is a positive stranded RNA virus belonging to the genus Sobemovirus. Construction of an infectious clone is an essential step for deciphering the virus gene functions in vivo. Using Agrobacterium based transient expression system we show that SeMV icDNA is infectious on Sesbania grandiflora and Cyamopsis tetragonoloba plants. The efficiency of icDNA infection was found to be significantly high on Cyamopsis plants when compared to that on Sesbania grandiflora. The coat protein could be detected within 6 days post infiltration in the infiltrated leaves. Different species of viral RNA (double stranded and single stranded genomic and subgenomic RNA) could be detected upon northern analysis, suggesting that complete replication had taken place. Based on the analysis of the sequences at the genomic termini of progeny RNA from SeMV icDNA infiltrated leaves and those of its 3' and 5' terminal deletion mutants, we propose a possible mechanism for 3' and 5' end repair in vivo. Mutation of the cleavage sites in the polyproteins encoded by ORF 2 resulted in complete loss of infection by the icDNA, suggesting the importance of correct polyprotein processing at all the four cleavage sites for viral replication. Complementation analysis suggested that ORF 2 gene products can act in trans. However, the trans acting ability of ORF 2 gene products was abolished upon deletion of the N-terminal hydrophobic domain of polyprotein 2a and 2ab, suggesting that these products necessarily function at the replication site, where they are anchored to membranes.