A Loss-of-Function Screen for Phosphatases that Regulate Neurite Outgrowth Identifies PTPN12 as a Negative Regulator of TrkB Tyrosine Phosphorylation

Alterations in function of the neurotrophin BDNF are associated with neurodegeneration, cognitive decline, and psychiatric disorders. BDNF promotes axonal outgrowth and branching, regulates dendritic tree morphology and is important for axonal regeneration after injury, responses that largely result from activation of its tyrosine kinase receptor TrkB. Although intracellular neurotrophin (NT) signaling presumably reflects the combined action of kinases and phosphatases, little is known about the contributions of the latter to TrkB regulation. The issue is complicated by the fact that phosphatases belong to multiple independently evolved families, which are rarely studied together. We undertook a loss-of-function RNA-interference-based screen of virtually all known (254) human phosphatases to understand their function in BDNF/TrkB-mediated neurite outgrowth in differentiated SH-SY5Y cells. This approach identified phosphatases from diverse families, which either positively or negatively modulate BDNF-TrkB-mediated neurite outgrowth, and most of which have little or no previously established function related to NT signaling. “Classical” protein tyrosine phosphatases (PTPs) accounted for 13% of the candidate regulatory phosphatases. The top classical PTP identified as a negative regulator of BDNF-TrkB-mediated neurite outgrowth was PTPN12 (also called PTP-PEST). Validation and follow-up studies showed that endogenous PTPN12 antagonizes tyrosine phosphorylation of TrkB itself, and the downstream activation of ERK1/2. We also found PTPN12 to negatively regulate phosphorylation of p130cas and FAK, proteins with previously described functions related to cell motility and growth cone behavior. Our data provide the first comprehensive survey of phosphatase function in NT signaling and neurite outgrowth. They reveal the complexity of phosphatase control, with several evolutionarily unrelated phosphatase families cooperating to affect this biological response, and hence the relevance of considering all phosphatase families when mining for potentially druggable targets.     

Tissue and cellular morphological changes in growth plate explants under compression

The mechanisms by which mechanical loading may alter bone development within growth plates are still poorly understood. However, several growth plate cell or tissue morphological parameters are associated with both normal and mechanically modulated bone growth rates. The aim of this study was to quantify in situ the three-dimensional morphology of growth plate explants under compression at both cell and tissue levels. Growth plates were dissected from ulnae of immature swine and tested under 15% compressive strain. Confocal microscopy was used to image fluorescently labeled chondrocytes in the three growth plate zones before and after compression. Quantitative morphological analyses at both cell (volume, surface area, sphericity, minor/major radii) and tissue (cell/matrix volume ratio) levels were performed. Greater chondrocyte bulk strains (volume decrease normalized to the initial cell volume) were found in the proliferative (35.4%) and hypertrophic (41.7%) zones, with lower chondrocyte bulk strains (24.7%) in the reserve zone. Following compression, the cell/matrix volume ratio decreased in the reserve and hypertrophic zones by 24.3% and 22.6%, respectively, whereas it increased by 35.9% in the proliferative zone. The 15% strain applied on growth plate explants revealed zone-dependent deformational states at both tissue and cell levels. Variations in the mechanical response of the chondrocytes from different zones could be related to significant inhomogeneities in growth plate zonal mechanical properties. The ability to obtain in situ cell morphometry and monitor the changes under compression will contribute to a better understanding of mechanisms through which abnormal growth can be triggered.     

Conformational study of calf thymus HMG14 nonhistone protein

Physicochemical study of nonhistone protein HMG14 from calf thymus has been undertaken. The protein has a random structure with a molecular weight of approximately 10,000. On interaction with DNA, it behaves like histones and nonhistone protein HMG17. Both circular dichroism and melting absorption technics show that the protein has an ionic interaction with DNA without causing significant changes in DNA structure. In conrast to HMG1 and HMG2 which reduce linking number of circular DNA, nonhistone protein HMG14 and HMG17 do not introduce any changes in topological winding number of DNA.     

Rank Covariance Methods for the Analysis of Survival Data

A procedure for comparing survival times between several groups of patients through rank analysis of covariance was introduced by WOOLSON and LACHENBRUCH (1983). It is a modification of Quade' rank analysis of covariance procedure (1967) and can be used for the analysis of right-censored data. In this paper, two additional modifications of Quade' original test statistic are proposed and compared to the original modification introduced by Woolson and Lachenbruch. These statistics are compared to one another and to the score test from Cox' proportional hazards model by way of a limited Monte Carlo study. One of the statistics, Q_R2, is recommended for general use for the rank analysis of covariance of right-censored survivorship data.     

Low Selection Pressure Aids the Evolution of Cooperative Ribozyme Mutations in Cells

Understanding the evolution of functional RNA molecules is important for our molecular understanding of biology. Here we tested experimentally how two evolutionary parameters, selection pressure and recombination, influenced the evolution of an evolving RNA population. This was done using four parallel evolution experiments that employed low or gradually increasing selection pressure, and recombination events either at the end or dispersed throughout the evolution. As model system, a trans-splicing group I intron ribozyme was evolved in Escherichia coli cells over 12 rounds of selection and amplification, including mutagenesis and recombination. The low selection pressure resulted in higher efficiency of the evolved ribozyme populations, whereas differences in recombination did not have a strong effect. Five mutations were responsible for the highest efficiency. The first mutation swept quickly through all four evolving populations, whereas the remaining four mutations accumulated later and more efficiently under low selection pressure. To determine why low selection pressure aided this evolution, all evolutionary intermediates between the wild type and the 5-mutation variant were constructed, and their activities at three different selection pressures were determined. The resulting fitness profiles showed a high cooperativity among the four late mutations, which can explain why high selection pressure led to inefficient evolution. These results show experimentally how low selection pressure can benefit the evolution of cooperative mutations in functional RNAs.     

Insulin production from hiPSC-derived pancreatic cells in a novel wicking matrix bioreactor

Clinical use of pancreatic β islets for regenerative medicine applications requires mass production of functional cells. Current technologies are insufficient for large-scale production in a cost-efficient manner. Here, we evaluate advantages of a porous cellulose scaffold and demonstrate scale-up to a wicking matrix bioreactor as a platform for culture of human endocrine cells. Scaffold modifications were evaluated in a multiwell platform to find the optimum surface condition for pancreatic cell expansion followed by bioreactor culture to confirm suitability. Preceding scale-up, cell morphology, viability, and proliferation of primary pancreatic cells were evaluated. Two optimal surface modifications were chosen and evaluated further for insulin secretion, cell morphology, and viable cell density for human-induced pluripotent stem cell-derived pancreatic cells at different stages of differentiation. Scale-up was accomplished with uncoated, amine-modified cellulose in a miniature bioreactor, and insulin secretion and cell metabolic profiles were determined for 13 days. We achieved 10-fold cell expansion in the bioreactor along with a significant increase in insulin secretion compared with cultures on tissue culture plastic. Our findings define a new method for expansion of pancreatic cells a on wicking matrix cellulose platform to advance cell therapy biomanufacturing for diabetes.     

A Truncated Proportional Hazards Test

We present two truncated proportional hazards tests. The first, which is applicable to those cases in which the time of action of an agent or treatment is known, was studied in two forms: One uses expected Fisher information and the other observed Fisher information. In this case the expected information statistic, C₁(a₀), had properties superior to the observed information statistic and is recommended. The use of the X² distribution with one degree of freedom for percentiles appears to be satisfactory. When the time of action is unknown, a statistic based on the maximum of the C₁(a) statistics is used. A simulation study gives empirical percentiles for the Max C₁(a) statistic which agree with those given in a study by Muenz, Green and Byar (1977).     

Human adipose derived stem cell exosomes enhance the neural differentiation of PC12 cells

Molecular Biology Reports - Human adipose stem cells (hADSCs) are proper cell sources for tissue regeneration. They mainly mediate their therapeutic effects through paracrine factors as exosomes....     

Isolation and identification of Amycolatopsis sp. strain 1119 with potential to improve cucumber fruit yield and induce plant defense responses in commercial greenhouse

Plant and Soil - The aims of this work were (i) to find a soil indicator to predict durum wheat yield response to Zn fertilization, (ii) to compare the effect of various Zn fertilization strategies...     

Antimicrobial Activity,Stability and Wound Healing Performances of Chitosan Nanoparticles Loaded Recombinant LL37 Antimicrobial Peptide

International Journal of Peptide Research and Therapeutics - Antimicrobial peptides have illustrated potent abilities in the elimination of several pathogens resistant to conventional antimicrobial...