Effect of gelling agents and antibiotics on adventitious bud regeneration from In vitro leaves of pear

Summary The effect of the type of gelling agent and of several antibiotics on the adventitious bud regeneration from in vitro leaves was tested on eight pear genotypes. The use of gellan gum (Phytagel™) in the medium instead of agar had a very strong positive effect on the rate of adventitious bud regeneration for all pear genotypes tested in this study. This gelling agent induced faster cell divisions than agar, thus more callus was produced on wound sites and subsequently more buds regenerated. Incubation on gellan gum medium during the first 20 d of bud induction was sufficient to induce a stimulatory effect on regeneration and limited the production of hyperhydric buds. In the prospect of Agrobacterium transformation, the effect of several antibiotics was tested. Cefotaxime (200 mg/l) plus ticarcillin/clavulanic acid (100 mg/l) could be used in the culture medium without affecting the frequency of bud regeneration. The inhibition of bud regeneration was obtained with different kanamycin concentrations according to the gelling agent in the medium. On gellan gum medium, a concentration of 100 mg/l of kanamycin was suitable. These conditions can be recommended for experiments on Agrobacterium-mediated transformation of pear, where bacterial inoculation and presence of antibiotics generally reduce and delay bud regeneration.     

The T-786C and C774T endothelial nitric oxide synthase gene polymorphisms independently affect the onset pattern of severe diabetic retinopathy.

Genetic factors could be implicated in the pathogenesis of severe diabetic retinopathy (DR). Recently, we reported a strong association between the eNOS4b/a endothelial nitric oxide synthase (eNOS) polymorphism and severe DR. To examine whether T-786C and C774T eNOS polymorphisms are involved in severe DR, 254 Caucasians with longstanding C-peptide-negative type 1 diabetes, 128 patients with absent/mild DR (control group), and 126 patients with preproliferative/proliferative DR (study group) were genotyped. The distribution of T-786C and C774T eNOS polymorphisms was in Hardy-Weinberg equilibrium and did not differ between the study and control groups. However, in case patients (n=126), T-786C and C774T polymorphisms influenced the onset pattern of severe DR (P=0.0169 and P=0.0257, respectively). The C-786C genotype was associated with early-onset severe DR (duration of diabetes: 15.2+/-5.9 vs. 19.4+/-6.3 years, P=0.0105), and the homozygous T774T genotype was associated with late-onset severe DR (24.3+/-7.0 vs. 18.4+/-6.2 years, P=0.0067). In the case of patients with high glycosylated hemoglobin levels (HbA1c >8%, n=88), the association between the T-786C polymorphism and early-onset severe DR was stronger (P=0.0068). Case patients carrying the C-786C genotype had higher HbA1c values (9.61+/-1.89%) than those carrying the T-786T genotype (8.93+/-1.47%, P=0.0173). Multivariate analysis showed that T-786C polymorphism was the best independent factor for onset pattern of severe DR (P<0.001). These findings, supported by previous associations between eNOS4b/a polymorphism and DR, suggest that T-786C and C774T eNOS polymorphisms affect the onset pattern of severe DR.     

Core protein domains involved in hepatitis C virus-like particle assembly and budding at the endoplasmic reticulum membrane

Hepatitis C virus (HCV) core protein, expressed with a Semliki forest virus (SFV) replicon, self-assembles into HCV-like particles (HCV-LPs) at the endoplasmic reticulum (ER) membrane, providing an opportunity to study HCV particle morphogenesis by electron microscopy. Various mutated HCV core proteins with engineered internal deletions were expressed with this system, to identify core domains required or dispensable for HCV-LP assembly. The HCV core protein sequence was compared with its counterpart in GB virus B (GBV-B), the virus most closely related to HCV, to identify conserved domains. GBV-B and HCV display similar tropism for liver hepatocytes and their core proteins are organized similarly into three main domains (I, II and III), although GBV-B core is smaller and lacks approximately 35 amino acids (aa) in domain I. The deletion of short hydrophobic domains (aa 133-152 and 153-167 in HCV core) that appear highly conserved in domain II of both GBV-B and HCV core proteins resulted in loss of HCV core ER anchoring and self-assembly into HCV-LPs. The deletion of short domains found within domain I of HCV core protein but not in the corresponding domain of GBV-B core according to sequence alignment had contrasting effects. Amino acids 15-28 and 60-66 were shown to be dispensable for HCV-LP assembly and morphogenesis, whereas aa 88-106 were required for this process. The production of GBV-B core protein from a recombinant SFV vector was associated with specific ER ultrastructural changes, but did not lead to the morphogenesis of GBV-B-LPs, suggesting that different budding mechanisms occur in members of the Flaviviridae family.     

Social familiarity and shoal formation in juvenile fishes

The potential influence of social familiarity in shoal‐choice decisions was investigated in two sympatric species of north temperate fishes, juvenile banded killifish Fundulus diaphanus and juvenile bluegill sunfish Lepomis macrochirus. Groups of socially familiar and unfamiliar conspecifics were formed in the laboratory using wild‐caught fishes. Juvenile F. diaphanus demonstrated a strong preference for familiar conspecific shoalmates, whereas juvenile L. macrochirus exhibited no preference for either unfamiliar or familiar conspecific shoalmates. The differential influence of familiarity on shoalmate choice in juveniles of these two species could be due to their different ecologies, local population densities and life histories.     

MALDI-TOF mass spectrometry: A powerful tool to study the internalization of cell-penetrating peptides

This review summarizes the contribution of MALDI-TOF mass spectrometry in the study of cell-penetrating peptide (CPP) internalization in eukaryote cells. This technique was used to measure the efficiency of cell-penetrating peptide cellular uptake and cargo delivery and to analyze carrier and cargo intracellular degradation. The impact of thiol-containing membrane proteins on the internalization of CPP–cargo disulfide conjugates was also evaluated by combining MALDI-TOF MS with simple thiol-specific reactions. This highlighted the formation of cross-linked species to cell-surface proteins that either remained trapped in the cell membrane or led to intracellular delivery. MALDI-TOF MS is thus a powerful tool to dissect CPP internalization mechanisms.     

Application of Deamidated Gliadin Antibodies in the Follow-Up of Treated Celiac Disease

Introduction

The role of serological tests such as IgA anti-transglutaminase autoantibodies has become increasingly important in celiac disease (CD) diagnosis. However, the efficiency of these tests for patient follow-up is controversial. We investigated the correlation of 12 different serological tests, including recent deamidated gliadin and actin IgA tests, with villous atrophy (VA) in a retrospective cohort of treated celiac patients.

Materials and Methods

Serum samples were collected from 100 treated CD patients who had intestinal biopsy in the course of their follow-up. Antibodies against transglutaminase, deamidated gliadin peptides, and native gliadin were measured, along with IgA anti-actin. The biopsy slides were all blind-reviewed and scored according to Marsh classification.

Results

For all deamidated gliadin and transglutaminase tests, we found that a positive result was significantly associated with persistence of intestinal VA, with a diagnostic efficacy up to 80%. Furthermore, antibodies titers directly correlated with the degree of VA, indicating a strong link between disease activity and presence of antibodies in the serum. Interestingly, the tests with the highest association with persistent VA were those for deamidated gliadin IgG. Using a test positivity pattern analysis, we were also able to identify several groups of patients with distinct antibody profiles that showed significant differences in intestinal damage and diet compliance.

Conclusions

Altogether, these results show that deamidated gliadin antibodies are strongly correlated with VA and should be considered valuable tools in CD follow-up and that multiplex serologic analysis for treated CD represents a promising tool for personalized patient management.     

Randomised controlled trial of enalapril and beta blockers in non-diabetic chronic renal failure.

OBJECTIVE--To compare the ability of angiotensin converting enzyme inhibitors and beta blockers to slow the development of end stage renal failure in non-diabetic patients with chronic renal failure. DESIGN--Open randomised multicentre trial with three year follow up. SETTING--Outpatient departments of six French hospitals. PATIENTS--100 hypertensive patients with chronic renal failure (initial serum creatinine 200-400 mumol/l. 52 randomised to enalapril and 48 to beta blockers (conventional treatment). INTERVENTIONS--Enalapril or beta blocker was combined with frusemide and, if necessary, a calcium blocker or centrally acting drug in patients whose diastolic pressure remained above 90 mm Hg. RESULTS--17 patients receiving conventional treatment and 10 receiving enalapril developed end stage renal failure. The cumulative renal survival rate was significantly better in the enalapril group than in the conventional group (P < 0.05). The slope of the reciprocal serum creatinine concentration was steeper in the conventionally treated patients (-6.89 x 10(-5)l/mumol/month) than in the enalapril group (-4.17 x 10(-5)l/mumol/month; P < 0.05). No difference in blood pressure was found between groups. CONCLUSION--In hypertensive patients with chronic renal failure enalapril slows progression towards end stage renal failure compared with beta blockers. This effect was probably not mediated through controlling blood pressure.     

In situ monitoring of the nascent Pseudomonas fluorescens biofilm response to variations in the dissolved organic carbon level in low-nutrient water by attenuated total reflectance-Fourier transform infrared spectroscopy

Drinking water quality management requires early warning tools which enable water supply companies to detect quickly and to forecast degradation of the microbial quality of drinking water during its transport throughout distribution systems. This study evaluated the feasibility of assessing, in real time, drinking water biostability by monitoring in situ the evolution of the attenuated total reflectance-Fourier transform infrared (ATR-FTIR) fingerprint of a nascent reference biofilm exposed to water being tested. For this purpose, the responses of nascent Pseudomonas fluorescens biofilms to variations in the dissolved organic carbon (DOC) level in tap water were monitored in situ and in real time by ATR-FTIR spectroscopy. Nascent P. fluorescens biofilms consisting of a monolayer of bacteria were formed on the germanium crystal of an ATR flowthrough cell by pumping bacterial suspensions in Luria-Bertani (LB) medium through the cell. Then they were exposed to a continuous flow of dechlorinated sterile tap water supplemented with appropriate amounts of sterile LB medium to obtain DOC concentrations ranging from 1.5 to 11.8 mg/liter. The time evolution of infrared bands related to proteins, polysaccharides, and nucleic acids clearly showed that changes in the DOC concentration resulted in changes in the nascent biofilm ATR-FTIR fingerprint within 2 h after exposure of the biofilm to the water being tested. The initial bacterial attachment, biofilm detachment, and regrowth kinetics determined from changes in the areas of bands associated with proteins and polysaccharides were directly dependent on the DOC level. Furthermore, they were consistent with bacterial adhesion or growth kinetic models and extracellular polymeric substance overproduction or starvation-dependent detachment mechanisms.