A comparative analysis shows morphofunctional differences between the rat and mouse melanin-concentrating hormone systems

Sub-populations of neurons producing melanin-concentrating hormone (MCH) are characterized by distinct projection patterns, birthdates and CART/NK3 expression in rat. Evidence for such sub-populations has not been reported in other species. However, given that genetically engineered mouse lines are now commonly used as experimental models, a better characterization of the anatomy and morphofunctionnal organization of MCH system in this species is then necessary. Combining multiple immunohistochemistry experiments with in situ hybridization, tract tracing or BrdU injections, evidence supporting the hypothesis that rat and mouse MCH systems are not identical was obtained: sub-populations of MCH neurons also exist in mouse, but their relative abundance is different. Furthermore, divergences in the distribution of MCH axons were observed, in particular in the ventromedial hypothalamus. These differences suggest that rat and mouse MCH neurons are differentially involved in anatomical networks that control feeding and the sleep/wake cycle.     

Short- and long-term skin graft survival in cattle clones with different mitochondrial haplotypes

In contrast to nuclear DNA, cytoplasmic genes may differ among cloned animals due to the presence of polymorphic mitochondrial DNA haplotypes in the host oocytes, raising doubts about histocompatibility among clones. Three bovine clones were generated by nuclear transfer; dermal fibroblasts from a fetus were used as donor cells, whereas oocytes from abbatoir-derived ovaries were used as recipient cells. The mitochondrial DNA (sequencing of coding and non-coding regions) and nuclear DNA (13 microsatellite markers) of cloned and control animals were characterized to identify potential polymorphisms. Skin auto- and allografts were transplanted on the adult clones and a non-related animal as a measure of immunological reactivity. Nuclear DNA of cloned animals was genetically identical but differed in all microsatellites of the non-related control. Amounts of donor cell mitochondrial DNA in the skin ranged from 1 to 2.6% among clones. Few differences in heteroplasmy were observed between skin and WBC of the clones, indicating limited mitochondrial DNA segregation in tissues during pre- and post-natal development to adulthood. Sequencing of the remaining oocyte-derived mitochondrial DNA haplotype identified polymorphisms in coding and non-coding regions, confirming their origin from unrelated maternal lineages. Nonetheless, skin transplants between clones were accepted for the 92 d study period, whereas third-party grafts were rejected. In conclusion, the nuclear transfer-generated adult bovine clones used in this study were immunologically compatible with one another despite differences in their mitochondrial DNA haplotypes.     

A novel approach to characterize clonality and differentiation of human melanoma-specific T cell responses: spontaneous priming and efficient boosting by vaccination

Despite major progress in T lymphocyte analysis in melanoma patients, TCR repertoire selection and kinetics in response to tumor Ags remain largely unexplored. In this study, using a novel ex vivo molecular-based approach at the single-cell level, we identified a single, naturally primed T cell clone that dominated the human CD8(+) T cell response to the Melan-A/MART-1 Ag. The dominant clone expressed a high-avidity TCR to cognate tumor Ag, efficiently killed tumor cells, and prevailed in the differentiated effector-memory T lymphocyte compartment. TCR sequencing also revealed that this particular clone arose at least 1 year before vaccination, displayed long-term persistence, and efficient homing to metastases. Remarkably, during concomitant vaccination over 3.5 years, the frequency of the pre-existing clone progressively increased, reaching up to 2.5% of the circulating CD8 pool while its effector functions were enhanced. In parallel, the disease stabilized, but subsequently progressed with loss of Melan-A expression by melanoma cells. Collectively, combined ex vivo analysis of T cell differentiation and clonality revealed for the first time a strong expansion of a tumor Ag-specific human T cell clone, comparable to protective virus-specific T cells. The observed successful boosting by peptide vaccination support further development of immunotherapy by including strategies to overcome immune escape.     

Identification of basic amino acids at the N-terminal end of the core protein that are crucial for hepatitis C virus infectivity

A major function of the hepatitis C virus (HCV) core protein is the interaction with genomic RNA to form the nucleocapsid, an essential component of the virus particle. Analyses to identify basic amino acid residues of HCV core protein, important for capsid assembly, were initially performed with a cell-free system, which did not indicate the importance of these residues for HCV infectivity. The development of a cell culture system for HCV (HCVcc) allows a more precise analysis of these core protein amino acids during the HCV life cycle. In the present study, we used a mutational analysis in the context of the HCVcc system to determine the role of the basic amino acid residues of the core protein in HCV infectivity. We focused our analysis on basic residues located in two clusters (cluster 1, amino acids [aa]6 to 23; cluster 2, aa 39 to 62) within the N-terminal 62 amino acids of the HCV core protein. Our data indicate that basic residues of the first cluster have little impact on replication and are dispensable for infectivity. Furthermore, only four basic amino acids residues of the second cluster (R50, K51, R59, and R62) were essential for the production of infectious viral particles. Mutation of these residues did not interfere with core protein subcellular localization, core protein-RNA interaction, or core protein oligomerization. Moreover, these mutations had no effect on core protein envelopment by intracellular membranes. Together, these data indicate that R50, K51, R59, and R62 residues play a major role in the formation of infectious viral particles at a post-nucleocapsid assembly step.     

Functional outcome and prognostic factors in anti-Jo1 patients with antisynthetase syndrome

Introduction

The aims of this present study were firstly to assess the outcome, including functional course, in anti-Jo1 positive patients with antisynthetase syndrome (ASS), and secondly to determine predictive parameters of poor outcome in these patients.

Methods

The medical records of 86 consecutive anti-Jo1 patients with ASS were reviewed in 4 academic centers.

Results

13 patients (15.1%) achieved remission of ASS, whereas 55 (63.9%) improved and 18 (20.9%) deteriorated in their clinical status. Both steroid and cytotoxic drugs could be discontinued in only 4.7% of patients. ASS was associated with decreased quality of life at long-term follow-up: only 69.2% of patients considered to be in remission experienced a return to previous normal activities; and 24.7% of other patients with non-remitting ASS still had a marked reduction of activities (as shown by the disability scale of the Health Assessment Questionnaire). Decreased quality of life was further due to calcinosis cutis (8.1%) and adverse effects of steroid therapy (36%). Factors associated with ASS deterioration were older age, pulmonary and esophageal involvement, calcinosis cutis and cancer. Higher anti-Jo1 levels were further associated with disease severity in ASS patients.

Conclusions

The present study shows high morbidity related to ASS. Furthermore, we suggest that patients with predictive factors of ASS deterioration may require more aggressive therapy. Our findings also suggest that in anti-Jo1 patients with severe esophageal manifestations, combined high dose steroids and intravenous immunoglobulins might be proposed as the first line therapy. Finally, as cancer occurred in 14% of anti-Jo1 patients, our findings underscore that the search for cancer should be performed in these patients.     

Analysis of humeral head displacements from sequences of biplanar X-rays: repeatability study and preliminary results in healthy subjects

This work presents an accurate method to measure gleno-humeral translations in a controlled pseudo-kinematic environment. Low-dose biplanar X-rays were acquired from nine healthy subjects at three elevations of the arm in the scapular plane. On each set of images, shoulder bony landmarks were manually located in 3D using a dedicated software. Intra-observer and inter-observer repeatability of landmark identification, as well as humeral head center (GH) translations, were studied. Repeatability for the identification of GH in the global coordinate system (CS) was good with 95% confidence intervals (CIs) ranging from 0.57 to 2.25 mm. Scapular landmark CIs ranged from 0.80 to 12 mm. Gleno-humeral translations of small amplitude ( < 6 mm) were detected in seven out of nine subjects. The results obtained here confirm that calibrated low-dose stereo-radiography is a promising tool for the functional analysis of the shoulder.     

MQ2: visualizing multi-trait mapped QTL results

Bacterial blight (BB) of rice (Oryza sativa L.) caused by Xanthomonas oryzae pv. oryzae (Xoo) is a destructive disease in rice worldwide. Xa3, a gene conferring resistance to BB at the booting stage of the rice plant, has been characterized previously using map-based cloning. We cloned and sequenced the Xa3/xa3 gene in the Korean cultivars Hwayeong, Ilmi, and Goun and conferred resistance or susceptibility to BB. We detected polymorphisms, and polymerase chain reaction-based functional markers were developed based on the single nucleotide polymorphism from the Xa3 and xa3 nucleotide sequence. Susceptible or resistant individuals from an F2 population developed from a cross between Milyang 244 and Ilmi, near-isogenic lines carrying BB resistance genes, were screened with functional markers. The BB3-RF and BB3-RR primers consistently amplified a resistance-specific fragment of 255 bp only in resistant plants, whereas the BB3-SF and BB3-SR primers were specific to susceptible plants. Genotyping results were co-segregated with phenotype by conducting the BB resistance test with the K₃ race. These markers could be effective for marker-assisted selection of the Xa3 gene in rice breeding programs.     

Core and panmetabolism in Escherichia coli

Escherichia coli exhibits a wide range of lifestyles encompassing commensalism and various pathogenic behaviors which its highly dynamic genome contributes to develop. How environmental and host factors shape the genetic structure of E. coli strains remains, however, largely unknown. Following a previous study of E. coli genomic diversity, we investigated its diversity at the metabolic level by building and analyzing the genome-scale metabolic networks of 29 E. coli strains (8 commensal and 21 pathogenic strains, including 6 Shigella strains). Using a tailor-made reconstruction strategy, we significantly improved the completeness and accuracy of the metabolic networks over default automatic reconstruction processes. Among the 1,545 reactions forming E. coli panmetabolism, 885 reactions were common to all strains. This high proportion of core reactions (57%) was found to be in sharp contrast to the low proportion (13%) of core genes in the E. coli pangenome, suggesting less diversity of metabolic functions compared to that of all gene functions. Core reactions were significantly overrepresented among biosynthetic reactions compared to the more variable degradation processes. Differences between metabolic networks were found to follow E. coli phylogeny rather than pathogenic phenotypes, except for Shigella networks, which were significantly more distant from the others. This suggests that most metabolic changes in non-Shigella strains were not driven by their pathogenic phenotypes. Using a supervised method, we were yet able to identify small sets of reactions related to pathogenicity or commensalism. The quality of our reconstructed networks also makes them reliable bases for building metabolic models.     

Hepatocyte-derived cultured cells with unusual cytoplasmic keratin-rich spheroid bodies

Cytoplasmic inclusions are found in a variety of diseases that are characteristic morphological features of several hepatic, muscular and neurodegenerative disorders. They display a predominantly filamentous ultrastructure that is also observed in malignant rhabdoid tumor (MRT). A cellular clone containing an intracytoplasmic body was isolated from hepatocyte cell culture, and in the present study we examined whether this body might be related or not to Mallory-Denk body (MDB), a well characterized intracytoplasmic inclusion, or whether this cellular clone was constituted by malignant rhabdoid tumor cells. The intracytoplasmic body was observed in electron microscopy (EM), confocal immunofluorescence microscopy and several proteins involved in the formation of its structure were identified. Using light microscopy, a spheroid body (SB) described as a single regular-shaped cytoplasmic body was observed in cells. During cytokinesis, the SB was disassembled and reassembled in a way to reconstitute a unique SB in each progeny cell. EM examination revealed that the SB was not surrounded by a limiting membrane. However, cytoplasmic filaments were concentrated in a whorled array. These proteins were identified as keratins 8 and 18 (K8/K18), which formed the central core of the SB surrounded by a vimentin cage-like structure. This structure was not related to Mallory-Denk body or aggresome since no aggregated proteins were located in SB. Moreover, the structure of SB was not due to mutations in the primary sequence of K8/K18 and vimentin since no difference was observed in the mRNA sequence of their genes, isolated from Huh-7 and Huh-7w7.3 cells. These data suggested that cellular factor(s) could be responsible for the SB formation process. Aggregates of K18 were relocated in the SB when a mutant of K18 inducing disruption of K8/K18 IF network was expressed in the cellular clone. Furthermore, the INI1 protein, a remodeling-chromatin factor deficient in rhabdoid cells, which contain a spheroid perinuclear inclusion body, was found in our cellular clone. In conclusion, our data suggest that Huh-7w7.3 cells constitute an excellent model for determining the cellular factor(s) involved in the process of spheroid perinuclear body formation.