Enzyme linked receptor protein signaling pathway is one of the ontology groups that are highly up-regulated in porcine oocytes before in vitro maturation.

Before being able to fully participate in the processes associated with its function as a female gamete, the oocyte needs to undergo a range of changes to achieve its mature form. These morphological, biochemical and metabolomic processes are induced by the somatic tissues surrounding the oocyte, through the expression of specific transcription and growth factors. The maturation of the oocyte is highly important for the proceedings that lead to successful fertilization, early embryonic development and implantation. Domestic pigs were used as models for our study, with the cumulus-oocyte complexes obtained from the ovaries that were recovered at slaughter. After shedding of the cumulus, oocytes were assessed with BCB test, with the viable ones chosen to undergo in vitro maturation. With the use of expression microarrays, we analyzed gene expression before and after IVM and detected major changes in both genes that were proven to be associated with oocyte maturation before (FOS, VEGFA, CHRDL1, TGFBR3, FST, INSR, ID1, TXNIP, SMAD4, MAP3K1, EIF2AK3 and KIT) and genes not previously linked with reproduction associated processes (MYO1E, PHIP, KLF10 and SHOC2). All the genes were briefly described, with consideration of possible involvement of the newly discovered elements of the transcriptome in the process of oocyte maturation.

Expression of cell mitotic progression proteins and keratinocyte markers in porcine buccal pouch mucosal cells during short-term, real-time primary culture.

The porcine model is often used in clinical trials. The pig has many fundamental anatomic, physiological and nutritional similarities to humans. Additionally, the European Medicines Agency (EMA) demands the use large animals in clinical studies. Oral mucosa has received special attention due to its regenerative properties. Oral tissue is composed of several types of cells including fibroblasts and keratinocytes. The porcine oral mucosa/buccal pouch mucosa has a cellular structure with defined proliferation and differentiated capability. In this study, we investigated the expression pattern of porcine buccal pouch mucosal cell proliferation and differentiation markers such as Ki-67, proliferating cell nuclear antigen (PCNA), and involucrin. We observed a clear monolayer culture of spindle-shaped, porcine buccal pouch mucosal cells during 168 h of real-time in vitro culture. The RTCA assays revealed parametric and progressive increases in proliferation after 72 h of IVC. We found an altered proliferation index (PI) in the replicated groups of experiments except through the 144-168 h proliferation period. The RT-qPCR results demonstrated a significant increase in Ki-67 and PCNA expression after 48, 120, and 168 h of IVC as compared to other culture periods (P<0.001). The involucrin mRNA displayed increased expression after 168 h of IVC as compared to other periods. We observed a lack of PCR product at 24 h in the case of Ki-67 and both before IVC (0h) and after 24 h of IVC for PCNA mRNA. When we analyzed the three transcripts together, we found the highest expression of involucrin during each of the culture periods. It has been suggested that Ki-67, PCNA, and involucrin may be successfully used as markers of porcine buccal pouch mucosal cell proliferation and differentiation capability in vitro.

The origin, in vitro differentiation, and stemness specificity of progenitor cells.

Since the successful collection of the first progenitor stem cells (SCs), there has been an increased interest in these cells as a model for undiscovered and unlimited potential of differentiation and development. Additionally, it was shown that SC populations display an ability to form pluripotent and/or totipotent cell populations. It was found that human ovarian granulosa cells (GCs) maintain a large capacity for differentiation into several other cell lineages, such as chondrogenic, osteogenic, neurogenic, and adipogenic, particularly during long-term, in vitro culture. In these cases, the specific media supplements that promote various pathways of differentiation, such as leukemia-inhibiting factor (LIF) and/or FSH, are well recognized. However, these are only some examples of the differentiation possibilities of human SCs in vitro and other pathways still require further investigation. Many SC populations, which are directed to differentiate into specific cell types, are also successfully used in several human disease therapies, e.g. leukemia. Moreover, SCs are used for tissue scaffold construction in patients with respiratory and cardiovascular diseases. In this review, the most recent knowledge about the in vitro growth and differentiation capacity of SCs is presented. Furthermore, we discuss the possible worldwide application of SCs in advanced cell and tissue bioengineering. In conclusion, it is suggested that, in the future, SCs will be a basic strategy in human therapy, and their use will open new gates in regenerative and reconstructive medicine in the 21st century.

Molecular basis of growth, proliferation, and differentiation of mammalian follicular granulosa cells.

For normal folliculogenesis and oogenesis to occur many intrinsic and extrinsic factors are needed, i.e. positive feedback of hormone secretion and local ovarian-follicular growth factors distribution. During follicle formation, granulosa cells (GCs) change their morphology and physiological properties. The factors needed for GCs to differentiate within each layer are transforming growth factor beta (TGFB) and insulin-like growth factor (IGF), as well as the activation and modification of biochemical pathways involved in folliculogenesis. Physiological alterations occur when GC genes are characterized by several differences in their gene expression profile. Studies in recent years indicate a variety of processes involved in follicle morphology and biochemical remodeling during growth and development. It was demonstrated that IGFs play a central role in the differentiation of GCs both in vivo and in vitro. Moreover, the primary role of FSH and LH in the formation of the ovarian follicle, was also described. Our review article characterizes the most important pathways involved in the differentiation of GCs and the effect of various factors on gene expression in GCs during folliculogenesis.

The biomedical aspects of oral mucosal epithelial cell culture in mammals.

In recent years, there has been a growing interest in epithelial cell tissue culture, particularly oral mucosa and its application utilizing in vitro cell culture in medicine. This involves tests using animal models to better understand oral mucosa function, and the differences in its construction in various animal models. The use of buccal pouch mucosal cell culture provides insight into the processes of trans mucosal transport and regeneration of the oral epithelium. The processes associated with epithelium regeneration is the base for stem cell research and/or oral cancer investigation. These artificially cultured tissue equivalents are used in transplant surgery for the treatment of a variety of tissue dysfunctions, i.e. eye, esophagus, or urethra. In this review, the most recent results from studies carried out on in animal models, which may be applied in areas such as regenerative medicine and reconstructive surgery, were explored.

Differential pro-apoptotic effects of synthetic 4-thiazolidinone derivative Les-3288, doxorubicin and temozolomide in human glioma U251 cells.

AIM: To compare various pro-apoptotic effects of synthetic 4-thiazolidinone derivative (Les-3288), doxorubicin (Dox) and temozolomide (TMZ) in the treatment of human glioma U251 cells to improve treatment outcomes of glioblastoma and avoid anticancer drug resistance. METHODS: The cytotoxic effects of drugs used in human glioma U251 cells were measured by cell viability and proliferation assay (MTT), Trypan blue exclusion test, and Western-blot analysis of the apoptosis-related proteins. In addition, flow cytometry study of reactive oxygen species (ROS) level in glioma cells was carried out. Cytomorphological changes in treated cells were monitored by fluorescent microscopy after cell staining with Hoechst 33342 and ethydium bromide. RESULTS: Half-maximal inhibitory concentration (IC50) of Les-3288, Dox, and TMZ was calculated for human glioblastoma U251 cells. The rating of the values of this indicator of cellular vitality was assessed. The results of MTT assay proved the superiority of Les-3288 vs Les-3288>Dox>TMZ, which is in agreement with the results of Trypan blue testing showing Les-3288≈Dox>TMZ. In general, such ranking corresponded to a scale of pro-apoptotic impairments in the morphology of glioma U251 cells and the results of Western-blot analysis of cleaved Caspase 3. Contrary to Dox, Les-3288 and TMZ did not affect significantly ROS levels in the treated cells. CONCLUSION: The effect of the synthetic 4-thiazolidinone derivative Les-3288 is realized via apoptosis mechanisms and does not involve ROS. In comparison with Dox and TMZ, it is more effective in destroying human glioblastoma U251 cells. Les-3288 compound has a potential as an anticancer drug for glioblastoma. Nevertheless, further preclinical studies of the blood-brain barrier are needed.

Putative anticancer potential of novel 4-thiazolidinone derivatives: cytotoxicity toward rat C6 glioma in vitro and correlation of general toxicity with the balance of free radical oxidation in rats.

AIM: To evaluate the cytotoxic action of 4-thiazolidinone derivatives (ID 3288, ID 3882, and ID 3833) toward rat glioma C6 cells and to compare the effects of these compounds and doxorubicin on the balance of free radical oxidation (FRO) and antioxidant activity (AOA) in the serum of rats. METHODS: Glioma cells were treated with ID 3882, ID 3288, ID 3833, and doxorubicin, and their cytotoxicity was studied using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and Trypan blue exclusion test, light and fluorescent microscopy, and flow cytometric study of cell cycling and apoptosis, including measuring of Annexin V-positive cells. The contents of superoxide radical, hydrogen peroxide, hydroxyl radical, malonic dialdehyde, and hydrogen sulfide were measured in the serum of rats. Enzymatic activity of superoxide dismutase (SOD), catalase (Cat), and glutathione peroxydase (GPO) was determined. RESULTS: Among novel 4-thiazolidinone derivatives, ID 3288 was most toxic toward rat glioma C6 cells, even compared with doxorubicin. All applied derivatives were less active than doxorubicin in inducing reactive oxygen species-related indicators in the serum of rats. A similar effect was observed when enzymatic indicators of AOA processes were measured. While doxorubicin inhibited the activity of SOD, GPO, and Cat, the effects of 4-thiazolidinone derivatives were less prominent. CONCLUSION: Novel 4-thiazolidinone derivatives differ in their antineoplastic action toward rat glioma C6 cells, and ID 3288 possesses the highest activity compared to doxorubicin. Measurement of indicators of FRO and AOA in the serum of rats treated with these compounds showed their lower general toxicity compared with doxorubicin's toxicity.

Negative pressure wound therapy in the treatment of diabetic foot ulcers may be mediated through differential gene expression.

AIMS: Negative pressure wound therapy (NPWT) has been successfully used as a treatment for diabetic foot ulceration (DFU). Its mechanism of action on the molecular level, however, is not fully understood. We assessed the effect of NPWT on gene expression in patients with type 2 diabetes (T2DM) and DFU. METHODS: We included two cohorts of patients-individuals treated with either NPWT or standard therapy. The assignment to NWPT was non-randomized and based on wound characteristics. Differential gene expression profiling was performed using Illumina gene expression arrays and R Bioconductor pipelines based on the 'limma' package. RESULTS: The final cohort encompassed 21 patients treated with NPWT and 8 with standard therapy. The groups were similar in terms of age (69.0 versus 67.5 years) and duration of T2DM (14.5 versus 14.4 years). We identified four genes differentially expressed between the two study arms post-treatment, but not pre-treatment: GFRA2 (GDNF family receptor alpha-2), C1QBP (complement C1q binding protein), RAB35 (member of RAS oncogene family) and SYNJ1 (synaptic inositol 1,4,5-trisphosphate 5-phosphatase 1). Interestingly, all four genes seemed to be functionally involved in wound healing by influencing re-epithelialization and angiogenesis. Subsequently, we utilized co-expression analysis in publicly available RNA-seq data to reveal the molecular functions of GFRA2 and C1QBP, which appeared to be through direct protein-protein interactions. CONCLUSIONS: We found initial evidence that the NPWT effect on DFUs may be mediated through differential gene expression. A discovery of the specific molecular mechanisms of NPWT is potentially valuable for its clinical application and development of new therapies.

DNA methylation analysis of negative pressure therapy effect in diabetic foot ulcers.

OBJECTIVE: Negative pressure wound therapy (NPWT) has been used to treat diabetic foot ulcerations (DFUs). Its action on the molecular level, however, is only partially understood. Some earlier data suggested NPWT may be mediated through modification of local gene expression. As methylation is a key epigenetic regulatory mechanism of gene expression, we assessed the effect of NPWT on its profile in patients with type 2 diabetes (T2DM) and neuropathic non-infected DFUs. METHODS: Of 36 included patients, 23 were assigned to NPWT and 13 to standard therapy. Due to ethical concerns, the assignment was non-randomized and based on wound characteristics. Tissue samples were obtained before and 8 ± 1 days after therapy initiation. DNA methylation patterns were checked by Illumina Methylation EPIC kit. RESULTS: In terms of clinical characteristics, the groups presented typical features of T2DM; however, the NPWT group had significantly greater wound area: 16.8 cm2 vs 1.4 cm2 (P = 0.0003). Initially only one region at chromosome 5 was differentially methylated. After treatment, 57 differentially methylated genes were found, mainly located on chromosomes 6 (chr6p21) and 20 (chr20p13); they were associated with DNA repair and autocrine signaling via retinoic acid receptor. We performed differential analyses pre treatment and post treatment. The analysis revealed 426 differentially methylated regions in the NPWT group, but none in the control group. The enrichment analysis showed 11 processes significantly associated with NPWT, of which 4 were linked with complement system activation. All but one were hypermethylated after NPWT. CONCLUSION: The NPWT effect on DFUs may be mediated through epigenetic changes resulting in the inhibition of complement system activation.

Negative-pressure wound therapy for management of chronic neuropathic noninfected diabetic foot ulcerations - short-term efficacy and long-term outcomes.

PURPOSE: Negative pressure wound therapy (NPWT) is an adjunct method used in the treatment of diabetic foot ulceration (DFU). Real world data on its effectiveness and safety is scarce. In this prospective observational study, we assessed the short-term efficacy, safety, and long-term outcomes of NPWT in patients with type 2 diabetes (T2DM) and neuropathic, noninfected DFUs. METHODS: Based on wound characteristics, mainly area (>1 vs. ≤1 cm2), 75 patients with DFUs treated in an outpatient clinic were assigned to NPWT (n = 53) or standard therapy (n = 22). Wound area reduction was evaluated after 8 ± 1 days. Long-term outcomes assessed included complete ulceration closure and recurrence rate. RESULTS: Patients assigned to NPWT were characterized by greater wound area (15.7 vs. 2.9 cm2). Reduction in wound area was found in both the NPWT (-1.1 cm2, -10.2%, p = 0.0001) and comparator group (-0.3 cm2, -18.0%, p = 0.0038). No serious adverse events related to NPWT were noted. Within 1 year, 55.1% (27/49) of DFUs were closed in the NPWT group and 73.7% (14/19) in the comparator group (p = 0.15). In the logistic regression, wound duration and smaller initial area, but not treatment mode, were associated with closure. One-year follow-up after DFU resolution revealed an ~30.0% recurrence rate in both groups (p = 0.88). CONCLUSIONS: NPWT is a safe treatment for neuropathic, nonischemic, and noninfected DFU in patients with T2DM, although this observational study did not prove its effectiveness over standard therapy. Additionally, we report a high rate of both closure and recurrence of ulcers, the latter irrespective of initial ulcer area.

Liver Transplantation for Incurable Alveolar Echinococcosis: An Analysis of Patients Hospitalized in Department of Tropical and Parasitic Diseases in Gdynia.

INTRODUCTION: Alveolar echinococcosis is a parasitic disease caused by the larval stage of tapeworm Echinococcus multilocularis. It usually involves the liver, but can spread to other organs. The treatment of choice is a surgical resection supported by antiparasitic drugs. In the advanced stages of the disease a liver transplantation is the only option. AIM: This article presents the problems related to care of patients after liver transplantation for advanced alveolar echinococcosis. MATERIAL: Sixty-seven patients with alveolar echinococcosis were hospitalized in our clinic in the years 2000-2015. Liver transplantation has been a therapeutic option for 9 patients. We retrospectively analyzed data of qualification for the liver transplantation and the postoperative treatment. RESULTS: Follow-up time after liver transplantation ranged from 7 months to 155 months (average, 6.4 years). One patient, with a history of advanced disease (P4N1M0), died due to liver failure. One patient was lost to follow-up. After liver transplantation all patients were receiving albendazole treatment. Two patients did not follow the medical recommendations. In 1 patient, who decided to stop therapy after 1 year, the relapse of alveolar echinococcosis in the left lobe of the transplanted liver passing through the diaphragm to the pericardium was detected. In another case we suspected a relapse of alveolar echinococcosis in transplanted liver due to positive serological tests. CONCLUSION: The prognosis of patient after liver transplantation for alveolar echinococcosis is good. The main problem caused by immunosuppressive therapy is a recurrence of disease in the transplanted liver.

AmpFlSTR Profiler Plus and AmpFlSTR COfiler analysis of tissues stored in GenoFix, a new tissue preservation solution for mass disaster DNA identification.

A preliminary study was conducted to assess the capability of a new alcohol-based tissue fixative, GenoFix, to preserve DNA from biopsy tissues stored at room temperature and/or -20 degrees C in a freezer, for subsequent short tandem repeat (STR) DNA typing analysis. Fresh human smooth muscle samples were stored at room temperature in GenoFix for one month and up to one year and seven months before being processed using the megaplex STR systems, AmpFlSTR Profiler Plus and AmpFlSTR COfiler. Alternatively, muscle tissues in GenoFix were placed at -20 degrees C in a freezer for up to 3 1/2 years following two to three months in the fixative at room temperature. DNA analysis was also carried out on tissues stored in GenoFix for one month at room temperature and subsequently paraffin-embedded and stored at room temperature for four years. The AmpFlSTR Profiler Plus and AmpFlSTR COfiler STR profiles produced, using DNA extracted from all fixed tissue samples, were of very good quality. The fluorescent signals were well balanced across the nine STR loci or six loci comprised in the megaplexes surveyed and profiles showed no differences with those observed for the control blood of the respective donor patients. Continuous exposure to GenoFix at room temperature (up to one year and seven months) did not compromise the STR typing analysis of the fixed tissues. No adverse effects were noted on the STR typeability of tissues fixed with GenoFix and stored at -20 degrees C in a freezer for up to 3 1/2 years. STR profiles generated from the paraffin-embedded tissues fixed in GenoFix were of excellent quality. This preliminary study suggests that GenoFix can be used to store tissue samples at room temperature for up to one year and seven months or at -20 degrees C in a freezer for longer storage (up to 3 1/2 years). This new and odorless tissue fixative promotes tissue and DNA preservation in a very effective manner and as such may prove useful in criminal investigations or mass disaster identifications carried out in remote locations and in which a small or large number of tissue samples are collected for further analyses.

Short-term retention in retarded adolescents as a function of load, delay, and interpolated activity.

The present study investigated certain variables influencing the short-term retention of retarded adolescents. Forty trainable and 40 educable retarded adolescents were shown slides containing arrays of 2, 3, 4, or 5 chromatic pictures to be recalled after varying periods (0, 18, 36, and 140 sec) of filled or unfilled activity. Results indicated that recall in the unfilled condition remained relatively stable over time, while in the filled condition recall was deleteriously affected by a distracter task (p less than .01). No significant retention slope differences were observed between the two IQ groups. As is typically found with nonretarded adults, the curve of forgetting for the retarded individuals described a negatively accelerated function. With increasing proactive interference there were increasing decrements in short-term recall, with neither IQ group differing from the other. It was suggested that both decay and interference contribute to the short-term forgetting of retarded people.

Effect of imposed vs. self-generated imagery and sentence mediation on the free recall of retarded adolescents.

The effectiveness of verbal and imaginal elaborations on the free recall of institutionalized EMR adolescents was investigated. Fifty subjects were assigned to one of five conditions: experimenter-imposed verbalization, experimenter-imposed imagery, subject-generated verbalization, subject-generated imagery, and control. The subjects saw 15 pictures, presented in sets of 3, for five trial bloks, and were asked to recall the 15 pictures after each trial block. Over all trials combined, it was found that recall in the experimenter-imposed verbalization and the experimenter-imposed imagery conditions was reliably superior to recall in the control and subject-generated verbalization conditions. Although retarded adolescents were capable of producing verbal or imaginal elaborations, as evidenced by post-experimental inquiry, elaborations facilitated recall only when they were supplied by the experimenter.

Kinetic-imagery ability of mentally retarded young adults.

Two kinetic-imagery tasks were administered to mentally retarded young adults. In one task they had to judge whether pairs of symmetric ice-cream cone figures were the same or different. The cone on the left was always upright, while the cone on the right was oriented either 0 degrees, 45 degrees, 90 degrees, 135 degrees, or 180 degrees clockwise from the upright. Only one-half of the subjects successfully passed a criterion pretest in which they were required to discriminate pairs of upright same--different cones. These successful subjects exhibited a linear increase in their reaction time judgments as the angular discrepancy between stimuli increased, presumably evidence that they were using an analogical mental rotation process. Although their performance was very good on this task, however, it was quite poor on the second task, in which they had to imagine the rotation of a three-block array, which apparently involves a more complex transformation. Subjects who failed to cone criterion task were unable to anticipate the outcome of the block array and produced primarily egocentric (simple reproduction) responses.

Factors influencing the interrogative strategies of mentally retarded and nonretarded students.

The interrogative strategies of institutionalized mentally retarded young adults and nonretarded first and fourth graders were investigated using a 20-questions type task. Subjects were presented with 8- and 16-cell matrices consisting of geometric forms, and the items that were eliminated by their questions were either covered or left uncovered or left uncovered. A variety of dependent measures was obtained, including information efficiency and proportion of redundant and constraint-seeking questions. In contrast to findings in previous research, results showed that reducing array size and covering eliminated items significantly improved various aspects of children's interrogative strategies, lending support to the idea that reducing cognitive strain can enhance information-processing ability. Despite task simplification, the performance of the fourth graders exceeded that of the first graders on most measures, and the performance of the retarded young adults was comparable to, or poorer than, that of the first graders, who were of appreciably lower MA.

Effects of temporal grouping and redundancy level on the paired-associate learning of retarded adolescents and nonretarded children.

Institutionalized adolescents and nonretarded third-grade children were presented with sets of paired associates which differed in redundancy level and temporal grouping. Redundancy level (33 and 50 percent) was embedded in a list of paired associates by varying the number of repetitions in the response terms. The paired associates were presented temporally in blocks of twos (33 percent) or threes (50 percent). One-half of the subjects received the paired associates highly organized; i.e., all response terms of a temporal block were the same. During testing, all stimulus terms were presented simultaneously for the free-ordered recall of the response terms. For both groups, performance was superior following the more highly organized (congruent) presentation, with the performance of the nonretarded subjects exceeding that of the retarded adolescents. Comparisons were made with previous studies in this series.