Myosin VIIa,harmonin and cadherin 23, three Usher I gene products that cooperate to shape the sensory hair cell bundle

Deaf-blindness in three distinct genetic forms of Usher type I syndrome (USH1) is caused by defects in myosin VIIa, harmonin and cadherin 23. Despite being critical for hearing, the functions of these proteins in the inner ear remain elusive. Here we show that harmonin, a PDZ domain-containing protein, and cadherin 23 are both present in the growing stereocilia and that they bind to each other. Moreover, we demonstrate that harmonin b is an F-actin-bundling protein, which is thus likely to anchor cadherin 23 to the stereocilia microfilaments, thereby identifying a novel anchorage mode of the cadherins to the actin cytoskeleton. Moreover, harmonin b interacts directly with myosin VIIa, and is absent from the disorganized hair bundles of myosin VIIa mutant mice, suggesting that myosin VIIa conveys harmonin b along the actin core of the developing stereocilia. We propose that the shaping of the hair bundle relies on a functional unit composed of myosin VIIa, harmonin b and cadherin 23 that is essential to ensure the cohesion of the stereocilia.     

Central neural mechanisms mediating human visceral hypersensitivity

Although visceral hypersensitivity is thought to be important in generating symptoms in functional gastrointestinal disorders, the neural mechanisms involved are poorly understood. We recently showed that central sensitization (hyperexcitability of spinal cord sensory neurones) may play an important role. In this study, we demonstrate that after a 30-min infusion of 0.15 M HCl acid into the healthy human distal esophagus, we see a reduction in the pain threshold to electrical stimulation of the non-acid-exposed proximal esophagus (9.6 +/- 2.4 mA) and a concurrent reduction in the latency of the N1 and P2 components of the esophageal evoked potentials (EEP) from this region (10.4 +/- 2.3 and 15.8 +/- 5.3 ms, respectively). This reduced EEP latency indicates a central increase in afferent pathway velocity and therefore suggests that hyperexcitability within the central visceral pain pathway contributes to the hypersensitivity within the proximal, non-acid-exposed esophagus (secondary hyperalgesia/allodynia). These findings provide the first electrophysiological evidence that central sensitization contributes to human visceral hypersensitivity.     

Genetic Variability of Vancomycin-Resistant Enterococcus faecium and Enterococcus faecalis Isolates from Humans,Chickens, and Pigs in Malaysia

Vancomycin-resistant enterococci (VRE) have been reported to be present in humans, chickens, and pigs in Malaysia. In the present study, representative samples of VRE isolated from these populations were examined for similarities and differences by using the multilocus sequence typing (MLST) method. Housekeeping genes of Enterococcus faecium (n = 14) and Enterococcus faecalis (n = 11) isolates were sequenced and analyzed using the MLST databases eBURST and goeBURST. We found five sequence types (STs) of E. faecium and six STs of E. faecalis existing in Malaysia. Enterococcus faecium isolates belonging to ST203, ST17, ST55, ST79, and ST29 were identified, and E. faecium ST203 was the most common among humans. The MLST profiles of E. faecium from humans in this study were similar to the globally reported nosocomial-related strain lineage belonging to clonal complex 17 (CC17). Isolates from chickens and pigs have few similarities to those from humans, except for one isolate from a chicken, which was identified as ST203. E. faecalis isolates were more diverse and were identified as ST4, ST6, ST87, ST108, ST274, and ST244, which were grouped as specific to the three hosts. E. faecalis, belonging to the high-risk CC2 and CC87, were detected among isolates from humans. In conclusion, even though one isolate from a chicken was found clonal to that of humans, the MLST analysis of E. faecium and E. faecalis supports the findings of others who suggest VRE to be predominantly host specific and that clinically important strains are found mainly among humans. The infrequent detection of a human VRE clone in a chicken may in fact suggest a reverse transmission of VRE from humans to animals.     

The role of stratifin in fibroblast–keratinocyte interaction

Stratifin is a member of 14-3-3 protein family, a highly conserved group of proteins constituted by seven isoforms. They are involved in numerous crucial intracellular functions such as cell cycle and apoptosis, regulation of signal transduction pathways, cellular trafficking, cell proliferation and differentiation, cell survival, and protein folding and processing, among others. At epidermal level, stratifin (also called 14-3-3 sigma) has been described as molecule with relevant functions. For instance, this isoform is a marker associated with keratinocyte differentiation. In this maturation process, the presence of dominant negative molecules of p53 induces a “stemness condition” of keratinocyte precursor cells and suppression of stratifin expression. In addition, the recently described keratinocyte-releasable form of stratifin is involved in dermal fibroblast MMP-1 over-expression through c-Fos and c-Jun activity. This effect is mediated, at least in part, by p38 mitogen-activated protein kinase (MAPK). Other MMP family members such as stromelysin-1 (MMP-3), stromelysin-2 (MMP-10), neutrophil collagenase (MMP-8), and membrane-type MMP-24 (MT5-MMP) are also up-regulated by stratifin. Within fibroproliferative disorder of skin, hypertrophic scar and keloids exhibit a high content of collagen, proteoglycans, and fibronectin. Thus, the MMP profile induced by stratifin is an interesting starting point to establish new therapeutic tools to control the process of wound healing. In this review, we will focus on site of synthesis and mode of action of stratifin in skin and wound healing.     

Stroma regulates increased epithelial lateral cell adhesion in 3D culture: a role for actin/cadherin dynamics

Background

Cell shape and tissue architecture are controlled by changes to junctional proteins and the cytoskeleton. How tissues control the dynamics of adhesion and cytoskeletal tension is unclear. We have studied epithelial tissue architecture using 3D culture models and found that adult primary prostate epithelial cells grow into hollow acinus-like spheroids. Importantly, when co-cultured with stroma the epithelia show increased lateral cell adhesions. To investigate this mechanism further we aimed to: identify a cell line model to allow repeatable and robust experiments; determine whether or not epithelial adhesion molecules were affected by stromal culture; and determine which stromal signalling molecules may influence cell adhesion in 3D epithelial cell cultures.

Methodology/Principal Findings

The prostate cell line, BPH-1, showed increased lateral cell adhesion in response to stroma, when grown as 3D spheroids. Electron microscopy showed that 9.4% of lateral membranes were within 20 nm of each other and that this increased to 54% in the presence of stroma, after 7 days in culture. Stromal signalling did not influence E-cadherin or desmosome RNA or protein expression, but increased E-cadherin/actin co-localisation on the basolateral membranes, and decreased paracellular permeability. Microarray analysis identified several growth factors and pathways that were differentially expressed in stroma in response to 3D epithelial culture. The upregulated growth factors TGFβ2, CXCL12 and FGF10 were selected for further analysis because of previous associations with morphology. Small molecule inhibition of TGFβ2 signalling but not of CXCL12 and FGF10 signalling led to a decrease in actin and E-cadherin co-localisation and increased paracellular permeability.

Conclusions/Significance

In 3D culture models, paracrine stromal signals increase epithelial cell adhesion via adhesion/cytoskeleton interactions and TGFβ2-dependent mechanisms may play a key role. These findings indicate a role for stroma in maintaining adult epithelial tissue morphology and integrity.     

The emerging role of miR-200 family in metastasis: focus on EMT,CSCs, angiogenesis,and anoikis

Introduction

Cancer is the second major threat to human society and one of the main challenges facing healthcare systems. One of the main problems of cancer care is the metastases of cancer cells that cause 90% of deaths due to cancer. Multiple molecular mechanisms are involved in cancer cell metastasis. Therefore, a better understanding of these molecular mechanisms is necessary for designing restrictive strategies against cancer cell metastasis. Accumulating data suggests that MicroRNAs (miRNAs) are involved in metastasis and invasion of human tumors through regulating multiple genes expression levels that are involved in molecular mechanisms of metastasis. The goal of this review is to present the molecular pathways by which the miR 200 family manifests its effects on EMT, cancer stem cells, angiogenesis, anoikis, and the effects of tumor cell metastases.

Methods

A detailed literature search was conducted to find information about the role of the miR-200 family in the processes involved in metastasis in various databases.

Results

Numerous lines of evidence revealed an association between the mir-200 family and metastasis of human tumors by impressing processes such as cancer stem cells, EMT, angiogenesis, and anoikis.

Conclusions

Understanding the molecular mechanisms associated with metastasis in which the miR-200 family is involved can be effective in treating metastatic cancers.

Graphic abstract

     

Oral Activated Charcoal Prevents Experimental Cerebral Malaria in Mice and in a Randomized Controlled Clinical Trial in Man Did Not Interfere with the Pharmacokinetics of Parenteral Artesunate

Background

Safe, cheap and effective adjunct therapies preventing the development of, or reducing the mortality from, severe malaria could have considerable and rapid public health impact. Oral activated charcoal (oAC) is a safe and well tolerated treatment for acute poisoning, more recently shown to have significant immunomodulatory effects in man. In preparation for possible efficacy trials in human malaria, we sought to determine whether oAC would i) reduce mortality due to experimental cerebral malaria (ECM) in mice, ii) modulate immune and inflammatory responses associated with ECM, and iii) affect the pharmacokinetics of parenteral artesunate in human volunteers.

Methods/Principal Findings

We found that oAC provided significant protection against P. berghei ANKA-induced ECM, increasing overall survival time compared to untreated mice (p<0.0001; hazard ratio 16.4; 95% CI 6.73 to 40.1). Protection from ECM by oAC was associated with reduced numbers of splenic TNF⁺ CD4⁺ T cells and multifunctional IFNγ⁺TNF⁺ CD4⁺ and CD8⁺ T cells. Furthermore, we identified a whole blood gene expression signature (68 genes) associated with protection from ECM. To evaluate whether oAC might affect current best available anti-malarial treatment, we conducted a randomized controlled open label trial in 52 human volunteers (ISRCTN NR. 64793756), administering artesunate (AS) in the presence or absence of oAC. We demonstrated that co-administration of oAC was safe and well-tolerated. In the 26 subjects further analyzed, we found no interference with the pharmacokinetics of parenteral AS or its pharmacologically active metabolite dihydroartemisinin.

Conclusions/Significance

oAC protects against ECM in mice, and does not interfere with the pharmacokinetics of parenteral artesunate. If future studies succeed in establishing the efficacy of oAC in human malaria, then the characteristics of being inexpensive, well-tolerated at high doses and requiring no sophisticated storage would make oAC a relevant candidate for adjunct therapy to reduce mortality from severe malaria, or for immediate treatment of suspected severe malaria in a rural setting.

Trial Registration

Controlled-Trials.com ISRCTN64793756     

Lithostratigraphic Context for Kln-1993.05-SNJ,a Fossil Colobine Maxilla from Jokotingkir,Sangiran Dome

Jablonski and Tyler (1999) announced a new subspecies of colobine monkey based on a fossil partial maxilla from the Sangiran dome. The specimen is easily assigned to a living leaf monkey species—most extant Southeast Asian catarrhines differ only subspecifically from their Middle Pleistocene earliest local fossil ancestors. Yet Jablonski and Tyler (1999) reported an improbable provenance for the specimen; a mass-flow volcanic breccia generally considered late Pliocene in age. We show that the Lower Lahar was laid down amidst a range of paludal habitats and that its deposition predates the appearance of all-but-now extinct, water-tolerant mammals on emergent Java. No other catarrhine fossil has been ascribed to the Lower Lahar, not even hominins, which are the most gregarious members of the group. More probable provenance lies in the upper Sangiran or the lower Bapang formations. Either alternative would associate the specimen with other catarrhine fossils in more tenable Pleistocene environments. We also unravel errors and inconsistencies in the contextual report and in the discussion of dome geochronology. The various radiometric, paleomagnetic, and paleontologic studies cited show a discordance of about 300 Ka (thousand years) across the lithostratigraphic sequence. Plio-Pleistocene biogeographic hypotheses for Java must work with short and long chronologies.     

Fasciola gigantica cathepsin L proteinase-based synthetic peptide for immunodiagnosis and prevention of sheep fasciolosis

Sheep fasciolosis is a devastating burden for the livestock industry. We herein report on immunodiagnosis of fasciolosis, and significant protection of sheep against challenge infection with Fasciola gigantica following immunization with a peptide based on the H-Asp(110)-Lys-Ile-Asp-Trp-Arg-Glu-Ser-Gly-Tyr-Val-Thr-Glu-Val(123)-OH (Fas14p) sequence of F. gigantica cathepsin L-cysteine proteinase. This sequence was synthesized in three different forms: as N(alpha) acetylated (Ac-Asp(110)-Lys-Ile-Asp-Trp-Arg-Glu-Ser-Gly-Tyr-Val-Thr-Glu-Val(123)-OH, FasAc14p), bearing at the amino-terminus an N(alpha) acetylated cystein (Ac-Cys-Asp(110)-Lys-Ile-Asp-Trp-Arg-Glu-Ser-Gly-Tyr-Val-Thr-Glu-Val(123)-OH, FasAcCys14p), and conjugated to sequential oligopeptide carrier Ac-[Lys-Aib-Gly](4)-OH (Ac-SOC(4)) through an amide bond formed between Val(123) carboxylic group of the epitope and the lysine N(epsilon) groups of the carrier (Ac-[Lys(Fas14p)-Aib-Gly](4)-OH). Ac-[Lys(Fas14p)-Aib-Gly](4)-OH failed to readily discriminate between na?ve and infected sheep. In contrast, the free peptides reproducibly differentiated between parasite-free sheep, sheep infected with parasites other than Fasciola, and experimentally Fasciola-infected sheep. The data together indicated that the peptides might be of considerable use for discriminating between early and late, and low and high burden, sheep infection with F. gigantica. FasAc14p was chosen to determine whether a peptide based on a critical enzymatic site of cathepsin L proteinase may induce protection against challenge infection. Sheep immunization with FasAc14p peptide induced significant expression of interleukin-4 mRNA, and humoral antibodies that bound to molecule(s) on the intact surface membrane of newly excysted juvenile worms, and mediated their attrition. The immune responses were associated with significant (P < 0.02) decrease of 23.1% in worm recovery, but with no decrease in the size or maturation of worms recovered.