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.

OpenSAFELY NHS Service Restoration Observatory 1: describing trends and variation in primary care clinical activity for 23.3 million patients in England during the first wave of COVID-19

BackgroundThe COVID-19 pandemic has disrupted healthcare activity globally. The NHS in England stopped most non-urgent work by March 2020, but later recommended that services should be restored to near-normal levels before winter where possible. The authors are developing the OpenSAFELY NHS Service Restoration Observatory, using data to describe changes in service activity during COVID-19, and reviewing signals for action with commissioners, researchers and clinicians. Here we report phase one: generating, managing, and describing the data. ObjectiveTo describe the volume and variation of coded clinical activity in English primary care across 23.8 million patients records, taking respiratory disease and laboratory procedures as key examples. MethodsWorking on behalf of NHS England we developed an open source software framework for data management and analysis to describe trends and variation in clinical activity across primary care EHR data on 23.8 million patients; and conducted a population cohort-based study to describe activity using CTV3 coding hierarchy and keyword searches from January 2019-September 2020. ResultsMuch activity recorded in general practice declined to some extent during the pandemic, but largely recovered by September 2020, with some exceptions. There was a large drop in coded activity for commonly used laboratory tests, with broad recovery to pre-pandemic levels by September. One exception was blood coagulation tests such as International Normalised Ratio (INR), with a smaller reduction (median tests per 1000 patients in 2020: February 8.0; April 6.2; September 7.0). The overall pattern of recording for respiratory symptoms was less affected, following an expected seasonal pattern and classified as "no change" from the previous year. Respiratory tract infections exhibited a sustained drop compared with pre-pandemic levels, not returning to pre-pandemic levels by September 2020. Various COVID-19 codes increased through the period. We observed a small decline associated with high level codes for long-term respiratory conditions such as chronic obstructive pulmonary disease (COPD) and asthma. Asthma annual reviews experienced a small drop but since recovered, while COPD annual reviews remain below baseline. ConclusionsWe successfully delivered an open source software framework to describe trends and variation in clinical activity across an unprecedented scale of primary care data. The COVD-19 pandemic led to a substantial change in healthcare activity. Most laboratory tests showed substantial reduction, largely recovering to near-normal levels by September 2020, with some important tests less affected. Records of respiratory infections decreased with the exception of codes related to COVID-19, whilst activity of other respiratory disease codes was mixed. We are expanding the NHS Service Restoration Observatory in collaboration with clinicians, commissioners and researchers and welcome feedback.

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.

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.

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.

Design and implementation of a 2-DOF PID compensation for magnetic levitation systems.

This paper employs a 2-DOF (degree of freedom) PID controller for compensating a physical magnetic levitation system. It is shown that because of having a feedforward gain in the proposed 2-DOF PID control, the transient performance of the compensated system can be changed in a desired manner unlike the conventional 1-DOF PID control. It is also shown that for a choice of PID parameters, although the theoretical loop robustness is the same for both the compensated systems, in real-time, 2-DOF PID control may provide superior robustness if a suitable choice of the feedforward parameter is made. The results are verified through simulations and experiments.

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.

The role of leukemia inhibitory factor in tubal ectopic pregnancy.

INTRODUCTION: Ectopic pregnancy is unique to humans and a leading cause of maternal morbidity and mortality. The etiology remains unknown however factors regulating embryo implantation likely contribute. Leukemia inhibitory factor (LIF) has roles in extravillous trophoblast adhesion and invasion and is present in ectopic implantation sites. We hypothesised that LIF facilitates blastocyst adhesion/invasion in the Fallopian tube, contributing to ectopic pregnancy. METHODS: We immunolocalised LIF receptor (R) in tubal ectopic pregnancy (N = 5). We used an oviduct cell line (OE-E6/E7) to model Fallopian tube epithelial cells and a trophoblast spheroid co-culture model (HTR-8/SVneo cell line formed spheroids) to model blastocyst attachment to the Fallopian tube. We examined LIF signaling pathways in OE-E6/E7 cells by Western blot. The effect of LIF and LIF inhibition (using a novel LIF inhibitor, PEGLA) on first-trimester placental outgrowth was determined. RESULTS: LIFR localised to villous and extravillous trophoblast and Fallopian tube epithelium in ectopic pregnancy. LIF activated STAT3 but not the ERK pathway in OE-E6/E7 cells. LIF stimulated HTR-8/SVneo spheroid adhesion to OE-E6/E7 cells which was significantly reduced after PEGLA treatment. LIF promoted placental explants outgrowth, while co-treatment with PEGLA blocked outgrowth. DISCUSSION: Our data suggests LIF facilitates the development of ectopic pregnancy by stimulating blastocyst adhesion and trophoblast outgrowth from placental explants. Ectopic pregnancy is usually diagnosed after 6 weeks of pregnancy, therefore PEGLA may be useful in targeting trophoblast growth/invasion. CONCLUSION: LIF may contribute to the development of ectopic pregnancies and that pharmacologically targeting LIF-mediated trophoblast outgrowth may be useful as a treatment for ectopic pregnancy.

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.

Predictors of outcome in fungal keratitis.

PURPOSE: To analyse predictors of clinical outcome in fungal keratitis. METHODS: Data was collected during a prospective, randomized, controlled, double-masked clinical trial of treatment for fungal keratitis. Clinical features at presentation and demographics were collected at the enrollment visit for all patients. Pre-specified clinical outcomes included 3-month visual acuity and infiltrate/scar size, time to re-epithelialization, and corneal perforation. A separate multivariable model with each outcome as the dependent variable included all predictor variables. RESULTS: Predictors for worse 3-month visual acuity include older age (P=0.024), worse presentation visual acuity (P<0.001), larger infiltrate size at presentation (P<0.001), and pigmented ulcer (P=0.030). Larger infiltrate size at presentation was a significant predictor of worse 3-month infiltrate/scar size (P<0.001). Larger epithelial defect size was a significant predictor of perforation (P=0.0013). Predictors of longer time to re-epithelialization include infiltrate size at presentation (P<0.001) and older age (P=0.025). CONCLUSION: Ulcer severity at presentation is highly predictive of worse outcomes. Presentation of clinical characteristics such as baseline acuity and infiltrate scar can provide important information to clinicians about prognosis, and may help guide management and treatment decisions. Prevention of corneal ulcer remains important, as it is difficult to change the course of the ulcer once it has begun.

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.

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.

Phacoemulsification versus manual small-incision cataract surgery for white cataract.

PURPOSE: To compare the safety and efficacy of phacoemulsification and manual small-incision cataract surgery (SICS) to treat white cataracts in southern India. SETTING: Aravind Eye Hospital, Pondicherry, India. DESIGN: Randomized prospective study. METHODS: Consecutive patients with white cataract were randomly assigned to have phacoemulsification or manual SICS by 1 of 3 surgeons experienced in both techniques. Surgical complications, operative time, uncorrected (UDVA) and corrected (CDVA) distance visual acuities, and surgically induced astigmatism were compared. RESULTS: On the first postoperative day, the UDVA was comparable in the 2 groups (P = .805) and the manual SICS group had less corneal edema (10.2%) than the phacoemulsification group (18.7%) (P = .047). At 6 weeks, the UDVA was 20/60 or better in 99 patients (87.6%) in the phacoemulsification group and 96 patients (82.0%) in the manual SICS group (P = .10) and the CDVA was 20/60 or better in 112 (99.0%) and 115 (98.2%), respectively (P = .59). The mean time was statistically significantly shorter in the manual SICS group (8.8 minutes ± 3.4 [SD]) than in the phacoemulsification group (12.2 ± 4.6 minutes) (P<.001). Posterior capsule rupture occurred in 3 eyes (2.2%) in the phacoemulsification group and 2 eyes (1.4%) in the manual SICS group (P = .681). CONCLUSIONS: Both techniques achieved excellent visual outcomes with low complication rates. Because manual SICS is significantly faster, less expensive, and less technology-dependent than phacoemulsification, it may be a more appropriate technique in eyes with mature cataract in the developing world.

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.

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.

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.

Secondary pseudomonas infection of fungal keratitis following use of contaminated natamycin eye drops: a case series.

AIM: To report a five case of secondary pseudomonas infection of fungal keratitis following use of contaminated natamycin eye drops. METHODS: A retrospective analysis of the course and clinical outcomes of five eyes of five patients with clinical and laboratory-confirmed fungal keratitis species was performed. Clinical worsening despite hourly topical 5% natamycin drops prompted a repeat corneal scraping and microbiological evaluation. RESULTS: The causative fungi for the initial keratitis were Fusarium and Aspergillus species. All the five specimens obtained from repeat scrapings revealed Pseudomonas aeruginosa. The cultures obtained from the natamycin eye drops being used by the patients also grew pseudomonas. On further evaluation, the source of contamination of the natamycin containers was obscure but speculated to be nosocomial, being within the hospital or the pharmacy. All patients had a poor visual outcome with one requiring evisceration because of panophthalmitis, whereas three underwent therapeutic keratoplasty. CONCLUSIONS: A high index of suspicion is recommended in all cases of worsening fungal keratitis to identify secondary contamination of antifungal agents with nosocomial infections.