Immunohistochemical distribution of S-100 protein and type IV collagen in human embryonic and fetal sympathetic neuroblasts

Summary The expression and distribution of S-100 protein and type IV collagen was studied immunohistochemically in sympathetic neuroblasts from the paravertebral region to the adrenal glands in human embryos and fetuses ranging from 7 to 12 weeks gestational age. Prom 7 weeks gestational age, S-100 protein was detected in round or oval cells mingling with sympathetic neuroblasts, and in spindle-shaped cells forming a continuous layer around them. The latter S-100 protein-positive cells were found in contact with the Schwann cells of nerve fibres entering the groups of sympathetic neuroblasts. Staining for type IV collagen showed that all groups of sympathetic neuroblasts were surrounded by a continuous basement membrane. By examining serial sections stained for type IV collagen and S-100 protein, a continuous basement membrane was found along the distribution pattern of the peripheral S-100 protein-positive spindle cells. The morphology of these cells, and their relationships with Schwann cells and with the basement membrane of the sympathetic neuroblasts, indicated that they were Schwann-like cells probably capable of synthesizing a continuous basement membrane separating the neuroblasts from the adjacent tissues. In contrast, the round or oval S-100 protein-positive cells, in contact with the sympathetic neuroblasts and not associated with nerve fibres, were considered as sustentacular or sustentacular precursor cells. At week 7 gestational age, the peri-adrenal sympathetic neuroblasts and their sustentacular and Schwann-like cells started to invade the adrenal glands and mingled with the adrenal cortical cells. These findings suggest the extra-adrenal origin of the sustentacular cells in embryonic and fetal adrenal glands.     

A Unifying Organ Model of Pancreatic Insulin Secretion

The secretion of insulin by the pancreas has been the object of much attention over the past several decades. Insulin is known to be secreted by pancreatic β-cells in response to hyperglycemia: its blood concentrations however exhibit both high-frequency (period approx. 10 minutes) and low-frequency oscillations (period approx. 1.5 hours). Furthermore, characteristic insulin secretory response to challenge maneuvers have been described, such as frequency entrainment upon sinusoidal glycemic stimulation; substantial insulin peaks following minimal glucose administration; progressively strengthened insulin secretion response after repeated administration of the same amount of glucose; insulin and glucose characteristic curves after Intra-Venous administration of glucose boli in healthy and pre-diabetic subjects as well as in Type 2 Diabetes Mellitus. Previous modeling of β-cell physiology has been mainly directed to the intracellular chain of events giving rise to single-cell or cell-cluster hormone release oscillations, but the large size, long period and complex morphology of the diverse responses to whole-body glucose stimuli has not yet been coherently explained. Starting with the seminal work of Grodsky it was hypothesized that the population of pancreatic β-cells, possibly functionally aggregated in islets of Langerhans, could be viewed as a set of independent, similar, but not identical controllers (firing units) with distributed functional parameters. The present work shows how a single model based on a population of independent islet controllers can reproduce very closely a diverse array of actually observed experimental results, with the same set of working parameters. The model’s success in reproducing a diverse array of experiments implies that, in order to understand the macroscopic behaviour of the endocrine pancreas in regulating glycemia, there is no need to hypothesize intrapancreatic pacemakers, influences between different islets of Langerhans, glycolitic-induced oscillations or β-cell sensitivity to the rate of change of glycemia.     

Potential advantages of acute kidney injury management by mesenchymal stem cells

Mesenchymal stem cells are currently considered as a promising tool for therapeutic application in acute kidney injury (AKI) management. AKI is characterized by acute tubular injury with rapid loss of renal function. After AKI, inflammation, oxidative stress and excessive deposition of extracellular matrix are the molecular events that ultimately cause the end-stage renal disease. Despite numerous improvement of supportive therapy, the mortality and morbidity among patients remain high. Therefore, exploring novel therapeutic options to treat AKI is mandatory. Numerous evidence in animal models has demonstrated the capability of mesenchymal stem cells (MSCs) to restore kidney function after induced kidney injury. After infusion, MSCs engraft in the injured tissue and release soluble factors and microvesicles that promote cell survival and tissue repairing. Indeed, the main mechanism of action of MSCs in tissue regeneration is the paracrine/endocrine secretion of bioactive molecules. MSCs can be isolated from several tissues, including bone marrow, adipose tissue, and blood cord; pre-treatment procedures to improve MSCs homing and their paracrine function have been also described. This review will focus on the application of cell therapy in AKI and it will summarize preclinical studies in animal models and clinical trials currently ongoing about the use of mesenchymal stem cells after AKI.     

Magnetic Resonance Imaging Allows the Evaluation of Tissue Damage and Regeneration in a Mouse Model of Critical Limb Ischemia

Magnetic resonance imaging (MRI) provides non-invasive, repetitive measures in the same individual, allowing the study of a physio-pathological event over time. In this study, we tested the performance of 7 Tesla multi-parametric MRI to monitor the dynamic changes of mouse skeletal muscle injury and regeneration upon acute ischemia induced by femoral artery dissection. T2-mapping (T2 relaxation time), diffusion-tensor imaging (Fractional Anisotropy) and perfusion by Dynamic Contrast-Enhanced MRI (K-trans) were measured and imaging results were correlated with histological morphometric analysis in both Gastrocnemius and Tibialis anterior muscles. We found that tissue damage positively correlated with T2-relaxation time, while myofiber regeneration and capillary density positively correlated with Fractional Anisotropy. Interestingly, K-trans positively correlated with capillary density. Accordingly, repeated MRI measurements between day 1 and day 28 after surgery in ischemic muscles showed that: 1) T2-relaxation time rapidly increased upon ischemia and then gradually declined, returning almost to basal level in the last phases of the regeneration process; 2) Fractional Anisotropy dropped upon ischemic damage induction and then recovered along with muscle regeneration and neoangiogenesis; 3) K-trans reached a minimum upon ischemia, then progressively recovered. Overall, Gastrocnemius and Tibialis anterior muscles displayed similar patterns of MRI parameters dynamic, with more marked responses and less variability in Tibialis anterior. We conclude that MRI provides quantitative information about both tissue damage after ischemia and the subsequent vascular and muscle regeneration, accounting for the differences between subjects and, within the same individual, between different muscles.     

Loss of proline-rich tyrosine kinase 2 function induces spreading and motility of epithelial prostate cells

Although prostate carcinoma is an aggressive cancer preferentially metastasizing to the bones, many prostate tumors remain localized and confined to the prostate indefinitely. Prediction of the behavior of anatomically localized and moderately differentiated prostate tumors remains difficult because of lack of prognostic markers. Cell motility is an important step in the progression of epithelial tumor toward invasive metastatic carcinomas and changes in the expression and function of adhesion molecules contribute to the acquisition of a more malignant phenotype. Proline-rich tyrosine kinase 2 (Pyk2) is implicated in regulating the organization of actin cytoskeleton, a process critical for cell migration, mitosis, and tumor metastasis. In this report, we investigated whether Pyk2 played a role in the acquisition of an aggressive phenotype in prostate cell. Data reported here demonstrate that loss of Pyk2 kinase function results in induction of cell motility and migration in EPN cells, a line of non-transformed epithelial cells derived from human normal prostate tissue. Changes in motility and migration of prostate cells were associated with changes in the expression of several proteins involved in cell adhesion and reorganization of actin cytoskeleton. Ablation of Pyk2 kinase activity caused a dramatic decrease of the expression of E-cadherin and IRS1 and an increase of the expression of alpha5-integrin. In addition, a massive reorganization of actin cytoskeleton was observed. Our data indicate that Pyk2 plays a central role in the mechanism that regulate cell-cell and cell-substrate interaction and lack of its kinase activity induces prostate cells to acquire a malignant, migrating phenotype.     

Pyrosequencing Unveils Cystic Fibrosis Lung Microbiome Differences Associated with a Severe Lung Function Decline

Chronic airway infection is a hallmark feature of cystic fibrosis (CF) disease. In the present study, sputum samples from CF patients were collected and characterized by 16S rRNA gene-targeted approach, to assess how lung microbiota composition changes following a severe decline in lung function. In particular, we compared the airway microbiota of two groups of patients with CF, i.e. patients with a substantial decline in their lung function (SD) and patients with a stable lung function (S). The two groups showed a different bacterial composition, with SD patients reporting a more heterogeneous community than the S ones. Pseudomonas was the dominant genus in both S and SD patients followed by Staphylococcus and Prevotella. Other than the classical CF pathogens and the most commonly identified non-classical genera in CF, we found the presence of the unusual anaerobic genus Sneathia. Moreover, the oligotyping analysis revealed the presence of other minor genera described in CF, highlighting the polymicrobial nature of CF infection. Finally, the analysis of correlation and anti-correlation networks showed the presence of antagonism and ecological independence between members of Pseudomonas genus and the rest of CF airways microbiota, with S patients showing a more interconnected community in S patients than in SD ones. This population structure suggests a higher resilience of S microbiota with respect to SD, which in turn may hinder the potential adverse impact of aggressive pathogens (e.g. Pseudomonas). In conclusion, our findings shed a new light on CF airway microbiota ecology, improving current knowledge about its composition and polymicrobial interactions in patients with CF.     

Functional insights from structural genomics

Structural genomics efforts have produced structural information, either directly or by modeling, for thousands of proteins over the past few years. While many of these proteins have known functions, a large percentage of them have not been characterized at the functional level. The structural information has provided valuable functional insights on some of these proteins, through careful structural analyses, serendipity, and structure-guided functional screening. Some of the success stories based on structures solved at the Northeast Structural Genomics Consortium (NESG) are reported here. These include a novel methyl salicylate esterase with important role in plant innate immunity, a novel RNA methyltransferase (H. influenzae yggJ (HI0303)), a novel spermidine/spermine N-acetyltransferase (B. subtilis PaiA), a novel methyltransferase or AdoMet binding protein (A. fulgidus AF_0241), an ATP:cob(I)alamin adenosyltransferase (B. subtilis YvqK), a novel carboxysome pore (E. coli EutN), a proline racemase homolog with a disrupted active site (B. melitensis BME11586), an FMN-dependent enzyme (S. pneumoniae SP_1951), and a 12-stranded β-barrel with a novel fold (V. parahaemolyticus VPA1032).     

Density gradient centrifugation of sperm from a subfertile stallion and effect of seminal plasma addition on fertility

Stallions are not selected for fertility but for other criteria (pedigree, conformation, performances, progeny), therefore valuable but subfertile stallions with poor semen quality are frequently used in commercial breeding programs. The object of this study was to evaluate whether sperm selection through a silane-coated silica colloid gradient centrifugation, with or without the addition of seminal plasma of a high fertile stallion, could improve the pregnancy rates of an oligospermic valuable stallion in a commercial breeding program. In 2008 breeding season (experiment 1, n=104 mares), simple centrifugation and density gradient centrifugation of the sperm were compared. In 2009 and 2010 breeding seasons (experiment 2, n=125 mares), the effect of the addition of 5% seminal plasma to the extender after sperm selection was evaluated. In all mares deep horn uterine insemination was performed with 1 ml containing 50×10(6) morphologically normal progressive motile spermatozoa, 24-30 h after induction of ovulation with hCG. Pregnancy diagnosis by ultrasonography was performed 14 days following ovulation. Results showed a higher per cycle pregnancy rate (P>0.05) when sperm selection through a density gradient was used (62% vs. 42.3%, exp 1), while the addition of 5% seminal plasma did not influence the outcome (45.9% vs. 47.6%, exp 2) (P>0.05). An age-related decrease in the fertility of the stallion was observed when comparing the results from the different breeding seasons (P<0.05). In conclusion, sperm selection through a discontinuous density gradient enabled a normal per cycle pregnancy rate to be achieved from an oligospermic-subfertile stallion in a commercial breeding program, and no differences were observed regarding the addition of seminal plasma.     

Crankshaft motions of the polypeptide backbone in molecular dynamics simulations of human type-α transforming growth factor

Summary Order parameters for the backbone N–H and C–H bond vectors have been calculated from a 150 ps molecular dynamics (MD) simulation of human type- transforming growth factor in H₂O solvent. Two kinds of crankshaft motions of the polypeptide backbone are observed in this MD trajectory. The first involves small-amplitude rocking of the rigid peptide bond due to correlated changes in the backbone dihedral angles _i–1 and _i. These high-frequency librational crankshaft motions are correlated with systematically smaller values of motional order parameters for backbone N–H bond vectors compared to C–H bond vectors. In addition, infrequent crankshaft flips of the peptide bond from one local minimum to another are observed for several amino acid residues. These MD simulations demonstrate that comparisons of N–H and C–H order parameters provide a useful approach for identifying crank-shaft librational motions in proteins.