Transition from curvature sensing to generation in a vesicle driven by protein binding strength and membrane tension.

The ability of proteins to sense and/or generate membrane curvature is crucial for many biological processes inside the cell. We introduce a model for the binding and unbinding of curvature inducing proteins on vesicles using Dynamic Triangulation Monte Carlo (DTMC) simulations. In our study, the interaction between membrane curvature and protein binding is characterised by the binding affinity parameter µ, which indicates the interaction strength. We demonstrate that both sensing and generation of curvature can be observed in the same system as a function of the protein binding affinity on the membrane. Our results show that at low µ values, proteins only sense membrane curvature, whereas at high µ values, they induce curvature. The transition between sensing and generation regimes is marked by a sharp change in the µ-dependence of the protein bound fraction. We present ways to quantitatively characterise these two regimes. We also observe that imposing tension on the membrane (through internal excess pressure for liposomes) extends the region of curvature sensing in the parameter space.

Membrane disruption and anti-quorum sensing effects of synergistic interaction between Lavandula angustifolia (lavender oil) in combination with antibiotic against plasmid-conferred multi-drug-resistant Escherichia coli.

AIM: The aim of this study was to investigate the mode of action of the lavender essential oil (LV) on antimicrobial activity against multi-drug-resistant Escherichia coli J53 R1 when used singly and in combination with piperacillin. METHOD AND RESULTS: In the time-kill analysis, a complete killing of bacteria was observed based on colony counts within 4 h when LV was combined with piperacillin during exposure at determined FIC concentrations. Analysis of the membrane permeabilizing effects of LV on treated cultures through their stability against sodium dodecyl sulphate revealed that the LV played a role in disrupting the bacterial cell membrane. The finding is further supported by scanning electron microscopy analysis and zeta potential measurement. In addition, reduction in light production expression of E. coli [pSB1075] by the LV showed the presence of potential quorum sensing (QS) inhibitors. CONCLUSIONS: These results indicated that the LV has the potential to reverse bacterial resistance to piperacillin in E. coli J53 R1. It may operate via two mechanisms: alteration of outer membrane permeability and inhibition of bacterial QS. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings offer a novel approach to develop a new option of phytopharmaceuticals against multi-drug-resistant E. coli.

Maternal Uterine Artery Adenoviral Vascular Endothelial Growth Factor (Ad.VEGF-A165) Gene Therapy Normalises Fetal Brain Growth and Microglial Activation in Nutrient Restricted Pregnant Guinea Pigs.

Fetal growth restriction (FGR) is associated with uteroplacental insufficiency, and neurodevelopmental and structural brain deficits in the infant. It is currently untreatable. We hypothesised that treating the maternal uterine artery with vascular endothelial growth factor adenoviral gene therapy (Ad.VEGF-A165) normalises offspring brain weight and prevents brain injury in a guinea pig model of FGR. Pregnant guinea pigs were fed a restricted diet before and after conception and received Ad.VEGF-A165 (1 × 1010 viral particles, n = 18) or vehicle (n = 18), delivered to the external surface of the uterine arteries, in mid-pregnancy. Pregnant, ad libitum-fed controls received vehicle only (n = 10). Offspring brain weight and histological indices of brain injury were assessed at term and 5-months postnatally. At term, maternal nutrient restriction reduced fetal brain weight and increased microglial ramification in all brain regions but did not alter indices of cell death, astrogliosis or myelination. Ad.VEGF-A165 increased brain weight and reduced microglial ramification in fetuses of nutrient restricted dams. In adult offspring, maternal nutrient restriction did not alter brain weight or markers of brain injury, whilst Ad.VEGF-A165 increased microglial ramification and astrogliosis in the hippocampus and thalamus, respectively. Ad.VEGF-A165 did not affect cell death or myelination in the fetal or offspring brain. Ad.VEGF-A165 normalises brain growth and markers of brain injury in guinea pig fetuses exposed to maternal nutrient restriction and may be a potential intervention to improve childhood neurodevelopmental outcomes in pregnancies complicated by FGR.

Effect of temperature, relative humidity and rainfall on rotavirus infections in Kolkata, India.

Rotavirus is a common viral cause of severe diarrhoea. For the underlying cause of rotavirus seasonality, the meteorological factor has been suspected, whereas quantitative correlation between seasonality and meteorological factor has not been fully investigated. In this study, we investigated the correlation of temporal patterns of the isolation rate of rotavirus with meteorological condition (temperature, relative humidity, rainfall) in Kolkata, India. We used time-series analysis combined with spectral analysis and least squares method. A 1-year cycle explained underlying variations of rotavirus and meteorological data. The 1-year cycle for rotavirus data was correlated with an opposite phase to that for meteorological data. Relatively high temperature could be associated with a low value of isolation rate of rotavirus in the monsoon season. Quantifying a correlation of rotavirus infections with meteorological conditions might prove useful in predicting rotavirus epidemics and health services could plan accordingly.

Molecular epidemiology of norovirus infections in children and adults: sequence analysis of region C indicates genetic diversity of NVGII strains in Kolkata, India.

Faecal specimens of diarrhoea cases (n=2495, collected between November 2007 and October 2009) from Infectious Diseases and Beliaghata General (ID&BG) Hospital, Kolkata, India, were screened by RT-PCR using specific primers targeting region C of the capsid gene of noroviruses (NoVs) to determine the seasonal distribution and clinical characteristics of NoVs associated with diarrhoea. NoV infection was detected in 78 cases, mostly in children aged <2 years. In 22/78 positive cases, the virus was detected as the sole agent; others were as mixed infections with other enteric pathogens. Sequencing of NVGII strains showed clustering with GII.4 NoVs followed by GII.13 and GII.6 NoVs. Clinical characteristics of the diarrhoeic children and adults in Kolkata indicated that NoV infections were detected throughout the year and were associated with a mild degree of dehydration.

Molecular characterization of the VP1, VP2, VP4, VP6, NSP1 and NSP2 genes of bovine group B rotaviruses: identification of a novel VP4 genotype.

Studies on bovine group B rotaviruses (GBRs) are limited. To date, only the VP6 gene of a single bovine GBR strain and the VP7 and NSP5 genes of a few bovine GBR strains have been sequenced and analyzed. In the present study, using a single-primer amplification method, we have determined the full-length nucleotide sequences of the VP1, VP2, VP4, VP6, NSP1 and NSP2 genes of three bovine GBR strains from eastern India. In all six of these genes, the bovine GBR strains shared high genetic relatedness among themselves but exhibited high genetic diversity with cognate genes of human, murine and ovine GBRs. Interestingly, as with group A rotaviruses, the bovine GBR VP1, VP2, VP6 and NSP2 genes appeared to be more conserved than the VP4 and NSP1 genes among strains of different species. The present study provides important insights into the genetic makeup and diversity of bovine GBRs, and also identifies a novel GBR VP4 genotype.

Full genomic analysis and possible origin of a porcine G12 rotavirus strain RU172.

Human group A rotavirus (GAR) G12 strains are regarded as potentially important pathogens for acute gastroenteritis. On the other hand, to date, the only report of detection of G12 in animals was that of a porcine G12P[7] strain RU172. Strain RU172 formed a separate G12 lineage, distinct from human G12 strains, and by analyses of deduced amino acid sequences, had a VP4, VP6, NSP4-5 of porcine origin. In the present study, we determined the full-length nucleotide sequences of VP1, VP3, and NSP1-3 genes and nearly full-length nucleotide sequence of VP2 gene of RU172. By nucleotide sequence identities and phylogenetic analyses, the VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5 genes of RU172 were assigned to G12-P[7]-I5-R1-C1-M1-A1-N1-T1-E1-H1 genotypes, respectively. Within their respective genotypes, (i) VP1 gene of RU172 exhibited higher genetic relatedness to Wa-like human G12 GARs than porcine strains, (ii) VP2-3 and NSP2 genes clustered separately from the Wa-like human (including G12) and porcine clusters, while (iii) the VP6, NSP1 and NSP3-5 genes clustered with porcine and porcine-like human strains. These observations suggested that (i) the porcine G12 strain might have originated from porcine-human reassortment events, or alternatively, (ii) the Wa-like human and porcine G12 strains might have originated from a common ancestor, and eventually evolved (by genetic drift and shift) with time. Our findings provided important insights into the possible patterns of evolution of the porcine G12 strain.

Emergence of Noroviruses homologous to strains reported from Djibouti (horn of Africa), Brazil, Italy, Japan and USA among children in Kolkata, India.

AIM: A total of 625 faecal specimens of diarrheic cases (n-313) and non diarrheic controls (n-312), were screened by RT-PCR to detect Noroviruses in children aged below 5 years in Kolkata, India. MATERIALS AND METHODS: Out of the 313 fecal specimens (cases) screened using CDC primer set, 10 (3.19%) showed amplification in reverse transcription-polymerase chain reaction (RT-PCR) for Norovirus. These included 5 of 260 (1.92%) from hospitalized and 5 of 53 (9.43%) from out patients departament (OPD) cases. RESULTS: Nine (90%) of Norovirus positive cases belonged to genogroup GII and one specimen (10%) was positive for genogroup GI. Among the 312 non diarrheic controls 2 (0.63%) were positive for Norovirus GII. Partial RNA dependent RNA polymerase gene (RdRp) sequences corresponding to the six Norovirus GII positive samples showed homology to the sequences of Djibouti (horn of Africa), Brazil, Italy, Japan and US norovirus strains. CONCLUSION: This study shows the detection of newly emerging Norovirus strains among diarrheic and non diarrheic children in Kolkata.

A study on the possibility of zoonotic infection in rotaviral diarrhoea among calves and buffalo calves in and around Kolkata, India.

Group A and group B rotaviruses are important diarrhea causing agents among calves and buffalo calves. Epidemiological studies in Indian calves revealed the predominance of group A rotavirus strains with G6, G8, and G10 specificity and group B rotaviruses. A total of 95 fecal samples were collected from calves and buffalo calves affected with diarrhea from an unorganized cattle farm and two cattle markets in and around Kolkata, in the state of West Bengal of Eastern India. Rotaviruses were detected in 23.15% (22/95) samples by polyacrylamide gel electrophoresis. Of 22 rotavirus positive cases, 10.52% (10/95) samples showed characteristic group A rotavirus-like long type electropherotype (e-type) pattern and 4.21% (4/95) samples showed the characteristic group B rotavirus long type of electropherotype pattern and in 8.42% (8/95) the electropherotype pattern could not be recorded. Out of 22 positive samples, 7 samples of group A rotaviruses were subjected to reverse transcription-polymerase chain reaction, using VP7 generic and genotype [G type] specific primers and 2 of 7 isolates were identified as G10.

Thermal and active fluctuations of a compressible bilayer vesicle.

We discuss thermal and active fluctuations of a compressible bilayer vesicle by using the results of hydrodynamic theory for vesicles. Coupled Langevin equations for the membrane deformation and the density fields are employed to calculate the power spectral density matrix of membrane fluctuations. Thermal contribution is obtained by means of the fluctuation dissipation theorem, whereas active contribution is calculated from exponentially decaying time correlation functions of active random forces. We obtain the total power spectral density as a sum of thermal and active contributions. An apparent response function is further calculated in order to compare with the recent microrheology experiment on red blood cells. An enhanced response is predicted in the low-frequency regime for non-thermal active fluctuations.

Changing pattern of human group A rotaviruses: emergence of G12 as an important pathogen among children in eastern India.

BACKGROUND: Rotavirus genotypes, G1-G4 and G9 are associated with childhood diarrhoea throughout the world. In our previous study, we detected G1, G2, G4 and three G12 strains from Kolkata, India. OBJECTIVES: To study the prevalence of G- and P-genotypes of rotaviruses associated with dehydrating diarrhoea in children admitted to two leading hospitals in eastern India. STUDY DESIGN: An active surveillance was conducted for elucidation of rotavirus infection in two leading hospitals in Kolkata, West Bengal and Berhampur (GM), Orissa, India, separated by 603km from January 2003 to April 2005. The rotaviruses were detected by RNA electrophoresis in polyacrylamide gels. G- and P-typing of the positive samples were accomplished by amplifying VP7 and VP4 genes by RT-PCR and genotyped by seminested multiplex PCR methods. Sequencing, sequence analysis and phylogenetic analysis of VP7 genes of G12 strains were carried out to understand the variations between the strains isolated from different parts of the world. RESULTS: The genotypic distribution varied remarkably from our earlier study period (1998-2001) with G1 (53.8%) being the most predominant strain followed by G2 (22.5%), G12 (17.1%), G9 (2.1%) and not a single G3 or G4 isolate was detected separately. 35.2% samples exhibited mixed P-types followed by P[4] (31.7%), P[8] (21.8%) and P[6] (9.8%). The phylogenetic analysis of G12 strains revealed that the G12 strains detected from different parts of the world clustered into three different lineages. Though VP7 sequences of G12 strains isolated from Kolkata and Berhampur are conserved, their P-types were different. CONCLUSION: During this study period we reported emergence of G12 strains as an important pathogen among children in eastern India, thus necessitating its inclusion in future polyvalent vaccine to control rotavirus diarrhoea.

RT-PCR based diagnosis revealed importance of human group B rotavirus infection in childhood diarrhoea.

BACKGROUND: Human group B rotavirus was first identified as causative agent of a large outbreak of severe gastroenteritis affecting more than 1 million people, predominantly adults in China in 1982-1983. In spite of serological evidences for the presence of group B rotavirus in many countries of the world, the virus has been detected only from China, India and Bangladesh, where most of the cases were from adults. OBJECTIVES: To ascertain the role of group B rotavirus as an aetiological agent of diarrhoea among children in Kolkata, India. STUDY DESIGN: An active surveillance was conducted for rotavirus infection in children in a leading referral paediatric hospital and a few samples were also collected from adults of another hospital in Kolkata, India over a period of 3 years (2002-2004). After primary screening of rotaviruses by RNA electrophoresis in polyacrylamide gel, 200 of 412 samples negative by PAGE were screened by reverse transcription polymerase chain reaction for group B rotaviruses. The group B rotavirus positives samples were also confirmed by dot-blot hybridization. RESULT: During the study period, we detected 37 (18.5%) sporadic cases of human group B rotavirus infection in children below 3 years of age of which 15 (7.5%) showed mixed infection with group A rotaviruses by RT-PCR. In dot-blot hybridization studies the RNA of all rotavirus positive samples hybridized with the nonisotopic psoralen-biotin labeled total RNA probe generated from a human group B rotavirus CAL-1 strain confirming the samples as group B rotaviruses. CONCLUSION: The shift in age preference of group B rotavirus infection from adult to children and mixed infection of group B and group A rotaviruses reveals the importance of group B rotavirus as an etiological agent of childhood diarrhoea. Therefore, future vaccination strategy should include both group A and B rotaviruses to control rotavirus diarrhoea.

Relaxation dynamics of a compressible bilayer vesicle containing highly viscous fluid.

We study the relaxation dynamics of a compressible bilayer vesicle with an asymmetry in the viscosity of the inner and outer fluid medium. First we explore the stability of the vesicle free energy which includes a coupling between the membrane curvature and the local density difference between the two monolayers. Two types of instabilities are identified: a small wavelength instability and a larger wavelength instability. Considering the bulk fluid viscosity and the inter-monolayer friction as the dissipation sources, we next employ Onsager's variational principle to derive the coupled equations both for the membrane and the bulk fluid. The three relaxation modes are coupled to each other due to the bilayer and the spherical structure of the vesicle. Most importantly, a higher fluid viscosity inside the vesicle shifts the crossover mode between the bending and the slipping to a larger value. As the vesicle parameters approach the unstable regions, the relaxation dynamics is dramatically slowed down, and the corresponding mode structure changes significantly. In some limiting cases, our general result reduces to the previously obtained relaxation rates.

Production of the beta-subunit of human chorionic gonadotropin in Escherichia coli and its export mediated by the heat-labile enterotoxin chain-B signal sequence.

The PCR-amplified beta-subunit of the human chorionic gonadotropin structural gene (betahCG) was cloned under the control of the tac promoter and the heat-labile enterotoxin chain B (LTB) signal sequence (LTBss). BetahCG was successfully produced, processed and exported to the periplasmic space in Escherichia coli. Expression of betahCG was confirmed by immunoblot analysis using an anti-betahCG polyclonal antibody. The processing of the protein was very efficient, as only the processed band could be detected at all time points during the course of induction. Expression was evident soon after the addition of the lactose analogue, IPTG. These results demonstrate that E. coli cells can synthesize, process and export betahCG using the LTBss.

Amplification of various genes of human group B rotavirus from stool specimens by RT-PCR.

BACKGROUND: The detection of the human group B rotavirus (HuGBR) CAL strain from India has given us an opportunity to design suitable primers for the detection of HuGBR since CAL is the second HuGBR detected until now, the Chinese Adult Diarrhoea Rotavirus (ADRV) being the first reported human pathogen belonging to this group of viruses. The primers described here may thus be used for the detection of human group B rotaviruses by reverse transcription-PCR (RT-PCR) in a diagnostic laboratory. OBJECTIVE: To establish a set of primers suitable for the detection of various genes of human group B rotaviruses using a rapid RT-PCR assay. STUDY DESIGN: Until recently, the Chinese ADRV strain was the only HuGBR strain that had been partially sequenced by cloning various viral genes using vector-specific primers. Consequently, there are very few reports in the literature describing primers that may be used for the detection of HuGBR viruses using RT-PCR in a clinical laboratory. The sequences of various genes from the ADRV strain that had been submitted to the nucleotide sequence database GenBank were analyzed in order to design several putative detection primer pairs for an RT-PCR assay. The rationale was to amplify the cognate genes from five isolates of the HuGBR CAL strain (CAL-1 to CAL-5) that have been detected to date from India. Primers that resulted in a specific product of the expected size from the CAL isolates were used to standardize a protocol for amplifying various genes of the CAL isolates under identical reaction conditions. RESULTS: Out of several synthetic oligonucleotides designed, 12 were found to be satisfactory for the amplification of gene segments 4, 5, 6, 7, and 9 from the five CAL isolates and are presented here. A set of previously described primers that have been shown to be specific for human group B rotavirus gene segment 8 were also found to amplify the cognate gene from the CAL isolates. All the reactions were carried out using the same thermal cycling conditions. CONCLUSIONS: The extreme virulence potential of HuGBR has been documented in several epidemics in China. Until recently, the Chinese ADRV strain was the only known HuGBr strain. As there have not been any reports of HuGBR infections outside China, there are no consensus nucleotide sequences available for HuGBR that may be used to validate primers for the detection of HuGBR. Here we report a set of 12 primer sequences that were designed from ADRV sequences and also found to amplify various genes from the different CAL isolates and hence may represent consensus primers suitable for the detection of HuGBR.

Transdermal delivery of cyclosporin-A using electroporation.

Topical delivery of cyclosporin A (CSA) is desirable for treating psoriasis, but it is hindered by the barrier property of stratum corneum, and the physicochemical properties of CSA. Attempts to deliver CSA from a solution prepared in 40% ethanol (EtOH) is phosphate buffered saline (PBS) using iontophoresis did not result in any significant increase in drug delivery, compared to passive. However, the use of electroporation pulses as a physical penetration enhancer enabled delivery of a significant amount of CSA. Single pulse electroporation study indicated that the amount of EtOH delivered across the skin increased as the applied electrode voltage (Uelectrode) was increased. However, it did not translate into a proportional increase in the delivery of CSA and only a three to four times increase, compared to passive delivery, was seen with the single purse electroporation. The drug contact duration had a varying effect in the efficiency of transdermal delivery of CSA. Four hour contact duration was chosen for the multiple pulse study. Use of multiple pulses (25 pulses, 10 ms each) at Uelectrode 200 V resulted in a sixty-fold increase, compared to passive, in the delivery of CSA to the skin. Transdermally delivered CSA was mostly bound to the skin and only a small amount was seen to cross the full skin into the receiver compartment. In a study of solvent transport, the flux of water was up to three times larger than that of EtOH after electroporation.

Evaluation of cytotoxicity and absorption enhancing effects of melittin - a novel absorption enhancer.

Melittin is the major active ingredient in bee venom and has been widely studied for its membrane-fusion property. We have explored the possibility of determining a concentration range of melittin where it is relatively safe to be used as an adsorption enhancer. Melittin's potential use as an adsorption enhancer for mannitol was determined using Caco-2 cells as the model epithelial membrane. The results indicated that at concentrations below 2.4 (M melittin is safe. Using a melittin concentration of 1.5 (M there was 3.5 times increased transepithelial transport of mannitol across Caco-2 cell monolayers.

Transdermal drug delivery using electroporation. I. Factors influencing in vitro delivery of terazosin hydrochloride in hairless rats.

The use of electroporation pulses as a physical means of enhancing the permeability of skin to deliver drugs is in the early stages of development. In this article, a systematic study examining the parameters influencing electroporative transdermal delivery of terazosin hydrochloride to hairless rat skin are reported. It was found that voltage, pulse length (tau), and number of pulses were the three most important parameters, in that order. For creating a significant enhancement in drug delivery to the skin, without causing any apparent change in its external appearance, it was necessary to deliver five or more exponentially decaying electroporation pulses, at 88 +/- 2.5 V (voltage across the skin), with a decay time constant of 20 ms. Electrodes with larger area could attain the same voltages across the skin with a much lower applied voltage and possessed other advantages with regard to performance of the drug delivery system.

Transdermal drug delivery using electroporation. II. Factors influencing skin reversibility in electroporative delivery of terazosin hydrochloride in hairless rats.

A previous study indicated that the parameters governing the performance of electroporative delivery to the skin, are voltage, pulse length, number of pulses and electrode area.1 This article describes a study in which the reversibility of the electroporation technique is evaluated with in vitro methods. The skin's reversal from an enhanced permeation mode as a result of electroporation to the base level was used as an index to understand the mechanism of drug delivery and also as a preliminary indicator of safety. Maximum delivery of the model drug, terazosin hydrochloride, occurred during the pulsing. Electroporative delivery with a wire electrode (small-area electrode, 0.56 cm(2)) using 20 pulses at U(skin,0) 88 V, and pulse length 20 ms, did not cause any damage to the skin. Increasing the pulse length to 60 ms, while keeping the rest of the parameters fixed, caused a visible change in the external appearance of the skin. However, with the use of a spiral electrode (large-area electrode, 2.74 cm(2)) at 60-ms pulse length, there was minimal damage to the skin. This may be attributed to the more uniform flow of current over the whole skin area. The large-area electrode required a smaller electrode voltage, U(electrode,0) for any given U(skin,0) and also delivered nearly double the instantaneous power density compared with the small-area electrode. These findings indicate that using shorter pulses and large-area electrodes is a safer technique than large pulses and small-area electrodes when electroporation is used to enhance skin's permeability for drug delivery.

Rotavirus vaccine: current status & future prospects.

A number of rotavirus vaccines, live attenuated, killed, and subunit, genetically engineered vaccines have been developed to control infantile diarrhoea. Field trials in several parts of the world have met with moderate or no success as the vaccinees failed to develop heterotypic protection. The failure of vaccine to control rotavirus diarrhoea may be due to the lack of understanding of the neonatal mucosal immune response, evolution of reassortant strains in nature and seasonal re-emergence of different types of strains in the field situation.