Immunogold silver staining associated with epi-fluorescence for cucumber mosaic virus localisation on semi-thin sections of banana tissues

The immunogold-silver staining (IGSS) technique in combination with epi-fluorescence detection was used to localise cucumber mosaic virus (CMV) particles within banana infected tissues. For this purpose, tissue samples (2 mm3) were excised from CMV-infected and highly proliferating meristem cultures of Williams BSJ banana (ITC. 0570, AAA, Cavendish subgroup). These samples were immediately fixed in a 2% paraformaldehyde/0.25% glutaraldehyde mixture, dehydrated in ethanol, and finally embedded in L.R.White resin. Semi-thin sections were cut, mounted on clean treated glass slides and immunostained for CMV particles using gold-labelled secondary antibodies and silver enhancement. Sections were counterstained with basic fuchsin and examined using laser scanning confocal microscopy. Negative controls included immuno-stained samples excised from non-virus infected material as well as infected material on which primary or secondary antibodies were not applied. Images of autofluorescence (in red) and of epi-reflectance of silver-enhanced immunogold particles (in green) were recorded separately and merged, allowing the specific localisation of CMV particles at the cellular level on semi-thin sections of aldehyde-fixed banana tissues. The main advantage of this analytical approach compared to previously published protocols is that it combines a fast staining procedure, stable preparation, a high resolution, and a narrow plane of focus with the flexibility in generation, processing and analysis of images offered by laser scanning confocal microscopy. Finally, the presence of numerous CMV particles within banana meristems constitutes a clear explanation of the very low CMV elimination efficiency when using meristem-tip culture alone.     

Treatment of tuberculosis in a region with high drug resistance: outcomes, drug resistance amplification and re-infection

Introduction

Emerging antituberculosis drug resistance is a serious threat for tuberculosis (TB) control, especially in Eastern European countries.

Methods

We combined drug susceptibility results and molecular strain typing data with treatment outcome reports to assess the influence of drug resistance on TB treatment outcomes in a prospective cohort of patients from Abkhazia (Georgia). Patients received individualized treatment regimens based on drug susceptibility testing (DST) results. Definitions for antituberculosis drug resistance and treatment outcomes were in line with current WHO recommendations. First and second line DST, and molecular typing were performed in a supranational laboratory for Mycobacterium tuberculosis (MTB) strains from consecutive sputum smear-positive TB patients at baseline and during treatment.

Results

At baseline, MTB strains were fully drug-susceptible in 189/326 (58.0%) of patients. Resistance to at least H or R (PDR-TB) and multidrug-resistance (MDR-TB) were found in 69/326 (21.2%) and 68/326 (20.9%) of strains, respectively. Three MDR-TB strains were also extensively resistant (XDR-TB). During treatment, 3/189 (1.6%) fully susceptible patients at baseline were re-infected with a MDR-TB strain and 2/58 (3.4%) PDR-TB patients became MDR-TB due to resistance amplification. 5/47 (10.6%) MDR- patients became XDR-TB during treatment. Treatment success was observed in 161/189 (85.2%), 54/69 (78.3%) and 22/68 (32.3%) of patients with fully drug susceptible, PDR- and MDR-TB, respectively. Development of ofloxacin resistance was significantly associated with a negative treatment outcome.

Conclusion

In Abkhazia, a region with high prevalence of drug resistant TB, the use of individualized MDR-TB treatment regimens resulted in poor treatment outcomes and XDR-TB amplification. Nosocomial transmission of MDR-TB emphasizes the importance of infection control in hospitals.     

Temporal requirement of Hoxa2 in cranial neural crest skeletal morphogenesis

Little is known about the spatiotemporal requirement of Hox gene patterning activity in vertebrates. In Hoxa2 mouse mutants, the hyoid skeleton is replaced by a duplicated set of mandibular and middle ear structures. Here, we show that Hoxa2 is selectively required in cranial neural crest cells (NCCs). Moreover, we used a Cre-ERT2 recombinase system to induce a temporally controlled Hoxa2 deletion in the mouse. Hoxa2 inactivation after cranial NCC migration into branchial arches resulted in homeotic transformation of hyoid into mandibular arch skeletal derivatives, reproducing the conventional Hoxa2 knockout phenotype, and induced rapid changes in Alx4, Bapx1, Six2 and Msx1 expression patterns. Thus, hyoid NCCs retain a remarkable degree of plasticity even after their migration in the arch, and require Hoxa2 as an integral component of their morphogenetic program. Moreover, subpopulations of postmigratory NCCs required Hoxa2 at discrete time points to pattern distinct derivatives. This study provides the first temporal inactivation of a vertebrate Hox gene and illustrates Hox requirement during late morphogenetic processes.     

Change in sugar, sterol and fatty acid composition in banana meristems caused by sucrose-induced acclimation and its effects on cryopreservation

To understand the mechanisms of sucrose‐induced acclimation in relation to plant cryopreservation, sugars, sterols, fatty acids of different lipid fractions (neutral lipids, glycolipids and sphingolipids and phospholipids), as well as free fatty acids were analyzed in proliferating meristem cultures of different banana varieties. The four banana varieties that were selected show different post‐thaw shoot regeneration rates (0–53.4%). All mentioned parameters were analyzed using (1) control meristems that were cultured on a normal sucrose concentration (0.09 M), which resulted in low survival after cryopreservation; and (2) 2‐week sucrose precultured meristems (0.4 M). This sucrose preculture, essential for regeneration after cryopreservation, resulted in a significant increase of each of seven sugars detected. The ratio of stigmasterol/sitosterol (St/Si) in sucrose‐pretreated meristems significantly increased. The sucrose pretreatment also resulted in a significant increase of total fatty acid content of the neutral lipid fraction and of the glycolipid and sphingolipid fraction, as well as the total free fatty acid content. The individual fatty acid content of the phospholipids was differently changed by the sucrose pretreatment for the given varieties studied. In most cases, sucrose pretreatment resulted in an increase of the double bond index (DBI) in the neutral lipids and a decrease of DBI in the glycolipids and sphingolipids, in phospholipids as well as in free fatty acids. Principal component analysis of all collected data revealed that (1) for the control material, sucrose and total sugar contents were closely linked to the post‐thaw shoot regeneration, suggesting that sucrose and total sugar may be main limiting factors to survive cryopreservation; (2) accumulation of large quantities of sugars (glucose, fructose, sucrose and total sugar) in sucrose‐pretreated material cannot explain the differences in survival after cryopreservation of the four banana varieties. We assume that a minimal amount of sugars is needed in meristem cultures to survive cryopreservation. Still, other limiting factors do influence the survival following the sucrose pretreatment. We observed that the parameters which are closely linked to the post‐thaw shoot regeneration are a minimal change in the ratios of St/Si, the minimal change of the DBI of phospholipids and free fatty acids, as well as linoleic acid content (C18:2); and (3) inositol, raffinose, myristic acid (C14:0) and oleic acid (C18:1) were present in small quantities; however, they could be correlated to survival after cryopreservation, suggesting that they may be also involved in cryopreservation process.     

Complete Genome Sequence of Caulobacter crescentus Bacteriophage {varphi}CbK

?CbK is a B3 morphotype bacteriophage of the Siphoviridae family that infects Caulobacter crescentus, the preeminent model system for bacterial cell cycle studies. The last 4 decades of research with ?CbK as a genetic and cytological tool to study the biology of the host warrant an investigation of the phage genome composition. Herein, we report the complete genome sequence of ?CbK and highlight unusual features that emerged from its annotation. The complete genome analysis of the ?CbK phage provides new insight into its characteristics and potential interactions with its Caulobacter crescentus host, setting the stage for future functional studies with ?CbK.     

Arbuscular mycorrhizal fungi affect both penetration and further life stage development of root-knot nematodes in tomato

The root-knot nematode Meloidogyne incognita poses a worldwide threat to agriculture, with an increasing demand for alternative control options since most common nematicides are being withdrawn due to environmental concerns. The biocontrol potential of arbuscular mycorrhizal fungi (AMF) against plant-parasitic nematodes has been demonstrated, but the modes of action remain to be unraveled. In this study, M. incognita penetration of second-stage juveniles at 4, 8 and 12 days after inoculation was compared in tomato roots (Solanum lycopersicum cv. Marmande) pre-colonized or not by the AMF Glomus mosseae. Further life stage development of the juveniles was also observed in both control and mycorrhizal roots at 12 days, 3 weeks and 4 weeks after inoculation by means of acid fuchsin staining. Penetration was significantly lower in mycorrhizal roots, with a reduction up to 32%. Significantly lower numbers of third- and fourth-stage juveniles and females accumulated in mycorrhizal roots, at a slower rate than in control roots. The results show for the first time that G. mosseae continuously suppresses root-knot nematodes throughout their entire early infection phase of root penetration and subsequent life stage development.     

Srf-dependent paracrine signals produced by myofibers control satellite cell-mediated skeletal muscle hypertrophy

Adult skeletal muscles adapt their fiber size to workload. We show that serum response factor (Srf) is required for satellite cell-mediated hypertrophic muscle growth. Deletion of Srf from myofibers and not satellite cells blunts overload-induced hypertrophy, and impairs satellite cell proliferation and recruitment to pre-existing fibers. We reveal a gene network in which Srf within myofibers modulates interleukin-6 and cyclooxygenase-2/interleukin-4 expressions and therefore exerts a paracrine control of satellite cell functions. In Srf-deleted muscles, in vivo overexpression of interleukin-6 is sufficient to restore satellite cell proliferation but not satellite cell fusion and overall growth. In contrast cyclooxygenase-2/interleukin-4 overexpression rescue satellite cell recruitment and muscle growth without affecting satellite cell proliferation, identifying altered fusion as the limiting cellular event. These findings unravel a role for Srf in the translation of mechanical cues applied to myofibers into paracrine signals, which in turn will modulate satellite cell functions and support muscle growth.