Extracellular enzymes of mycobacteria

Abstract Extracellular enzymes were studied in different mycobacteria using a plate substrate assay. All the pathogenic mycobacteria included in the study showed the presence of protease, while lipase, ribonuclease, mucinase and β-lactamase could also be detected in some strains. In contrast, no protease was detected in the 3 saprophytic mycobacteria studied. DNase was not detected in any of the species studied. Thus, the demonstration of extracellular enzymes, in particular of protease, in mycobacteria may be relevant in understanding their role in pathogenicity.     

Augmented Dried versus Cryopreserved Amniotic Membrane as an Ocular Surface Dressing

Purpose

Dried amniotic membrane (AM) can be a useful therapeutic adjunct in ophthalmic surgery and possesses logistical advantages over cryopreserved AM. Differences in preservation techniques can significantly influence the biochemical composition and physical properties of AM, potentially affecting clinical efficacy. This study was established to investigate the biochemical and structural effects of drying AM in the absence and presence of saccharide lyoprotectants and its biocompatibility compared to cryopreserved material.

Methods

AM was cryopreserved or dried with and without pre-treatment with trehalose or raffinose and the antioxidant epigallocatechin (EGCG). Structural and visual comparisons were assessed using electron microscopy. Localisation, expression and release of AM biological factors were determined using immunoassays and immunofluorescence. The biocompatibility of the AM preparations co-cultured with corneal epithelial cell (CEC) or keratocyte monolayers were assessed using cell proliferation, cytotoxicity, apoptosis and migration assays.

Results

Drying devitalised AM epithelium, but less than cryopreservation and cellular damage was reduced in dried AM pre-treated with trehalose or raffinose. Dried AM alone, and with trehalose or raffinose showed greater factor retention efficiencies and bioavailability compared to cryopreserved AM and demonstrated a more sustained biochemical factor time release in vitro. Cellular health assays showed that dried AM with trehalose or raffinose are compatible and superior substrates compared to cryopreserved AM for primary CEC expansion, with increased proliferation and reduced LDH and caspase-3 levels. This concept was supported by improved wound healing in an immortalised human CEC line (hiCEC) co-cultured with dried and trehalose or raffinose membranes, compared to cryopreserved and fresh AM.

Conclusions

Our modified preservation process and our resultant optimised dried AM has enhanced structural properties and biochemical stability and is a superior substrate to conventional cryopreserved AM. In addition this product is stable and easily transportable allowing it to be globally wide reaching for use in clinical and military sectors.     

Inner Nuclear Layer Thickening Is Inversley Proportional to Retinal Ganglion Cell Loss in Optic Neuritis

Aim

To examine the relationship between retinal ganglion cell loss and changes in the inner nuclear layer (INL) in optic neuritis (ON).

Methods

36 multiple sclerosis (MS) patients with a history of ON and 36 age and sex-matched controls underwent Optical Coherence Tomography. The paramacular retinal nerve fiber layer (RNFL), combined ganglion cell and inner plexiform layers (GCL/IPL) and inner nuclear layer (INL) thickness were measured at 36 points around the fovea. To remove inter-subject variability, the difference in thickness of each layer between the ON and fellow eye of each patient was calculated. A topographic analysis was conducted.

Results

The INL of the ON patients was thicker than the controls (42.9µm versus 39.6µm, p=0.002). ON patients also had a thinner RNFL (27.8µm versus 32.2µm, p<0.001) and GCL/IPL (69.3µm versus 98.1µm, p<0.001). Among the controls, there was no correlation between RNFL and GCL/IPL as well as RNFL and INL, but a positive correlation was seen between GCL/IPL and INL (r=0.65, p<0.001). In the ON group, there was a positive correlation between RNFL and GCL/IPL (r=0.80, p<0.001) but a negative correlation between RNFL and INL (r=-0.61, p<0.001) as well as GCL/IPL and INL (r=-0.44, p=0.007). The negative correlation between GCL/IPL and INL strengthened in the ON group when inter-subject variability was removed (r=-0.75, p<0.001). Microcysts within the INL were present in 5 ON patients, mainly in the superior and infero-nasal paramacular regions. While patients with microcysts lay at the far end of the correlation curve between GCL/IPL and INL (i.e. larger INL and smaller GCL/IPL compared to other patients), their exclusion did not affect the correlation (r= -0.76, p<0.001).

Conclusions

INL enlargement in MS-related ON is associated with the severity of GCL loss. This is a continuous relationship and patients with INL microcysts may represent the extreme end of the scale.     

Comparison of SSR and cytochrome P-450 markers for estimating genetic diversity in Picrorhiza kurrooa L.

Picrorhiza kurrooa L., a high altitude medicinal plant, is known for its drug content called Kutkin. In the present study, DNA-based molecular marker techniques, viz. simple sequence repeats (SSR) and cytochrome P-450 markers were used to estimate genetic diversity in Picrorhiza kurrooa. Twenty five accessions of Picrorhiza kurrooa, collected from ten different eco-geographical locations were subjected to 22 SSR and eight cytochrome P-450 primer pairs, out of which 13 SSR markers detected mean 5.037 alleles with a mean polymorphic information content (PIC) of 0.7718, whereas eight cytochrome P-450 markers detected mean 5.0 alleles with a mean PIC of 0.7596. Genetic relationship among the accessions was estimated by constructing the dendrograms using SSR and cytochrome P-450 data. There was a clear consistency between SSR and cytochrome P-450 trees in terms of positioning of most Picrorhiza accessions. SSR markers could cluster various Picrorhiza kurrooa accessions based on their geographical locations whereas cytochrome P-450 markers could cluster few accessions as per their geographical locations. The Mantel test between SSR and cytochrome P-450 markers revealed a good fit correlation (r = 0.6405). The dendrogram constructed using the combined data of SSR and cytochrome P-450s depicted two clusters of accessions based on its eco-geographical locations whereas two clusters contained the accessions from mixed eco-geographical locations. Overall, the results of the present study point towards quiet high degree of genetic variation among the accessions of each eco-geographic region.     

Potential cotton aphid,Aphis gossypii,population suppression by arthropod predators in upland cotton

The cotton aphid, Aphis gossypii Glover, predation rate of convergent lady beetle, Hippodamia convergens Guerin‐Meneville, was determined by assigning a single predator randomly to each of four prey density treatments in the laboratory. Prey densities included 25, 50, 100, and 200 aphids per Petri dish arena. Predation response was recorded at 1, 4, 8, 16, 24, and 48 h after assigning predators to their prey treatments. Rate of consumption increased through time, with all 25 aphids consumed during the first 4 h of the experiment. At the highest density, adult lady beetle consumed on average 49, 99, 131, 163, 183, and 200 aphids within 1, 4, 8, 16, 24 and 48 h, respectively. Predators showed a curvilinear feeding response in relation to total available time, indicating that convergent lady beetles have the potential to suppress larger populations of aphids through continuous feeding by regulating their predation efficiency during feeding. The analysis of age‐specific mortality in absence of prey revealed that lady beetles could survive for an extended period of time (more than 2 weeks) without prey. The ability of a predator to survive without prey delays or prevents the rebound of pest populations that is a significant factor in natural biological control. A two‐year field sampling of 10 cotton arthropod predator species showed that spiders (27%) were the most dominant foliage dwelling predators in the Texas High Plains cotton followed by convergent lady beetles (23.5%), hooded beetles (13.5%), minute pirate bugs (11%), green lacewings (9.5%), bigeyed bugs (7.5%), scymnus beetles (3%), soft‐winged flower beetles (2%), damsel bugs (1.5%), and assassin bugs (1.5%). A field cage study showed that one H. convergens adult per plant released at prey density of one aphid per leaf kept the aphid population below economic threshold for the entire growing season.     

Formin-mediated actin polymerization at cell–cell junctions stabilizes E-cadherin and maintains monolayer integrity during wound repair

Cadherin-mediated cell–cell adhesion is required for epithelial tissue integrity in homeostasis, during development, and in tissue repair. E-cadherin stability depends on F-actin, but the mechanisms regulating actin polymerization at cell–cell junctions remain poorly understood. Here we investigated a role for formin-mediated actin polymerization at cell–cell junctions. We identify mDia1 and Fmnl3 as major factors enhancing actin polymerization and stabilizing E-cadherin at epithelial junctions. Fmnl3 localizes to adherens junctions downstream of Src and Cdc42 and its depletion leads to a reduction in F-actin and E-cadherin at junctions and a weakening of cell–cell adhesion. Of importance, Fmnl3 expression is up-regulated and junctional localization increases during collective cell migration. Depletion of Fmnl3 or mDia1 in migrating monolayers results in dissociation of leader cells and impaired wound repair. In summary, our results show that formin activity at epithelial cell–cell junctions is important for adhesion and the maintenance of epithelial cohesion during dynamic processes, such as wound repair.     

Molecular and genomic studies of IMMP2L and mutation screening in autism and Tourette syndrome

We recently reported the disruption of the inner mitochondrial membrane peptidase 2-like (IMMP2L) gene by a chromosomal breakpoint in a patient with Gilles de la Tourette syndrome (GTS). In the present study we sought to identify genetic variation in IMMP2L, which, through alteration of protein function or level of expression might contribute to the manifestation of GTS. We screened 39 GTS patients, and, due to the localization of IMMP2L in the critical region for the autistic disorder (AD) locus on chromosome 7q (AUTS1), 95 multiplex AD families; however, no coding mutations were found in either GTS or AD patients. In addition, no parental-specific expression of IMMP2L was detected in somatic cell hybrids containing human chromosome 7 and human cell lines carrying a maternal uniparental disomy for chromosome 7 (mUPD7). Despite the fact that no deleterious mutations in IMMPL2 (other than the inverted duplication identified previously) were identified in either GTS or AD, this gene cannot be excluded as a possible rare cause of either disorder.     

A novel steroidal spin label for membrane structure studies: synthesis and applications

2,2,6,6-Tetramethyl piperidine-N-oxyl nitroxyls are known to partition between aqueous and lipid phases, thus serving as probes to study membrane dynamics. The synthesis of a novel steroidal spin label, 3alpha-hydroxycholan-24-yl-(2",2",6",6"-tetramethyl-N-oxyl)p iperidyl butan-1',4'-dioate, containing 2,2,6,6-tetramethylpiperidine-N-oxyl moiety covalently bonded to the side chain in 3,24-caprostan-diol has been described. The localization of this spin label in model biomembranes has been studied by using electron spin resonance, differential scanning calorimetry, and 1H and 31P NMR spectroscopic techniques. Its applicability in studying the phase transition properties of model membrane L-alpha-dipalmitoyl phosphatidyl choline in the presence and absence of drugs has been described by using electron spin resonance. The label has also been used to study the permeability of epinephrine into membrane. The results have shown the applicability of the spin label as a potential spin probe in the study of biomembranes.     

Global Spectrum of Copy Number Variations Reveals Genome Organizational Plasticity and Proposes New Migration Routes

Global spectrum of CNVs is required to catalog variations to provide a high-resolution on the dynamics of genome-organization and human migration. In this study, we performed genome-wide genotyping using high-resolution arrays and identified 44,109 CNVs from 1,715 genomes across 12 populations. The study unraveled the force of independent evolutionary dynamics on genome-organizational plasticity across populations. We demonstrated the use of CNV tool to study human migration and identified a second major settlement establishing new migration routes in addition to existing ones.