Bacterial communities of plum phyllosphere and characterization of indigenous antagonistic Bacillus thuringiensis R3/3 isolate.

AIMS: The characterization of bacterial communities diversity on four local plum cultivars in two phenological stages using culture-dependent and culture-independent methods and screening among culturable plum community for indigenous bacteria active against phytopathogens. METHODS AND RESULTS: The bacterial communities associated with leaves and fruits of four local Serbian plum cultivars (Pozegaca, Ranka, Cacanska Lepotica and Cacanska Rodna) were investigated in two phenological stages during early (May) and late (July) fruit maturation. Metagenomic approach revealed Methylobacterium, Sphingomonas and Hymenobacter as dominant genera. The most frequently isolated representatives with cultivable approach were pseudomonads with Pseudomonas syringae and Pseudomonas graminis, the most likely resident species of plum community. Antagonistic Bacillus thuringiensis R3/3 isolate from plum phyllosphere had ability to produce exoenzymes, reduce the growth of phytopathogenic bacteria in co-culture environment and show quorum quenching activity. CONCLUSIONS: Plum cultivar and growth season contribute to the structure of the bacterial community associated with plum. Plum phyllosphere is good source of antagonists effective against phytopathogens. SIGNIFICANCE AND IMPACT OF STUDY: Knowledge of bacterial communities on plum will have an impact on studies related to phyllosphere ecology and biocontrol. The indigenous antagonistic isolate, B. thuringiensis R3/3, from plum could be further investigated for its potential use in biological control of plum diseases.

Seeding hESCs to achieve optimal colony clonality.

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have promising clinical applications which often rely on clonally-homogeneous cell populations. To achieve this, it is important to ensure that each colony originates from a single founding cell and to avoid subsequent merging of colonies during their growth. Clonal homogeneity can be obtained with low seeding densities; however, this leads to low yield and viability. It is therefore important to quantitatively assess how seeding density affects clonality loss so that experimental protocols can be optimised to meet the required standards. Here we develop a quantitative framework for modelling the growth of hESC colonies from a given seeding density based on stochastic exponential growth. This allows us to identify the timescales for colony merges and over which colony size no longer predicts the number of founding cells. We demonstrate the success of our model by applying it to our own experiments of hESC colony growth; while this is based on a particular experimental set-up, the model can be applied more generally to other cell lines and experimental conditions to predict these important timescales.

Gamma-irradiated human amniotic membrane decellularised with sodium dodecyl sulfate is a more efficient substrate for the ex vivo expansion of limbal stem cells.

The gold standard substrate for the ex vivo expansion of human limbal stem cells (LSCs) remains the human amniotic membrane (HAM) but this is not a defined substrate and is subject to biological variability and the potential to transmit disease. To better define HAM and mitigate the risk of disease transmission, we sought to determine if decellularisation and/or γ-irradiation have an adverse effect on culture growth and LSC phenotype. Ex vivo limbal explant cultures were set up on fresh HAM, HAM decellularised with 0.5M NaOH, and 0.5% (w/v) sodium dodecyl sulfate (SDS) with or without γ-irradiation. Explant growth rate was measured and LSC phenotype was characterised by histology, immunostaining and qRT-PCR (ABCG2, ΔNp63, Ki67, CK12, and CK13). Ƴ-irradiation marginally stiffened HAM, as measured by Brillouin spectromicroscopy. HAM stiffness and γ-irradiation did not significantly affect the LSC phenotype, however LSCs expanded significantly faster on Ƴ-irradiated SDS decellularised HAM (p<0.05) which was also corroborated by the highest expression of Ki67 and putative LSC marker, ABCG2. Colony forming efficiency assays showed a greater yield and proportion of holoclones in cells cultured on Ƴ-irradiated SDS decellularised HAM. Together our data indicate that SDS decellularised HAM may be a more efficacious substrate for the expansion of LSCs and the use of a γ-irradiated HAM allows the user to start the manufacturing process with a sterile substrate, potentially making it safer. STATEMENT OF SIGNIFICANCE: Despite its disadvantages, including its biological variability and its ability to transfer disease, human amniotic membrane (HAM) remains the gold standard substrate for limbal stem cell (LSC) culture. To address these disadvantages, we used a decellularised HAM sterilised by gamma-irradiation for LSC culture. We cultured LSCs on fresh HAM, HAM decellularised with NaOH, HAM decellularised with sodium dodecyl sulfate (SDS) and HAM decellularised with SDS and sterilised with gamma-irradiation. We demonstrated that although HAM decellularised with SDS and sterilised with gamma-irradiation is significantly stiffer this does not affect LSC culture growth rate or the phenotype of cultured LSCs. We therefore recommend the use of SDS decellularised gamma-irradiated HAM in future LSC clinical trials.

Electrophysiological findings in patients with low density lipoprotein receptor related protein 4 positive myasthenia gravis.

BACKGROUND AND PURPOSE: The aim was to determine the electrophysiological profile of our cohort of low density lipoprotein receptor related protein 4 (LRP4) positive myasthenia gravis (MG) patients. METHODS: A repetitive nerve stimulation (RNS) test and jitter analysis using a concentric needle electrode were performed in 17 LRP4 positive MG patients. The results were compared to 31 muscle-specific tyrosine kinase (MuSK) positive and 28 acetylcholine receptor (AChR) positive MG patients. RESULTS: The RNS test was negative in almost all patients belonging to the LRP4/seronegative and LRP4/MuSK groups. It was positive most frequently in the AChR MG patients, especially those without anti-LRP4 antibodies. The presence of anti-LRP4 antibodies was connected to lower decrement values, whilst the independent presence of anti-AChR or anti-MuSK antibodies was connected to higher decrement values. Lowest jitter was recorded in patients with LRP4/seronegative MG. The highest percentage of pathological jitter analysis test results was present in MuSK and AChR MG patients. The isolate presence of anti-LRP4 antibodies did not influence the mean consecutive difference values, whilst mean consecutive difference values were higher in the presence of anti-AChR or anti-MuSK antibodies. CONCLUSIONS: Low density lipoprotein receptor related protein 4 positive patients make a distinct MG subgroup with rarely detected pathological electrophysiological test results. The lack of influence of anti-LRP4 antibodies on the different electrophysiological parameters brings into question the pathogenic role of anti-LRP4 antibodies in MG.

Human leukocyte antigen class II (DRB1 and DQB1) alleles and haplotypes frequencies in patients with pemphigus vulgaris among the Serbian population.

The etiology of pemphigus vulgaris (PV) is multifactorial and includes genetic, environmental, hormonal, and immunological factors. Inheritance of certain Human class II leukocyte antigen (HLA) alleles is by far the best-established predisposing factor for the development of PV. Class II HLA alleles vary among racial/ethnic backgrounds. We have determined an association between HLA class II alleles and PV among the Serbian population. A total of 72 patients with confirmed diagnosis of PV were genotyped for HLA class II alleles. HLA frequencies were compared with unrelated healthy bone marrow donors. The statistical significance of differences between patients and controls was evaluated using Fisher's exact test. The DRB1*04 and DRB1*14 allelic groups were associated with PV (P adj = 4.45 × 10(-13) and 4.06 × 10(-19) respectively), while HLA-DRB1*11 was negatively associated with PV (P adj = 0.0067) suggesting a protective role. DRB1*04:02, DRB1*14:04, DQB1*03:02 and DQB1*05:03 alleles were shown to be strongly associated with PV (P adj = 1.63 × 10(-12), 5.20 × 10(-7), 1.28 × 10(-6), and 4.44 × 10(-5), respectively). The frequency of HLA DRB1*04-DQB1*03 and HLA DRB1*14-DQB1*05 haplotypes in PV patients was significantly higher than in controls (31.3% vs 8.8%, P adj =7.66 × 10(-8) and 30.6% vs 6.3%, P adj = 3.22 × 10(-10), respectively). At high-resolution level, statistical significance was observed in HLA-DRB1*04:02-DQB1*03:02 and HLA-DRB1*14:04-DQB1*05:03 haplotypes (P adj = 5.55 × 10(-12), and P adj = 3.91 × 10(-6), respectively). Our findings suggest that HLA-DRB1*04:02, DRB1*14:04, HLA-DQB1* 03:02 and DQB1*05:03 alleles and HLA-DRB1*04:02-DQB1*03:02 and HLA-DRB1*14:04-DQB1*05:03 haplotypes are genetic markers for susceptibility for PV, while DRB1*11 allelic group appears protective in Serbian population.

High frequency of DQB1*05 and absolute absence of DRB1*13 in muscle-specific tyrosine kinase positive myasthenia gravis.

BACKGROUND AND PURPOSE: Myasthenia gravis (MG) is an autoimmune disease but certain genetic factors predispose its development. Since susceptibility to different forms of MG is linked to a number of allelic variants, the aim of this study was to explore the human leukocyte antigen (HLA) profile of our patients with muscle-specific tyrosine kinase (MuSK) MG. METHODS: Human leukocyte antigen (HLA) typing was performed in our cohort of 31 MuSK MG patients available for the study. The allele groups of DRB1* and DQB1* loci were typed with sequence-specific oligonucleotide probes and high resolution typing for DQB1* was performed using sequence-specific primers. HLA frequencies were compared with unrelated healthy bone marrow donors. RESULTS: Significant association of MuSK MG with alleles DRB1*14 [odds ratio (OR) 3.8], DRB1*16 (OR 3.3) (P < 0.01) and DQB1*05 (OR 2.2) (P < 0.05) was found. In our patients the most frequent DQB1* allele was DQB1*05:02. An absolute absence of DRB1*13 in our cohort of MuSK MG patients was also found, whilst this allele was present in 25% (495/1992) of control subjects (OR 0) (P < 0.01). The HLA DRB1*16-DQB1*05 (OR 2.9) haplotype was found to be associated with MuSK MG (P < 0.05). CONCLUSIONS: The strong association of MuSK MG with DQB1*05 alleles observed in patient series from other countries was confirmed. The novel finding in our cohort of MuSK MG patients was the absolute absence of DRB1*13 allele, which might have a protective role in the development of MuSK MG, at least in our population.