Gamma-Smooth Muscle Actin Expression Is Associated with Epithelial-Mesenchymal Transition and Stem-Like Properties in Hepatocellular Carcinoma

Background and Aims

The prognosis of hepatocellular carcinoma (HCC) is hampered by frequent tumour recurrence and metastases. Epithelial-Mesenchymal Transition (EMT) is now recognized as a key process in tumour invasion, metastasis and the generation of cancer initiating cells. The morphological identification of EMT in tumour samples from the expression of novel mesenchymal markers could provide relevant prognostic information and aid in understanding the metastatic process.

Methods

The expression of Smooth Muscle Actins was studied using immunofluorescence and immunohistochemistry assays in cultured liver cells during an induced EMT process and in liver specimens from adult and paediatric HCC series.

Results

We report here that in HCC cell lines treated with TGF-β and in HCC specimens, the expression of αSMA, a known mesenchymal marker of EMT, could never be detected. In addition, our in vitro studies identified the enteric form of SMA, γSMA, as being a marker of EMT. Moreover, this SMA isoform was expressed in 46% of 58 tumours from 42 adult HCC patients and in 90% of 16 tumours from 12 paediatric HCC patients. Interestingly, this expression was significantly correlated with poor tumour differentiation and progenitor cell features characterized by the expression of EpCAM and K19.

Conclusion

Taken together, our results support the conclusion that γSMA expression in HCC is strongly correlated with the EMT process, HCC aggressiveness and the identification of cancer stem cells. This correlation suggests that γSMA represents a novel and powerful marker to predict HCC progression.     

Study of the Histology of Leafy Axes and Male Cones of Glenrosa carentonensis sp. nov. (Cenomanian Flints of Charente-Maritime,France) Using Synchrotron Microtomography Linked with Palaeoecology

We report exceptionally well-preserved plant remains ascribed to the extinct conifer Glenrosa J. Watson et H.L. Fisher emend. V. Srinivasan inside silica-rich nodules from the Cenomanian of the Font-de-Benon quarry, Charente-Maritime, western France. Remains are preserved in three dimensions and mainly consist of fragmented leafy axes. Pollen cones of this conifer are for the first time reported and in some cases remain connected to leafy stems. Histology of Glenrosa has not previously been observed; here, most of internal tissues and cells are well-preserved and allow us to describe a new species, Glenrosa carentonensis sp. nov., using propagation phase-contrast X-ray synchrotron microtomography, a non-destructive technique. Leafy axes consist of characteristic helically arranged leaves bearing stomatal crypts. Glenrosa carentonensis sp. nov. differs from the other described species in developing a phyllotaxy 8/21, claw-shaped leaves, a thicker cuticle, a higher number of papillae and stomata per crypt. Pollen cones consist of peltate, helically arranged microsporophylls, each of them bearing 6–7 pollen sacs. The new high resolution tomographic approach tested here allows virtual palaeohistology on plants included inside a dense rock to be made. Most tissues of Glenrosa carentonensis sp. nov. are described. Lithological and palaeontological data combined with xerophytic features of Glenrosa carentonensis sp. nov. suggest that this conifer has been adapted to survive in harsh and instable environments such as coastal area exposed to hot, dry conditions.     

The Corynebacterium glutamicum mycothiol peroxidase is a reactive oxygen species‐scavenging enzyme that shows promiscuity in thiol redox control

Cysteine glutathione peroxidases (CysGPxs) control oxidative stress levels by reducing hydroperoxides at the expense of cysteine thiol (‐SH) oxidation, and the recovery of their peroxidatic activity is generally accomplished by thioredoxin (Trx). Corynebacterium glutamicum mycothiol peroxidase (Mpx) is a member of the CysGPx family. We discovered that its recycling is controlled by both the Trx and the mycothiol (MSH) pathway. After H₂O₂ reduction, a sulfenic acid (‐SOH) is formed on the peroxidatic cysteine (Cys36), which then reacts with the resolving cysteine (Cys79), forming an intramolecular disulfide (S‐S), which is reduced by Trx. Alternatively, the sulfenic acid reacts with MSH and forms a mixed disulfide. Mycoredoxin 1 (Mrx1) reduces the mixed disulfide, in which Mrx1 acts in combination with MSH and mycothiol disulfide reductase as a biological relevant monothiol reducing system. Remarkably, Trx can also take over the role of Mrx1 and reduce the Mpx‐MSH mixed disulfide using a dithiol mechanism. Furthermore, Mpx is important for cellular survival under H₂O₂ stress, and its gene expression is clearly induced upon H₂O₂ challenge. These findings add a new dimension to the redox control and the functioning of CysGPxs in general.     

Haploinsufficiency of Dmxl2, Encoding a Synaptic Protein,Causes Infertility Associated with a Loss of GnRH Neurons in Mouse

Characterization of the genetic defects causing gonadotropic deficiency has made a major contribution to elucidation of the fundamental role of Kisspeptins and Neurokinin B in puberty onset and reproduction. The absence of puberty may also reveal neurodevelopmental disorders caused by molecular defects in various cellular pathways. Investigations of these neurodevelopmental disorders may provide information about the neuronal processes controlling puberty onset and reproductive capacity. We describe here a new syndrome observed in three brothers, which involves gonadotropic axis deficiency, central hypothyroidism, peripheral demyelinating sensorimotor polyneuropathy, mental retardation, and profound hypoglycemia, progressing to nonautoimmune insulin-dependent diabetes mellitus. High-throughput sequencing revealed a homozygous in-frame deletion of 15 nucleotides in DMXL2 in all three affected patients. This homozygous deletion was associated with lower DMXL2 mRNA levels in the blood lymphocytes of the patients. DMXL2 encodes the synaptic protein rabconnectin-3α, which has been identified as a putative scaffold protein for Rab3-GAP and Rab3-GEP, two regulators of the GTPase Rab3a. We found that rabconnectin-3α was expressed in exocytosis vesicles in gonadotropin-releasing hormone (GnRH) axonal extremities in the median eminence of the hypothalamus. It was also specifically expressed in cells expressing luteinizing hormone (LH) and follicle-stimulating hormone (FSH) within the pituitary. The conditional heterozygous deletion of Dmxl2 from mouse neurons delayed puberty and resulted in very low fertility. This reproductive phenotype was associated with a lower number of GnRH neurons in the hypothalamus of adult mice. Finally, Dmxl2 knockdown in an insulin-secreting cell line showed that rabconnectin-3α controlled the constitutive and glucose-induced secretion of insulin. In conclusion, this study shows that low levels of DMXL2 expression cause a complex neurological phenotype, with abnormal glucose metabolism and gonadotropic axis deficiency due to a loss of GnRH neurons. Our findings identify rabconectin-3α as a key controller of neuronal and endocrine homeostatic processes.     

Effects of Quadriceps Muscle Fatigue on Stiff-Knee Gait in Patients with Hemiparesis

The relationship between neuromuscular fatigue and locomotion has never been investigated in hemiparetic patients despite the fact that, in the clinical context, patients report to be more spastic or stiffer after walking a long distance or after a rehabilitation session. The aim of this study was to evaluate the effects of quadriceps muscle fatigue on the biomechanical gait parameters of patients with a stiff-knee gait (SKG). Thirteen patients and eleven healthy controls performed one gait analysis before a protocol of isokinetic quadriceps fatigue and two after (immediately after and after 10 minutes of rest). Spatiotemporal parameters, sagittal knee and hip kinematics, rectus femoris (RF) and vastus lateralis (VL) kinematics and electromyographic (EMG) activity were analyzed. The results showed that quadriceps muscle weakness, produced by repetitive concentric contractions of the knee extensors, induced an improvement of spatiotemporal parameters for patients and healthy subjects. For the patient group, the increase in gait velocity and step length was associated with i) an increase of sagittal hip and knee flexion during the swing phase, ii) an increase of the maximal normalized length of the RF and VL and of the maximal VL lengthening velocity during the pre-swing and swing phases, and iii) a decrease in EMG activity of the RF muscle during the initial pre-swing phase and during the latter 2/3 of the initial swing phase. These results suggest that quadriceps fatigue did not alter the gait of patients with hemiparesis walking with a SKG and that neuromuscular fatigue may play the same functional role as an anti-spastic treatment such as botulinum toxin-A injection. Strength training of knee extensors, although commonly performed in rehabilitation, does not seem to be a priority to improve gait of these patients.     

Did Vaccination Slow the Spread of Bluetongue in France?

Vaccination is one of the most efficient ways to control the spread of infectious diseases. Simulations are now widely used to assess how vaccination can limit disease spread as well as mitigate morbidity or mortality in susceptible populations. However, field studies investigating how much vaccines decrease the velocity of epizootic wave-fronts during outbreaks are rare. This study aimed at investigating the effect of vaccination on the propagation of bluetongue, a vector-borne disease of ruminants. We used data from the 2008 bluetongue virus serotype 1 (BTV-1) epizootic of southwest France. As the virus was newly introduced in this area, natural immunity of livestock was absent. This allowed determination of the role of vaccination in changing the velocity of bluetongue spread while accounting for environmental factors that possibly influenced it. The average estimated velocity across the country despite restriction on animal movements was 5.4 km/day, which is very similar to the velocity of spread of the bluetongue virus serotype 8 epizootic in France also estimated in a context of restrictions on animal movements. Vaccination significantly reduced the propagation velocity of BTV-1. In comparison to municipalities with no vaccine coverage, the velocity of BTV-1 spread decreased by 1.7 km/day in municipalities with immunized animals. For the first time, the effect of vaccination has been quantified using data from a real epizootic whilst accounting for environmental factors known to modify the velocity of bluetongue spread. Our findings emphasize the importance of vaccination in limiting disease spread across natural landscape. Finally, environmental factors, specifically those related to vector abundance and activity, were found to be good predictors of the velocity of BTV-1 spread, indicating that these variables need to be adequately accounted for when evaluating the role of vaccination on bluetongue spread.     

Trypanosoma brucei FKBP12 Differentially Controls Motility and Cytokinesis in Procyclic and Bloodstream Forms

FKBP12 proteins are able to inhibit TOR kinases or calcineurin phosphatases upon binding of rapamycin or FK506 drugs, respectively. The Trypanosoma brucei FKBP12 homologue (TbFKBP12) was found to be a cytoskeleton-associated protein with specific localization in the flagellar pocket area of the bloodstream form. In the insect procyclic form, RNA interference-mediated knockdown of TbFKBP12 affected motility. In bloodstream cells, depletion of TbFKBP12 affected cytokinesis and cytoskeleton architecture. These last effects were associated with the presence of internal translucent cavities limited by an inside-out configuration of the normal cell surface, with a luminal variant surface glycoprotein coat lined up by microtubules. These cavities, which recreated the streamlined shape of the normal trypanosome cytoskeleton, might represent unsuccessful attempts for cell abscission. We propose that TbFKBP12 differentially affects stage-specific processes through association with the cytoskeleton.     

Revisiting spatial distribution and biochemical composition of calcium-containing crystals in human osteoarthritic articular cartilage

Introduction

Calcium-containing (CaC) crystals, including basic calcium phosphate (BCP) and calcium pyrophosphate dihydrate (CPP), are associated with destructive forms of osteoarthritis (OA). We assessed their distribution and biochemical and morphologic features in human knee OA cartilage.

Methods

We prospectively included 20 patients who underwent total knee replacement (TKR) for primary OA. CaC crystal characterization and identification involved Fourier-transform infra-red spectrometry and scanning electron microscopy of 8 to 10 cartilage zones of each knee, including medial and lateral femoral condyles and tibial plateaux and the intercondyle zone. Differential expression of genes involved in the mineralization process between cartilage with and without calcification was assessed in samples from 8 different patients by RT-PCR. Immunohistochemistry and histology studies were performed in 6 different patients.

Results

Mean (SEM) age and body mass index of patients at the time of TKR was 74.6 (1.7) years and 28.1 (1.6) kg/m², respectively. Preoperative X-rays showed joint calcifications (chondrocalcinosis) in 4 cases only. The medial femoro-tibial compartment was the most severely affected in all cases, and mean (SEM) Kellgren-Lawrence score was 3.8 (0.1). All 20 OA cartilages showed CaC crystals. The mineral content represented 7.7% (8.1%) of the cartilage weight. All patients showed BCP crystals, which were associated with CPP crystals for 8 joints. CaC crystals were present in all knee joint compartments and in a mean of 4.6 (1.7) of the 8 studied areas. Crystal content was similar between superficial and deep layers and between medial and femoral compartments. BCP samples showed spherical structures, typical of biological apatite, and CPP samples showed rod-shaped or cubic structures. The expression of several genes involved in mineralization, including human homolog of progressive ankylosis, plasma-cell-membrane glycoprotein 1 and tissue-nonspecific alkaline phosphatase, was upregulated in OA chondrocytes isolated from CaC crystal-containing cartilages.

Conclusions

CaC crystal deposition is a widespread phenomenon in human OA articular cartilage involving the entire knee cartilage including macroscopically normal and less weight-bearing zones. Cartilage calcification is associated with altered expression of genes involved in the mineralisation process.     

Synthesis and Evaluation Of 2″-Modified MMI Linked Dimers in Antisense Constructs

Abstract

Synthesis of four methylene(methylimino) (MMI) linked dimers modifed at the 2′-position with fluoro and/or methoxy groups and their incorporation into different sequences has been accomplished. From these dimers, bis 2′-OMe MMI dimer was selected for further studies based on its synthetic accessibility, conformational study by NMR, and Tm analysis. Several chimeric antisense oligomers containing bis 2′-OMe dimers have been synthesized on a 10 μmol scale for in vivo studies.