Osteoblasts derived from induced pluripotent stem cells form calcified structures in scaffolds both in vitro and in vivo

Reprogramming somatic cells into an ESC-like state, or induced pluripotent stem (iPS) cells, has emerged as a promising new venue for customized cell therapies. In this study, we performed directed differentiation to assess the ability of murine iPS cells to differentiate into bone, cartilage, and fat in vitro and to maintain an osteoblast phenotype on a scaffold in vitro and in vivo. Embryoid bodies derived from murine iPS cells were cultured in differentiation medium for 8–12 weeks. Differentiation was assessed by lineage-specific morphology, gene expression, histological stain, and immunostaining to detect matrix deposition. After 12 weeks of expansion, iPS-derived osteoblasts were seeded in a gelfoam matrix followed by subcutaneous implantation in syngenic imprinting control region (ICR) mice. Implants were harvested at 12 weeks, histological analyses of cell and mineral and matrix content were performed. Differentiation of iPS cells into mesenchymal lineages of bone, cartilage, and fat was confirmed by morphology and expression of lineage-specific genes. Isolated implants of iPS cell-derived osteoblasts expressed matrices characteristic of bone, including osteocalcin and bone sialoprotein. Implants were also stained with alizarin red and von Kossa, demonstrating mineralization and persistence of an osteoblast phenotype. Recruitment of vasculature and microvascularization of the implant was also detected. Taken together, these data demonstrate functional osteoblast differentiation from iPS cells both in vitro and in vivo and reveal a source of cells, which merit evaluation for their potential uses in orthopedic medicine and understanding of molecular mechanisms of orthopedic disease.     

Participation of TRPV1 in the activity of the GnRH system in male rats

GnRH neuron activity is under the influence of multiple stimuli, including those coming from the endocannabinoid and the immune systems. Since it has been previously suggested that some of the main elements controlling the GnRH pulse generator possess the TRPV1 receptor, the aim of the present study was to evaluate the participation of the hypothalamic TRPV1, through its pharmacological blockade, in the activity of the hypothalamic‐pituitary‐testicular axis in male rats under basal or acute inflammatory conditions. Our hypothesis was based on the idea that the hypothalamic TRPV1 participates in the synthesis of the main neuromodulatory signals controlling GnRH, and therefore the reproductive axis. Our results showed that the hypothalamic TRPV1 blockade induced pro‐inflammatory effects by increasing Tnfα and Il‐1β mRNA hypothalamic levels and inhibited the reproductive axis by affecting Gnrh, Kiss1 and Rfrp3 mRNA levels and decreasing plasma levels of luteinizing hormone and testosterone under basal conditions, without significant additive effects in rats exposed to systemic LPS. Altogether, these results suggest that the hypothalamic TRPV1 receptor participates in the regulation of the GnRH system, probably by modulating immune‐dependent mechanisms.     

High grade prostate intraepithelial neoplasia (PIN) is a PSA-independent risk factor for prostate cancer in African American men: Results from a pilot study

African American men (AAM) demonstrate increased prostate cancer incidence and mortality rates. We investigated known prostate cancer risk factors in AAM. Prostate specific antigen (PSA) and diagnosis of high grade prostatic intraepithelial neoplasia (PIN) were significant prostate cancer predictors. However, even including AAM with low PSA (<4 ng/ml), those with PIN had significantly elevated risk, compared to men without PIN (83.3% vs. 6.9%, p < 0.0001). In AAM diagnosed with PIN, PSA level was no longer significant (83.3% vs. 92.3%, p = 0.593 respectively). Our results suggest that a history of PIN is highly predictive of prostate cancer in AAM, and help provide PSA-independent venues for screening.     

Dietary habits and nutritional status of children from Ukraine during the first 3 years of life

Aim: The aim of our study was to assess the nutritional status and eating behavior of young children and identify the prevalence of macro- and micronutrient diet deficiencies in Ukraine. Materials and methods: Three hundred and fifty children aged from 9 months to 3 years from central, eastern and western regions of Ukraine were involved in the cross-sectional study. Basic child's data were collected, health status was assessed by a physician, and parents used a 3-day food diary and a food questionnaire for self-completion. Data from the diaries and questionnaires were analyzed with DietPlan 6 software. Results: The diet composition was mostly adequate for age. Overall average provision with energy (1165.67 [29.67–4951.33] kcal/day), protein (40.53 [0.63–230.37] g/day) and carbohydrates (153.63 [3.53–708.7] g/day) exceeded the corresponding standards. The diet of the majority of children did not comply with the recommended intake of zinc (91%), iron (68%), calcium (61%), iodine (49%), vitamins A (99%), D (97%), B₆ (89%), B₁₂ (71%), E (70%) and B₁ (61%). Excessive weight was significantly associated with higher levels of energy, protein, carbohydrates and fat consumption. Overweight was reliably correlated with a higher diet's energy and carbohydrates content. Conclusions: The contemporary diet of young children in Ukraine, like in many other developed countries, is generally unbalanced, containing an excess of energy and protein as well as inadequate amount of many minerals and vitamins.     

Strengthening of lateral activation in adult rat visual cortex after retinal lesions captured with voltage-sensitive dye imaging in vivo

Sensory deprivation caused by peripheral injury can trigger functional cortical reorganization across the initially silenced cortical area. It is proposed that intracortical connectivity enables recovery of function within such a lesion projection zone (LPZ), thus substituting lost subcortical input. Here, we investigated retinal lesion-induced changes in the function of lateral connections in the primary visual cortex of the adult rat. Using voltage-sensitive dye recordings, we visualized in millisecond-time resolution spreading synaptic activity across the LPZ. Shortly after lesion, the majority of neurons within the LPZ were subthresholdly activated by delayed propagation of activity that originated from unaffected cortical regions. With longer recovery time, latencies within the LPZ gradually decreased, and activation reached suprathreshold levels. Targeted electrode recordings confirmed that receptive fields of intra-LPZ neurons were displaced to the retinal lesion border while displaying normal orientation and direction selectivity. These results corroborate the view that cortical horizontal connections have a central role in functional reorganization, as revealed here by progressive facilitation of synaptic activity and the traveling wave of excitation that propagates horizontally into the deprived cortical region.     

Generation and characterization of monoclonal antibodies to TDRD7 protein

TDRD7 is a scaffold protein whose specific function is unknown. It has been identified in complexes with proteins that regulate cytoskeleton dynamics and centrosomal movements, mRNA transport, and protein translation apparatus. Here we report the generation and characterization of monoclonal antibodies against TDRD7 protein. Bacterially expressed His-tagged fragments of TDRD7 were used as antigens. Spleen cells from immunized mice were collected and fused with SP2/0 myeloma cells using PEG 2000. High titer anti-TDRD7 antibody-producing hybridoma cell lines were identified by enzyme-linked immunosorbent assay (ELISA) and then subcloned by limiting dilution. Antibodies produced by E6 clone were further tested for their reactivity with the TDRD7 recombinant proteins. The results obtained clearly indicate that E6 anti-TDRD7 antibodies recognize specifically recombinant 6His-tagged TDRD7 proteins and endogenous TDRD7 in Western blotting, immunoprecipitation, and immunocytochemistry. In summary, these antibodies will be useful for researchers investigating TDRD7 and molecular complexes involving this protein.