Serum-Starved Apoptotic Fibroblasts Reduce Blastocyst Production but Enable Development to Term after SCNT in Cattle

Cell cycle synchronization by serum starvation (SS) induces apoptosis in somatic cells. This side effect of SS is hypothesized to negatively affect the outcome of somatic cell nuclear transfer (SCNT). We determined whether apoptotic fibroblasts affect SCNT yields. Serum-starved, adult, bovine fibroblasts were stained with annexin V-FITC/propidium iodide to allow apoptosis detection by flow cytometry. Positive and negative cells sorted by fluorescence activated cell sorting (FACS) and an unsorted control group were used as nuclear donors for SCNT. Reconstructed embryos were cultured in vitro and transferred to synchronized recipients. Apoptosis had no effect on fusion and cleavage rates; however, it resulted in reductions in blastocyst production and quality measured by apoptotic index. However, reconstructed embryos with apoptotic cells resulted in pregnancy rates similar to that of the control on day 30, and generated one live female calf. In conclusion, we showed that apoptotic cells present in serum-starved cultures negatively affect embryo production after SCNT without compromising full-term development. Further studies will evaluate the ability of the oocyte to reprogram cells in specific phases of apoptosis.

Random X Inactivation and Extensive Mosaicism in Human Placenta Revealed by Analysis of Allele-Specific Gene Expression along the X Chromosome

Imprinted inactivation of the paternal X chromosome in marsupials is the primordial mechanism of dosage compensation for X-linked genes between females and males in Therians. In Eutherian mammals, X chromosome inactivation (XCI) evolved into a random process in cells from the embryo proper, where either the maternal or paternal X can be inactivated. However, species like mouse and bovine maintained imprinted XCI exclusively in extraembryonic tissues. The existence of imprinted XCI in humans remains controversial, with studies based on the analyses of only one or two X-linked genes in different extraembryonic tissues. Here we readdress this issue in human term placenta by performing a robust analysis of allele-specific expression of 22 X-linked genes, including XIST, using 27 SNPs in transcribed regions. We show that XCI is random in human placenta, and that this organ is arranged in relatively large patches of cells with either maternal or paternal inactive X. In addition, this analysis indicated heterogeneous maintenance of gene silencing along the inactive X, which combined with the extensive mosaicism found in placenta, can explain the lack of agreement among previous studies. Our results illustrate the differences of XCI mechanism between humans and mice, and highlight the importance of addressing the issue of imprinted XCI in other species in order to understand the evolution of dosage compensation in placental mammals.

Human fallopian tube: a new source of multipotent adult mesenchymal stem cells discarded in surgical procedures

Background: The possibility of using stem cells for regenerative medicine has opened a new field of investigation. The search for sources to obtain multipotent stem cells from discarded tissues or through non-invasive procedures is of great interest. It has been shown that mesenchymal stem cells (MSCs) obtained from umbilical cords, dental pulp and adipose tissue, which are all biological discards, are able to differentiate into muscle, fat, bone and cartilage cell lineages. The aim of this study was to isolate, expand, characterize and assess the differentiation potential of MSCs from human fallopian tubes (hFTs). Methods: Lineages of hFTs were expanded, had their karyotype analyzed, were characterized by flow cytometry and underwent in vitro adipogenic, chondrogenic, osteogenic, and myogenic differentiation. Results: Here we show for the first time that hFTs, which are discarded after some gynecological procedures, are a rich additional source of MSCs, which we designated as human tube MSCs (htMSCs). Conclusion: Human tube MSCs can be easily isolated, expanded in vitro, present a mesenchymal profile and are able to differentiate into muscle, fat, cartilage and bone in vitro.

Human Dental Pulp Cells: A New Source of Cell Therapy in a Mouse Model of Compressive Spinal Cord Injury

Strategies aimed at improving spinal cord regeneration after trauma are still challenging neurologists and neuroscientists throughout the world. Many cell-based therapies have been tested, with limited success in terms of functional outcome. In this study, we investigated the effects of human dental pulp cells (HDPCs) in a mouse model of compressive spinal cord injury (SCI). These cells present some advantages, such as the ease of the extraction process, and expression of trophic factors and embryonic markers from both ecto-mesenchymal and mesenchymal components. Young adult female C57/BL6 mice were subjected to laminectomy at T9 and compression of the spinal cord with a vascular clip for 1 min. The cells were transplanted 7 days or 28 days after the lesion, in order to compare the recovery when treatment is applied in a subacute or chronic phase. We performed quantitative analyses of white-matter preservation, trophic-factor expression and quantification, and ultrastructural and functional analysis. Our results for the HDPC-transplanted animals showed better white-matter preservation than the DMEM groups, higher levels of trophic-factor expression in the tissue, better tissue organization, and the presence of many axons being myelinated by either Schwann cells or oligodendrocytes, in addition to the presence of some healthy-appearing intact neurons with synapse contacts on their cell bodies. We also demonstrated that HDPCs were able to express some glial markers such as GFAP and S-100. The functional analysis also showed locomotor improvement in these animals. Based on these findings, we propose that HDPCs may be feasible candidates for therapeutic intervention after SCI and central nervous system disorders in humans.

Genetic Variation among Major Human Geographic Groups Supports a Peculiar Evolutionary Trend in PAX9

A total of 172 persons from nine South Amerindian, three African and one Eskimo populations were studied in relation to the Paired box gene 9 (PAX9) exon 3 (138 base pairs) as well as its 5' and 3' flanking intronic segments (232 bp and 220 bp, respectively) and integrated with the information available for the same genetic region from individuals of different geographical origins. Nine mutations were scored in exon 3 and six in its flanking regions; four of them are new South American tribe-specific singletons. Exon3 nucleotide diversity is several orders of magnitude higher than its intronic regions. Additionally, a set of variants in the PAX9 and 101 other genes related with dentition can define at least some dental morphological differences between Sub-Saharan Africans and non-Africans, probably associated with adaptations after the modern human exodus from Africa. Exon 3 of PAX9 could be a good molecular example of how evolvability works.

Chrysotile effects on human lung cell carcinoma in culture: 3-D reconstruction and DNA quantification by image analysis

Background: Chrysotile is considered less harmful to human health than other types of asbestos fibers. Its clearance from the lung is faster and, in comparison to amphibole forms of asbestos, chrysotile asbestos fail to accumulate in the lung tissue due to a mechanism involving fibers fragmentation in short pieces. Short exposure to chrysotile has not been associated with any histopathological alteration of lung tissue. Methods: The present work focuses on the association of small chrysotile fibers with interphasic and mitotic human lung cancer cells in culture, using for analyses confocal laser scanning microscopy and 3D reconstructions. The main goal was to perform the analysis of abnormalities in mitosis of fibers-containing cells as well as to quantify nuclear DNA content of treated cells during their recovery in fiber-free culture medium. Results: HK2 cells treated with chrysotile for 48 h and recovered in additional periods of 24, 48 and 72 h in normal medium showed increased frequency of multinucleated and apoptotic cells. DNA ploidy of the cells submitted to the same chrysotile treatment schedules showed enhanced aneuploidy values. The results were consistent with the high frequency of multipolar spindles observed and with the presence of fibers in the intercellular bridge during cytokinesis. Conclusion: The present data show that 48 h chrysotile exposure can cause centrosome amplification, apoptosis and aneuploid cell formation even when long periods of recovery were provided. Internalized fibers seem to interact with the chromatin during mitosis, and they could also interfere in cytokinesis, leading to cytokinesis failure which forms aneuploid or multinucleated cells with centrosome amplification.

Modulation of small leucine-rich proteoglycans (SLRPs) expression in the mouse uterus by estradiol and progesterone

Background: We have previously demonstrated that four members of the family of small leucine-rich-proteoglycans (SLRPs) of the extracellular matrix (ECM), named decorin, biglycan, lumican and fibromodulin, are deeply remodeled in mouse uterine tissues along the estrous cycle and early pregnancy. It is known that the combined action of estrogen (E2) and progesterone (P4) orchestrates the estrous cycle and prepares the endometrium for pregnancy, modulating synthesis, deposition and degradation of various molecules. Indeed, we showed that versican, another proteoglycan of the ECM, is under hormonal control in the uterine tissues. Methods: E2 and/or medroxiprogesterone acetate (MPA) were used to demonstrate, by real time PCR and immunoperoxidase staining, respectively, their effects on mRNA expression and protein deposition of these SLRPs, in the uterine tissues. Results: Decorin and lumican were constitutively expressed and deposited in the ECM in the absence of the ovarian hormones, whereas deposition of biglycan and fibromodulin were abolished from the uterine ECM in the non-treated group. Interestingly, ovariectomy promoted an increase in decorin, lumican and fibromodulin mRNA levels, while biglycan mRNA conspicuously decreased. Hormone replacement with E2 and/or MPA differentially modulates their expression and deposition. Conclusions: The patterns of expression of these SLRPs in the uterine tissues were found to be hormone-dependent and uterine compartment-related. These results reinforce the existence of subpopulations of endometrial fibroblasts, localized into distinct functional uterine compartments, resembling the organization into basal and functional layers of the human endometrium.

The Thermal Effects of Therapeutic Lasers with 810 and 904 nm Wavelengths on Human Skin

Objective: To investigate the effect of therapeutic infrared class 3B laser irradiation on skin temperature in healthy participants of differing skin color, age, and gender. Background: Little is known about the potential thermal effects of Low Level Laser Therapy (LLLT) irradiation on human skin. Methods: Skin temperature was measured in 40 healthy volunteers with a thermographic camera at laser irradiated and control (non-irradiated) areas on the skin. Six irradiation doses (2-12 J) were delivered from a 200mW, 810nm laser and a 60mW, 904nm laser, respectively. Results: Thermal effects of therapeutic LLLT using doses recommended in the World Association for Laser Therapy (WALT) guidelines were insignificant; below 1.5 degrees C in light, medium, and dark skin. When higher irradiation doses were used, the 60mW, 904 nm laser produced significantly (p < 0.01) higher temperatures in dark skin (5.7, SD +/- 1.8 degrees C at 12 J) than in light skin, although no participants requested termination of LLLT. However, irradiation with a 200mW, 810nm laser induced three to six times more heat in dark skin than in the other skin color groups. Eight of 13 participants with dark skin asked for LLLT to be stopped because of uncomfortable heating. The maximal increase in skin temperature was 22.3 degrees C. Conclusions: The thermal effects of LLLT at doses recommended by WALT-guidelines for musculoskeletal and inflammatory conditions are negligible (< 1.5 degrees C) in light, medium, and dark skin. However, higher LLLT doses delivered with a strong 3B laser (200mW) are capable of increasing skin temperature significantly and these photothermal effects may exceed the thermal pain threshold for humans with dark skin color.

Low-Level Laser Irradiation (InGaAlP-660 nm) Increases Fibroblast Cell Proliferation and Reduces Cell Death in a Dose-Dependent Manner

Background and Objective: Impaired cell metabolism and increased cell death in fibroblast cells are physiological features of chronic tendinopathy. Although several studies have shown that low-level laser therapy (LLLT) at certain parameters has a biostimulatory effect on fibroblast cells, it remains uncertain if LLLT effects depend on the physiological state. Study Design/Material and Methods: High-metabolic immortal cell culture and primary human keloid fibroblast cell culture were used in this study. Trypan blue exclusion and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test were used to determine cell viability and proliferation. Propidium iodide stain was used for cell-cycle analysis by flow cytometry. Laser irradiation was performed daily on three consecutive days with a GaAlAs 660-nm laser (mean output: 50 mW, spot size 2 mm(2), power density = 2.5 W/cm(2)) and a typical LLLT dose and a high LLLT dose (irradiation times: 60 or 420 s; fluences: 150 or 1050 J/cm(2); energy delivered: 3 or 21 J). Results: Primary fibroblast cell culture from human keloids irradiated with 3 J showed significant proliferation by the trypan blue exclusion test (p < 0.05), whereas the 3T3 cell culture showed no difference using this method. Propidium iodide staining flow cytometry data showed a significant decrease in the percentage of cells being in proliferative phases of the cell cycle (S/g(2)/M) when irradiated with 21 J in both cell types (hypodiploid cells increased). Conclusions: Our data support the hypothesis that the physiological state of the cells affects the LLLT results, and that high-metabolic rate and short-cell-cycle 3T3 cells are not responsive to LLLT. In conclusion, LLLT with a dose of 3 J reduced cell death significantly, but did not stimulate cell cycle. A LLLT dose of 21 J had negative effects on the cells, as it increased cell death and inhibited cell proliferation.

Induction of Heme Oxygenase-1 Can Halt and Even Reverse Renal Tubule-Interstitial Fibrosis

Background: The tubule-interstitial fibrosis is the hallmark of progressive renal disease and is strongly associated with inflammation of this compartment. Heme-oxygenase-1 (HO-1) is a cytoprotective molecule that has been shown to be beneficial in various models of renal injury. However, the role of HO-1 in reversing an established renal scar has not yet been addressed. Aim: We explored the ability of HO-1 to halt and reverse the establishment of fibrosis in an experimental model of chronic renal disease. Methods: Sprague-Dawley male rats were subjected to unilateral ureteral obstruction (UUO) and divided into two groups: non-treated and Hemin-treated. To study the prevention of fibrosis, animals were pre-treated with Hemin at days -2 and -1 prior to UUO. To investigate whether HO-1 could reverse established fibrosis, Hemin therapy was given at days 6 and 7 post-surgery. After 7 and/or 14 days, animals were sacrificed and blood, urine and kidney tissue samples were collected for analyses. Renal function was determined by assessing the serum creatinine, inulin clearance, proteinuria/creatininuria ratio and extent of albuminuria. Arterial blood pressure was measured and fibrosis was quantified by Picrosirius staining. Gene and protein expression of pro-inflammatory and pro-fibrotic molecules, as well as HO-1 were performed. Results: Pre-treatment with Hemin upregulated HO-1 expression and significantly reduced proteinuria, albuminuria, inflammation and pro-fibrotic protein and gene expressions in animals subjected to UUO. Interestingly, the delayed treatment with Hemin was also able to reduce renal dysfunction and to decrease the expression of pro-inflammatory molecules, all in association with significantly reduced levels of fibrosis-related molecules and collagen deposition. Finally, TGF-beta protein production was significantly lower in Hemin-treated animals. Conclusion: Treatment with Hemin was able both to prevent the progression of fibrosis and to reverse an established renal scar. Modulation of inflammation appears to be the major mechanism behind HO-1 cytoprotection.

Positive Selection Results in Frequent Reversible Amino Acid Replacements in the G Protein Gene of Human Respiratory Syncytial Virus

Human respiratory syncytial virus (HRSV) is the major cause of lower respiratory tract infections in children under 5 years of age and the elderly, causing annual disease outbreaks during the fall and winter. Multiple lineages of the HRSVA and HRSVB serotypes co-circulate within a single outbreak and display a strongly temporal pattern of genetic variation, with a replacement of dominant genotypes occurring during consecutive years. In the present study we utilized phylogenetic methods to detect and map sites subject to adaptive evolution in the G protein of HRSVA and HRSVB. A total of 29 and 23 amino acid sites were found to be putatively positively selected in HRSVA and HRSVB, respectively. Several of these sites defined genotypes and lineages within genotypes in both groups, and correlated well with epitopes previously described in group A. Remarkably, 18 of these positively selected tended to revert in time to a previous codon state, producing a ""flipflop'' phylogenetic pattern. Such frequent evolutionary reversals in HRSV are indicative of a combination of frequent positive selection, reflecting the changing immune status of the human population, and a limited repertoire of functionally viable amino acids at specific amino acid sites.

Three-dimensional visualization of human hemoglobin phenotypes with HPLC

Hemoglobinopathies were included in the Brazilian Neonatal Screening Program on June 6, 2001. Automated high-performance liquid chromatography (HPLC) was indicated as one of the diagnostic methods. The amount of information generated by these systems is immense, and the behavior of groups cannot always be observed in individual analyses. Three-dimensional (3-D) visualization techniques can be applied to extract this information, for extracting patterns, trends or relations from the results stored in databases. We applied the 3-D visualization tool to analyze patterns in the results of hemoglobinopathy based on neonatal diagnosis by HPLC. The laboratory results of 2520 newborn analyses carried out in 2001 and 2002 were used. The ""Fast"", ""F1"", ""F"" and ""A"" peaks, which were detected by the analytical system, were chosen as attributes for mapping. To establish a behavior pattern, the results were classified into groups according to hemoglobin phenotype: normal (N = 2169), variant (N = 73) and thalassemia (N = 279). 3-D visualization was made with the FastMap DB tool; there were two distribution patterns in the normal group, due to variation in the amplitude of the values obtained by HPLC for the F1 window. It allowed separation of the samples with normal Hb from those with alpha thalassemia, based on a significant difference (P < 0.05) between the mean values of the ""Fast"" and ""A"" peaks, demonstrating the need for better evaluation of chromatograms; this method could be used to help diagnose alpha thalassemia in newborns.

A Draft of the Human Septin Interactome

Background: Septins belong to the GTPase superclass of proteins and have been functionally implicated in cytokinesis and the maintenance of cellular morphology. They are found in all eukaryotes, except in plants. In mammals, 14 septins have been described that can be divided into four groups. It has been shown that mammalian septins can engage in homo- and heterooligomeric assemblies, in the form of filaments, which have as a basic unit a hetero-trimeric core. In addition, it has been speculated that the septin filaments may serve as scaffolds for the recruitment of additional proteins. Methodology/Principal Findings: Here, we performed yeast two-hybrid screens with human septins 1-10, which include representatives of all four septin groups. Among the interactors detected, we found predominantly other septins, confirming the tendency of septins to engage in the formation of homo- and heteropolymeric filaments. Conclusions/Significance: If we take as reference the reported arrangement of the septins 2, 6 and 7 within the heterofilament, (7-6-2-2-6-7), we note that the majority of the observed interactions respect the ""group rule"", i.e. members of the same group (e. g. 6, 8, 10 and 11) can replace each other in the specific position along the heterofilament. Septins of the SEPT6 group preferentially interacted with septins of the SEPT2 group (p<0.001), SEPT3 group (p<0.001) and SEPT7 group (p<0.001). SEPT2 type septins preferentially interacted with septins of the SEPT6 group (p<0.001) aside from being the only septin group which interacted with members of its own group. Finally, septins of the SEPT3 group interacted preferentially with septins of the SEPT7 group (p<0.001). Furthermore, we found non-septin interactors which can be functionally attributed to a variety of different cellular activities, including: ubiquitin/sumoylation cycles, microtubular transport and motor activities, cell division and the cell cycle, cell motility, protein phosphorylation/signaling, endocytosis, and apoptosis.

Identification of the Schistosoma mansoni TNF-Alpha Receptor Gene and the Effect of Human TNF-Alpha on the Parasite Gene Expression Profile

Background: Schistosoma mansoni is the major causative agent of schistosomiasis. The parasite takes advantage of host signals to complete its development in the human body. Tumor necrosis factor-alpha (TNF-alpha) is a human cytokine involved in skin inflammatory responses, and although its effect on the adult parasite's metabolism and egg-laying process has been previously described, a comprehensive assessment of the TNF-alpha pathway and its downstream molecular effects is lacking. Methodology/Principal Findings: In the present work we describe a possible TNF-alpha receptor (TNFR) homolog gene in S. mansoni (SmTNFR). SmTNFR encodes a complete receptor sequence composed of 599 amino acids, and contains four cysteine-rich domains as described for TNFR members. Real-time RT-PCR experiments revealed that SmTNFR highest expression level is in cercariae, 3.5 (+/- 0.7) times higher than in adult worms. Downstream members of the known human TNF-alpha pathway were identified by an in silico analysis, revealing a possible TNF-alpha signaling pathway in the parasite. In order to simulate parasite's exposure to human cytokine during penetration of the skin, schistosomula were exposed to human TNF-alpha just 3 h after cercariae-to-schistosomula in vitro transformation, and large-scale gene expression measurements were performed with microarrays. A total of 548 genes with significantly altered expression were detected, when compared to control parasites. In addition, treatment of adult worms with TNF-alpha caused a significantly altered expression of 1857 genes. Interestingly, the set of genes altered in adults is different from that of schistosomula, with 58 genes in common, representing 3% of altered genes in adults and 11% in 3 h-old early schistosomula. Conclusions/Significance: We describe the possible molecular elements and targets involved in human TNF-alpha effect on S. mansoni, highlighting the mechanism by which recently transformed schistosomula may sense and respond to this host mediator at the site of cercarial penetration into the skin.

Crystallization and preliminary X-ray diffraction analysis of human IL-22 bound to its soluble decoy receptor IL-22BP

Interleukin-22 (IL-22) is a pleiotropic cytokine that is involved in inflammatory responses. Human IL-22 was incubated with its soluble decoy receptor IL-22BP (IL-22 binding protein) and the IL-22 -IL-22BP complex was crystallized in hanging drops using the vapour-diffusion method. Suitable crystals were obtained from polyethylene glycol solutions and diffraction data were collected to 2.75 angstrom resolution. The crystal belonged to the tetragonal space group P41, with unit-cell parameters a = b = 67.9, c = 172.5 angstrom, and contained two IL-22-IL- 22BP complexes per asymmetric unit.