Leu-7-positive cells with monocyte phenotype show different ultrastructural features in comparison to Leu-7-positive cells with T-cell phenotype

Summary The ultrastructural features of the Leu-7-positive — Leu-M3-positive cell subpopulation and the Leu-7-positive — Leu-4-positive cell subpopulation were characterized and compared using immunogold-immunoperoxidase double labelling with immunoelectron microscopy. The majority of Leu-7-positive cells coexpressed a monocyte phenotype and showed an ultrastructural pattern specific for functional natural killer (NK) cells, i.e. a low nuclear/cytoplasmic (N/C) ratio, an irregular outline, many cytoplasmic organelles and electron-dense granules. In contrast, only a minority of Leu-7-positive cells coexpressed a T phenotype, and these were characterized by a high N/C ratio, an even surface and the absence of electron-dense granules. Thus, Leu-7-positive — Leu-4-positive cells may by an immature form of NK cells, and Leu-7-positive—Leu-4-positive and Leu-7-positive — Leu-M3-positive cell subpopulations may represent different stages of Leu-7-positive cell differentiation.     

The p53/p21Cip1/ Waf1 pathway mediates the effects of SPARC on melanoma cell cycle progression

Secreted protein acidic and rich in cysteine (SPARC), or osteonectin, belongs to the family of matricellular proteins that modulate cell-matrix interactions and cellular functions. SPARC is highly expressed in melanoma, and we reported that SPARC promotes epithelial/mesenchymal-like changes and cell migration. Here, we used siRNA and conditional shRNA to investigate the contribution of tumor-derived SPARC to melanoma cell growth in vitro and in vivo. We found that depletion of SPARC induces G2/M cell cycle arrest and tumor growth inhibition with activation of p53 and induction of p21(Cip1/Waf1) acting as a checkpoint, preventing efficient mitotic progression. In addition, we demonstrate that reduced mesenchymal features and the invasive potential of SPARC-silenced cells are independent of p21(Cip1/Waf1) induction and cell cycle arrest. Importantly, overexpression of SPARC reduces p53 protein levels and leads to an increase in cell number during exponential growth. Our findings indicate that in addition to its well-known function as a mediator of melanoma cell migration and tumor-host interactions, SPARC regulates, in a cell-autonomous manner, cell cycle progression and proliferation through the p53/p21(Cip1/Waf1) pathway.     

OGX-427 inhibits tumor progression and enhances gemcitabine chemotherapy in pancreatic cancer

Despite many advances in oncology, almost all patients with pancreatic cancer (PC) die of the disease. Molecularly targeted agents are offering hope for their potential role in helping translate the improved activity of combination chemotherapy into improved survival. Heat shock protein 27 (Hsp27) is a chaperone implicated in several pathological processes such as cancer. Further, Hsp27 expression becomes highly upregulated in cancer cells after chemotherapy. Recently, a modified antisense oligonucleotide that is complementary to Hsp27 (OGX-427) has been developed, which inhibits Hsp27 expression and enhances drug efficacy in cancer xenograft models. Phase II clinical trials using OGX-427 in different cancers like breast, ovarian, bladder, prostate and lung are in progress in the United States and Canada. In this study, we demonstrate using TMA of 181 patients that Hsp27 expression and phosphorylation levels increase in moderately differentiated tumors to become uniformly highly expressed in metastatic samples. Using MiaPaCa-2 cells grown both in vitro and xenografted in mice, we demonstrate that OGX-427 inhibits proliferation, induces apoptosis and also enhances gemcitabine chemosensitivity via a mechanism involving the eukaryotic translation initiation factor 4E. Collectively, these findings suggest that the combination of Hsp27 knockdown with OGX-427 and chemotherapeutic agents such as gemcitabine can be a novel strategy to inhibit the progression of pancreas cancer.     

Recurrent BRAF kinase fusions in melanocytic tumors offer an opportunity for targeted therapy

BRAF is the most prevalent oncogene and an important therapeutic target in melanoma. In some cancers, BRAF is activated by rearrangements that fuse its kinase domain to 5′ partner genes. We examined 848 comparative genomic hybridization profiles of melanocytic tumors and found copy number transitions within BRAF in 10 tumors, of which six could be further characterized by sequencing. In all, the BRAF kinase domain was fused in‐frame to six N‐terminal partners. No other mutations were identified in melanoma oncogenes. One of the seven melanoma cell lines without known oncogenic mutations harbored a similar BRAF fusion, which constitutively activated the MAP kinase pathway. Sorafenib, but not vemurafenib, could block MAP kinase pathway activation and proliferation of the cell line at clinically relevant concentrations, whereas BRAF^V600E mutant melanoma cell lines were significantly more sensitive to vemurafenib. The patient from whom the cell line was derived showed a durable clinical response to sorafenib.     

Intranasal Infection with Chlamydia abortus Induces Dose-Dependent Latency and Abortion in Sheep

Background

Latency is a key feature of the animal pathogen Chlamydia abortus, where infection remains inapparent in the non-pregnant animal and only becomes evident during a subsequent pregnancy. Often the first sign that an animal is infected is abortion occurring late in gestation. Despite this, little is understood of the underlying mechanisms that control latency or the recrudescence of infection that occurs during subsequent pregnancy. The aim of this study was to develop an experimental model of latency by mimicking the natural route of infection through the intranasal inoculation of non-pregnant sheep with C. abortus.

Methodology/Principal Findings

Three groups of sheep (groups 1, 2 and 3) were experimentally infected with different doses of C. abortus (5×10³, 5×10⁵ and 5×10⁷ inclusion forming units (IFU), respectively) prior to mating and monitored over 2 breeding cycles for clinical, microbiological, pathological, immunological and serological outcomes. Two further groups received either negative control inoculum (group 4a,b) or were inoculated subcutaneously on day 70 of gestation with 2×10⁶ IFU C. abortus (group 5). Animals in groups 1, 2 and 5 experienced an abortion rate of 50–67%, while only one animal aborted in group 3 and none in group 4a,b. Pathological, microbiological, immunological and serological analyses support the view that the maternal protective immune response is influenced by initial exposure to the bacterium.

Conclusions/Significance

The results show that intranasal administration of non-pregnant sheep with a low/medium dose of C. abortus results in a latent infection that leads in a subsequent pregnancy to infection of the placenta and abortion. In contrast a high dose stimulates protective immunity, resulting in a much lower abortion rate. This model will be useful in understanding the mechanisms of infection underlying latency and onset of disease, as well as in the development of novel therapeutics and vaccines for controlling infection.     

Accurate and Precise DNA Quantification in the Presence of Different Amplification Efficiencies Using an Improved Cy0 Method

Quantitative real-time PCR represents a highly sensitive and powerful technology for the quantification of DNA. Although real-time PCR is well accepted as the gold standard in nucleic acid quantification, there is a largely unexplored area of experimental conditions that limit the application of the Ct method. As an alternative, our research team has recently proposed the Cy0 method, which can compensate for small amplification variations among the samples being compared. However, when there is a marked decrease in amplification efficiency, the Cy0 is impaired, hence determining reaction efficiency is essential to achieve a reliable quantification. The proposed improvement in Cy0 is based on the use of the kinetic parameters calculated in the curve inflection point to compensate for efficiency variations. Three experimental models were used: inhibition of primer extension, non-optimal primer annealing and a very small biological sample. In all these models, the improved Cy0 method increased quantification accuracy up to about 500% without affecting precision. Furthermore, the stability of this procedure was enhanced integrating it with the SOD method. In short, the improved Cy0 method represents a simple yet powerful approach for reliable DNA quantification even in the presence of marked efficiency variations.     

The role of sensory input in motor neuron sprouting control

Primary sensory neurons project to motor neurons directly or through interneurons and affect their activity. In our previous paper we showed that intramuscular sprouting can be affected by changing the sensory synaptic input to motor neurons. In this work, motor axon sprouting within a peripheral nerve (extramuscular sprouting) was induced by nerve injury at such a distance from muscle so as not to allow nerve-muscle trophic interactions. Two different procedures were carried out: (1) sciatic nerve crush and (2) sciatic nerve crush with homosegmental ipsilateral L3-L5 dorsal rhizotomy. The number of regenerating motor axons innervating extensor digitorum longus muscle was determined by in vivo muscle tension recordings and an index of their individual conduction rate was obtained by in vitro intracellular recordings of excitatory postsynaptic end-plate potentials in muscle fibers. The main findings were: (1) there are more regenerated axons distally from the lesion than parent axons proximally to the lesion (sprouting at the lesion); (2) sprouting at the lesion was negatively affected by homosegmental ipsilateral dorsal rhizotomy; (3) the number of motor axons innervating extensor digitorum longus muscle extrafusal fibers counted proximally to the lesion increased following nerve injury and regeneration but this did not occur when sensory input was lost. A transient innervation of extrafusal fibers by &#110 motor neurons may explain the increase of motor axons counted proximally to the lesion.     

NMES superimposed on movement is equally effective as heavy slow resistance training in patellar tendinopathy

Objective:This study aimed at investigating the effectiveness of an 8-week training protocol, based on neuromuscular electrical stimulation of the quadriceps, which was superimposed onto voluntary exercise (NMES+), in comparison to a traditional heavy slow resistance training (HSRT), in individuals with patellar tendinopathy.Methods:Thirty-two physically active participants, aged: 33.6±10.2 years, were divided into two groups: NMES+ or HSRT. Maximal voluntary isometric contraction (MVIC) of knee extensor and flexor muscles, power during a countermovement jump (CMJ), and VISA-p questionnaire scores were recorded at the start(T0), 2-weeks(T1), 4-weeks(T2), 6-weeks(T3), 8-weeks(T4) and 4-months post-training (T5). Knee pain and rate of perceived exertion (RPE) were recorded at each training session with a 0-10 scale.Results:Knee pain was significantly lower in NMES+ compared to HSRT during all training sessions. No significant between-group differences were found for VISA-p scores and forces recorded during MVICs at T0,T1,T2,T3,T4 and T5. A significant increase of VISA-p and peak forces during MVIC was recorded across-time in both groups. No significant between-group or across-time differences were found for RPE and CMJ parameters.Conclusions:NMES+ and HSRT were equally effective in decreasing tendinopathy symptoms and increasing strength, with NMES+ having the advantage to be a pain-free resistance training modality.     

Evolutionary history of chromosome 11 featuring four distinct centromere repositioning events in Catarrhini

Panels of BAC clones used in FISH experiments allow a detailed definition of chromosomal marker arrangement and orientation during evolution. This approach has disclosed the centromere repositioning phenomenon, consisting in the activation of a novel, fully functional centromere in an ectopic location, concomitant with the inactivation of the old centromere. In this study, appropriate panels of BAC clones were used to track the chromosome 11 evolutionary history in primates and nonprimate boreoeutherian mammals. Chromosome 11 synteny was found to be highly conserved in both primate and boreoeutherian mammalian ancestors. Amazingly, we detected four centromere repositioning events in primates (in Old World monkeys, in gibbons, in orangutans, and in the Homo-Pan-Gorilla (H-P-G) clade ancestor), and one in Equidae. Both H-P-G and Lar gibbon novel centromeres were flanked by large duplicons with high sequence similarity. Outgroup species analysis revealed that this duplicon was absent in phylogenetically more distant primates. The chromosome 11 ancestral centromere was probably located near the HSA11q telomere. The domain of this inactivated centromere, in humans, is almost devoid of segmental duplications. An inversion occurred in chromosome 11 in the common ancestor of H-P-G. A large duplicon, again absent in outgroup species, was found located adjacent to the inversion breakpoints. In Hominoidea, almost all the five largest duplicons of this chromosome appeared involved in significant evolutionary architectural changes.