Positive Alkaline Phosphatase Research Articles

Influence and Optimization of Diverse Culture Systems on Chicken Embryonic Stem Cell Culture

Background: The importance of embryonic stem cells (ESCs) in chickens is undeniable, as they can be applied across various fields, including animal modeling, developmental biology, cell fate research, drug screening, toxicity testing, and gene function studies. However, a widely applicable culture system for chicken ESCs has yet to be developed. Objectives: This study aimed to investigate the effects of different culture systems on the derivation and maintenance of chicken ESCs, with a focus on optimizing the selected culture conditions. Methods: To achieve this, we tested the effectiveness of various species-specific ESC media in the derivation and culture of chicken PGCs, while incorporating different small molecule compounds to optimize the process. The pluripotency and differentiation potential of the resulting ESC-like cells were also evaluated. Results: The combination of PD0325901, SB431542, and LIF (R2i+LIF system) was found to be effective in generating chicken ESC-like clones. Further experiments showed that enhancing the R2i+LIF system with cytokines such as SCF and FGF2 significantly extended the culture period and increased the passage number of chicken ESC-like cells. These ESC-like cells were characterized through positive alkaline phosphatase staining and the expression of pluripotency markers POUV, NANOG, and SOX2. Additionally, differentiation assays confirmed their ability to form the three germ layers. Conclusions: The newly developed culture system provides suitable conditions for the short-term culture of chicken ESCs. However, further optimization is required to establish a system that can sustain long-term maintenance.

Bacterial Ribosomes Induce Plasticity in Mouse Adult Fibroblasts.

The incorporation of bacterial ribosome has been reported to induce multipotency in somatic and cancer cells which leads to the conversion of cell lineages. Queried on its universality, we observed that bacterial ribosome incorporation into trypsinized mouse adult fibroblast cells (MAF) led to the formation of ribosome-induced cell clusters (RICs) that showed strong positive alkaline phosphatase staining. Under in vitro differentiation conditions, RICs-MAF were differentiated into adipocytes, osteoblasts, and chondrocytes. In addition, RICs-MAF were able to differentiate into neural cells. Furthermore, RICs-MAF expressed early senescence markers without cell death. Strikingly, no noticeable expression of renowned stemness markers like Oct4, Nanog, Sox2, etc. was observed here. Later RNA-sequencing data revealed the expression of rare pluripotency-associated markers, i.e., Dnmt3l, Sox5, Tbx3 and Cdc73 in RICs-MAF and the enrichment of endogenous ribosomal status. These observations suggested that RICs-MAF might have experienced a non-canonical multipotent state during lineage conversion. In sum, we report a unique approach of an exo-ribosome-mediated plastic state of MAF that is amenable to multi-lineage conversion.

Biofabrication of an in-vitro bone model for Gaucher disease

Gaucher disease (GD), the most prevalent lysosomal disorder, is caused by GBA1 gene mutations, leading to deficiency of glucocerebrosidase, and accumulation of glycosphingolipids in cells of the mononuclear phagocyte system. While skeletal diseases are the leading cause of morbidity and reduced quality of life in GD, the pathophysiology of bone involvement is not yet fully understood, partly due to lack of relevant human model systems. In this work, we present the first 3D human model of GD using aspiration-assisted freeform bioprinting, which enables a platform tool with a potential for decoding the cellular basis of the developmental bone abnormalities in GD. In this regard, human bone marrow-derived mesenchymal stem cells (obtained commercially) and peripheral blood mononuclear cells derived from a cohort of GD patients, at different severities, were co-cultured to form spheroids and differentiated into osteoblast and osteoclast lineages, respectively. Co-differentiated spheroids were then 3D bioprinted into rectangular tissue patches as a bone tissue model for GD. The results revealed positive alkaline phosphatase (ALP) and tartrate-resistant ALP activities, with multi-nucleated cells demonstrating the efficacy of the model, corroborating with gene expression studies. There were no significant changes in differentiation to osteogenic cells but pronounced morphological deformities in spheroid formation, more evident in the ‘severe’ cohort, were observed. Overall, the presented GD model has the potential to be adapted to personalized medicine not only for understanding the GD pathophysiology but also for personalized drug screening and development.

Establishment and Characterization of a Spermatogonial Stem Cell Line from Tiger Puffer Fish (Takifugu rubripes).

Tiger puffer fish (Takifugu rubripes) has become the main fish species cultured in China since the last century because of its high economic value. Male and female tiger puffer fish need 2 and 3 years each to reach sexual maturity, which limits the development of breeding research for this species. In recent years, in vitro culture of fish spermatogonial stem cells (SSCs) have shown potential in aquaculture. In the present study, we established a spermatogenic stem cell line from T. rubripes (TrSSCs). TrSSCs were characterized by polygonal morphology, predominantly retained 44 chromosomes, and grew rapidly at 26 °C and in L-15. TrSSCs were still able to grow stably after more than one year of in vitro culture. TrSSCs showed positive alkaline phosphatase staining. TrSSCs expressed germ cell-associated genes, including dnd, ddx4, piwil, gfra1b, sox2, myca, nanog, ly75, and dazl, as determined by semiquantitative assays, and almost all cells were found to express the germ cell genes ddx4 and gfra1b in a fluorescence in situ hybridization assay. In vitro, induction experiments demonstrated the TrSSCs possessed the ability to differentiate into other types of cells. Our research has enriched the fish spermatogonial stem cell resource bank, which will provide an efficient research model for sex determination and sex control breeding in fish, establishing a foundation for subsequent breeding research.

Blastocyst Stage Affects the Isolation and Culture of Buffalo Naive/Primed Embryonic Stem-Like Cells.

Since embryonic stem cells (ESCs) were first identified, significant progress has been achieved. However, the establishment of buffalo ESCs (bESCs) is still unclear. This study was undertaken to explore the effect of the blastocyst stage on the isolation of bESCs. Firstly, our results indicated that the pluripotent genes were mainly expressed at the early stages of blastocyst, and the attachment and colony formation rates of bESCs derived from expanded blastocyst and hatched blastocyst were significantly higher than early blastocyst and blastocyst. In the meantime, bESCs showed positive alkaline phosphatase activity and expressed genes like OCT4, NANOG, SOX2, c-MYC, CDH1, KLF4, and TBX3. Immunofluorescence also confirmed the expression of OCT4, SOX2. Embryoid bodies expressing three marker genes were generated from the differentiation experiment, and fibroblast, epithelial, and neuron-like cells were induced. Moreover, naive-related genes KLF4, TBX3, primed-related genes FGF5, ACTA2 were expressed in the cells, but not REX-1. Immunofluorescence and western blot confirmed the FGF5 expression. Furthermore, bESCs could maintain pluripotency with the signal of LIF and bFGF. In summary, our results indicated that expanded blastocyst and hatched blastocyst are more suitable for bESCs isolation.

A multi-omics study to characterize the transdifferentiation of human dermal fibroblasts to osteoblast-like cells.

Background: Various skeletal disorders display defects in osteoblast development and function. An in vitro model can help to understand underlying disease mechanisms. Currently, access to appropriate starting material for in vitro osteoblastic studies is limited. Native osteoblasts and their progenitors, the bone marrow mesenchymal stem cells, (MSCs) are problematic to isolate from affected patients and challenging to expand in vitro. Human dermal fibroblasts in vitro are a promising substitute source of cells. Method: We developed an in vitro culturing technique to transdifferentiate fibroblasts into osteoblast-like cells. We obtained human fibroblasts from forearm skin biopsy and differentiated them into osteoblast-like cells with ß-glycerophosphate, ascorbic acid, and dexamethasone treatment. Osteoblastic phenotype was confirmed by staining for alkaline phosphatase (ALP), calcium and phosphate deposits (Alizarin Red, Von Kossa) and by a multi-omics approach (transcriptomic, proteomic, and phosphoproteomic analyses). Result: After 14days of treatment, both fibroblasts and MSCs (reference cells) stained positive for ALP together with a significant increase in bone specific ALP (p = 0.04 and 0.004, respectively) compared to untreated cells. At a later time point, both cell types deposited minerals, indicating mineralization. In addition, fibroblasts and MSCs showed elevated expression of several osteogenic genes (e.g. ALPL, RUNX2, BMPs and SMADs), and decreased expression of SOX9. Ingenuity Pathways Analysis of RNA sequencing data from fibroblasts and MSCs showed that the osteoarthritis pathway was activated in both cell types (p_adj. = 0.003 and 0.004, respectively). Discussion: These data indicate that our in vitro treatment induces osteoblast-like differentiation in fibroblasts and MSCs, producing an in vitro osteoblastic cell system. This culturing system provides an alternative tool for bone biology research and skeletal tissue engineering.

Identification of potential regulatory genes for embryonic stem cell self-renewal and pluripotency by random forest

To identify novel genes associated with self-renewal and pluripotency of mouse embryonic stem cells(mESCs)by integrating multiomics data based on machine learning methods. We integrated multiomics information of mESCs involving transcriptome, histone modifications, chromatin accessibility, transcription factor binding and architectural protein binding, and compared the signal differences between known stem cell self-renewal and pluripotency genes and other genes.By integrating these multiomics data, we established prediction models based on several machine learning classifiers including random forests and performed 5-fold cross validations.The model was trained using the training dataset containing two thirds of the input samples, and the remaining one third of the input samples were used as the test dataset to assess the performance of the model in independent tests.Finally, the results predicted by the model were validated through gene function annotation and cell function experiments including cell viability assay, colony formation assay and cell cycle analysis. Compared with the random genes, the genes known to be associated with self-renewal and pluripotency of mESCs in the multiomics data showed significantly different features.Random forest outperformed the other machine learning algorithms tested on these multiomics data, with an area under the curve (AUC) of 0.883±0.018 for cross validation and an AUC of 0.880±0.028 for independent test.Based on this model, we identified 893 potential regulatory genes associated wwith self-renewal and pluripotency of mESCs, which were similar to the known genes in functional annotation.Known-down of the predicted novel regulator gene Cct6a resulted in significant decreases in the cell viability of mESCs (P < 0.0001) and the number of cell clones (P < 0.01), significantly increased the number of cells in G1 phase (P < 0.01) and decreasedthe number of S phase cells (P < 0.05).Knockdown of Cct6a also led to failure of positive alkaline phosphatase staining of the mESCs. Machine learning model based on multiomics data can be used to predict potential self-renewal and pluripotency regulators with high performance.By using this model, we predicted potential self-renewal and pluripotency regulatory genes including Cct6a and applied experimental validation.This model provides new insights into the regulatory mechanism of mESCs and contribute to stem cell research.

In Vitro Induction of Pluripotency from Equine Fibroblasts in 20% or 5% Oxygen.

The cellular reprogramming into pluripotency is influenced by external and internal cellular factors, such as in vitro culture conditions (e.g., environmental oxygen concentration), and the aging process. Herein, we aimed to generate and maintain equine iPSCs (eiPSCs) derived from fibroblasts of a horse older than 20 years and to evaluate the effect of different levels of oxygen tension (atmospheric 20% O2, 5% O2, or 20% to 5% O2) on these cells. Fibroblasts were reprogrammed, and putative eiPSCs were positive for positive alkaline phosphatase detection; they were positive for pluripotency-related genes OCT4, REX1, and NANOG; immunofluorescence-positive staining was presented for OCT4 and NANOG (all groups), SOX2 (groups 5% O2 and 20% to 5% O2), and TRA-1-60, TRA-1-81, and SSEA-1 (only in 20% O2); they formed embryoid bodies; and there is spontaneous differentiation in mesoderm, endoderm, and ectoderm embryonic germ layers. In addition to the differences in immunofluorescence analysis results, the eiPSC colonies generated at 20% O2 presented a more compact morphology with a well-defined border than cells cultured in 5% O2 and 20% to 5% O2. Significant differences were also observed in the expression of genes related to glucose metabolism, mitochondrial fission, and hypoxia (GAPDH, GLUT3, MFN1, HIF1α, and HIF2α), after reprogramming. Our results show that the derivation of eiPSCs was not impaired by aging. Additionally, this study is the first to compare high and low oxygen cultures of eiPSCs, showing the generation of pluripotent cells with different profiles. Under the tested conditions, the lower oxygen tension did not favor the pluripotency of eiPSCs. This study shows that the impact of oxygen atmosphere has to be considered when culturing eiPSCs, as this condition influences the pluripotency characteristics.

Histological primary biliary cholangitis changes in patients with positive serology and normal alkaline phosphatase

Histological primary biliary cholangitis changes in patients with positive serology and normal alkaline phosphatase

Generation of sepsis encephalopathy patient-specific inducible pluripotent stem cells with urine cells

To recombine the induced pluripotent stem cells (iPSC) derived from the urine of septic encephalopathy (SE) patients, and provided a specificity cell model to explore the mechanism of the neuronal damage and treatment for SE patients. Urine of SE patient was collected, and tubular epithelial cells were isolated and cultured from the urine. iPSC were derived from SE patient by introducing 4 transcription factors OCT4, Klf4, Sox2, c-Myc (OKSM) into patient-specific urine cells by Millipore's Human STEMCCATM Constitutive Polycistronic (OKSM) Lentivirus Kit. Colony morphology, alkaline phosphatase (AKP) activity, immunofluorescence staining, quantitative reverse transcription-polymerase chain reaction (RT-qPCR), and differentiation ability were used to identify the pluripetency of these iPSC lines. In addition, neurons were derived from these iPSC by inhibiting transforming growth factor-β (TGF-β) pathway. The SE-iPSC exhibited morphological and growth characteristics of human embryonic stem cell (hES), showed positivity for AKP by histochemical staining, and expressed embryonic stem cell (ESC) marker genes. There was a significant statistical difference in ESC-marker mRNA expression between the SE-iPSC and the urine cells [NANOG mRNA (2-ΔΔCt): 1.153±0.142 vs. 0.126±0.024, t = -10.688; REX1 mRNA (2-ΔΔCt): 1.419±0.206 vs. 0.103±0.066, t = -14.245; OCT4 mRNA (2-ΔΔCt): 1.233±0.176 vs. 0.201±0.022, t = -9.028; Sox2 mRNA (2-ΔΔCt): 1.334±0.119 vs. 0.159±0.017, t = -12.653, all P < 0.01]. Subcutaneous injection of iPSC into NOD-SCID mice resulted in teratomas containing tissues from all the 3 germ layers. Furthermore, neurons were successfully induced from SE-iPSC. The SE patient-specific iPSC could be generated from urine cells and differentiated into neurons, furthermore, the SE-iPSC cell line can be used as models for further elucidating the cellular pathology and developing therapeutic strategies for SE.

The Subacromial Bursa is a Viable Source of Autologous Mesenchymal Stem Cells for Rotator Cuff Repair

Objectives: Chronic rotator cuff tears still represent a significant source of morbidity and functional decline in the general population. The purpose of this study was to establish protocols for isolation and expansion of bursa-derived mesenchymal stem cells (BDSCs) and to evaluate their differentiation capacity, including tenogenesis. We hypothesized that BDSCs would be capable of multilineage differentiation (including tenogenesis) and represent an important source for autologous stem cells for patients undergoing rotator cuff repair. Methods: After IRB approval, 10 patients (ages 43-65 years) scheduled to undergo arthroscopic repair for chronic rotator cuff tears were enrolled. During diagnostic arthroscopy, subacromial bursa tissue was harvested using an arthroscopic shaver and collected by attaching the outflow tubing to a specialized specimen cup. Tissue specimens were transported to our laboratory for analysis. BDSCs were isolated via adherent culture and plated in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS). Chondrogenic, adipogenic, and osteogenic induction media were used to induce differentiation. Tenogenic induction was performed using DMEM supplemented with varying concentrations of BMP-12, ascorbic acid, and human tenocyte-conditioned media. Alcian Blue staining was used to evaluate chondrogenesis, Oil Red O staining for adipogenesis, and Alkaline Phosphatase staining for osteogenesis. Gene expression markers for adipogenesis (ADIPOQ, FABP4, PPARγ), chondrogenesis (COL2A1 and SOX5), and osteogenesis (osteocalcin, osterix), along with primary antibodies to tenogenic markers (scleraxis, tenomodulin), were used to verify each cell lineage. Results: BDSCs isolated by adherent culture without collagen exhibited a spindle-shaped morphology characteristic of mesenchymal stem cells (MSCs), formed colonies, and demonstrated great expandability for six to eight passages without morphology changes (Figure 1A). After 3 weeks of culture, 95% (p&lt;0.0001) of the BDSCs expressed the MSC surface marker CD90 and were negative for non-MSC cell markers (CD45, CD146, CD31, and CD34) (Figure 1B and C). These BDSCs demonstrated a capacity for adipogenesis (positive Oil Red O staining, ADIPOQ, FABP4, PPARγ expression), osteogenesis (positive alkaline phosphatase staining, positive osteocalcin and scleraxis expression), chondrogenesis (positive Alcian Blue staining, positive COL2A1 and SOX5 expression), and tenogenesis (scleraxis and tenomodulin expression) (Figure 2). The results indicate that BDSCs are multipotent as evidenced by their differentiation into fat, bone, cartilage, and tendon cells. For tenogenesis, we found that 7-day incubation in DMEM supplemented with 100 ng/mL of BMP-12 and 50 μg/mL of ascorbic acid produced superior tenogenic induction. These BDSCs adapted an elongated morphology combined with the expression of both scleraxis and tenomodulin - a unique characteristic of native tenocytes. Conclusion: Our results demonstrate that subacromial bursa represents a viable source of mesenchymal stem cells. We developed a reliable protocol for isolation of BDSCs from patient bursa samples. We show that BDSCs in the presence of BMP-12 and ascorbic acid can differentiate toward a tenogenic lineage. Our work provides strong evidence that BDSCs may be a potent tool for cellular therapy and may benefit future patients who undergo surgical repair of chronic rotator cuff tears. [Figure: see text][Figure: see text]

The modification of mitochondrial energy metabolism and histone of goat somatic cells under small molecules compounds induction.

In recent years, induced pluripotent stem cells (iPSCs) technique is able to allow us to generate pluripotency from somatic cells in vitro through the over expression of several transcription factors. Normally, viral vectors and transcription factors are commonly used on iPSC technique, which could cause many barriers on further application. In this study, we attempt to process a new method to obtain pluripotency from goat somatic cells in vitro under fully chemically defined condition. The results showed that chemically induced pluripotent stem cells-like cells (CiPSC-like cells) colonies were generated from goat ear fibroblasts by fully small-molecule compounds. Those three dimensions colonies were similar with mouse iPSCs in morphology and had strong positive alkaline phosphatase (AP) activity and expressed pluripotency related genes OCT4, SOX2, NANOG, CDH1, TDGF, GDF3, DAX1, REX1, which determined by RT-PCR. Those colonies could also differentiate into different cell types derived from three germ layers proved by RT-PCR and immunofluorescence assays. The expression of glycolysis-related genes about PGAM1, KPYM2 and HXK2 in CiPSC-like colonies formation groups was significantly higher than their parental fibroblasts, but not in the non-CiPSC-like colonies formation group. The expression of histone acetylation and methylation-related genes, HAT1 and SMYD3, was not significantly up-regulated within different groups compared to their parental fibroblasts, respectively. Yet, the expression of histone methylation-related gene, KDM5B, was significantly up-regulated on the cells from non-colonies formation group compared to parental fibroblasts, but the expression of KDM5B of the cells from CiPSC-like cell colonies was not significantly difference compared to that of parental fibroblasts. In conclusion, this is the first report that CiPSC-like cells could be generated in vitro from goat rather than just mouse under fully chemically defined condition. The generation of CiPSC-like colonies may be depended on the correct modification of energy metabolism and histone epigenetic during the reprogramming, rather than just the over-expression of those pluripotency-related genes. This study will strongly support us to further establish the stable goat CiPSC lines without any integration of exogenous genes.

Development of the Rabbit (Oryctolagus cuniculus) Metanephros

Thirty-seven rabbit embryos at em- bryonic days (E) 10-30 were used to study the morphogenesis of the metanephros by light microscope. The primordium of the metanephros appears at E 12 within the pelvic cavity as a spherical ureteric bud capped by metanephrogenic tissue. The nephrogenic spherules and the renal vesicles develop at E 15 while formation of the renal tubules and differentiation of the glomerular capsule take place at E 16. At E 18, differentiation of the glomerular ca- pillaries occurs and the proximal and distal convoluted tubules are clearly first time demarcated. Positive alkaline phosphatase activity and PAS-positive material are confined to the metanephric corpuscles and the luminal border of the proxi- mal convoluted tubules. In addition, the collecting tubules show large masses of intracytoplasmic PAS- positive granules. The rabbit metanephros is smooth unipapillary with indistinct renal columns and pyramids. It undergoes migration until reaches its permanent position at E 30 where the right metanephros is located opposite to the level of the 2nd – 4th lumbar vertebrae and the left one is situated opposite to the level of the 3rd – 5th lumbar verte- brae. This migration is accompanied by rotation for about 135 degrees resulting in a dorsomedially located hilus. In conclusion, the stages of development of the rabbit metanephros are similar to those in other mammals but they maintain a de- layed sequence.

Induction of reprogramming of human amniotic epithelial cells into iPS cells by overexpression of Yap, Oct4, and Sox2 through the activation of the Hippo-Yap pathway

The present study has reported a novel method for producing induced pluripotent stem (iPS) cells. Primary human amniotic epithelial cells (HuAECs) were isolated from the amniotic membranes of pregnant women who received Cesarean sections. These cells were infected with retroviruses carrying octamer-binding transcription factor 4 (Oct4), (sex determining region Y)-box 2 (Sox2) and Yes-associated protein (Yap) (OSY). Following in vitro culture for ~14 days, epithelial-like HuAECs exhibited several iPS clone-like cell colonies (OSY-iPS). These cell clones presented positive alkaline phosphatase features and expressed high levels of embryonic stem cell-like markers (Nanog homeobox, Sox2, Oct4, reduced expression protein 1, and SSES3/4). Additionally, epigenetic analysis results indicated that the methylation of CpG islands on endogenous Oct4 and Sox2 promoters was reduced in OSY-iPS cells. Furthermore, the majority of the histone H3 at lysine 9 sites that interacted with the Oct4 and Sox2 promoters were acetylated, suggesting that the transcription activities of the above two transcription factors significantly increased. In vivo and in vitro induced differentiation experiments demonstrated that OSY-iPS could develop into embryoid bodies in vitro, and express numerous cellular markers in the three germ layers. Furthermore, OSY-iPS could form teratomas in immunodeficient mice. The pathological detection results suggest that these teratomas contain numerous types of cells from the three germ layers. However, the results from the quantitative polymerase chain reaction and western blot analyses suggest that the Hippo-Yap signaling pathway was significantly activated in OSY-iPS cells. In conclusion, a novel method for iPS induction was established in the present study. HuAECs were successfully induced to reprogram iPS cells through the introduction of OSY to activate the Hippo-Yap signaling pathway.

Activation-Induced Cytidine Deaminase Induces DNA Demethylation of Pluripotency Genes in Bovine Differentiated Cells.

Activation-induced cytidine deaminase (AID) is the only enzyme that has been suggested as a putative DNA demethylase in mammals. However, very little is known about AID function as DNA demethylase of bovine differentiated cells toward pluripotent state. To investigate the effect of AID on DNA demethylation, bovine AID complementary DNAs were transfected into bovine differentiated cells, which were mostly methylated in the promoter regions of pluripotency genes. As a result, AID-transfected bovine cells started to transform into colonies at day 19 of transfection. The colonies derived from the transfected cells showed positive alkaline phosphatase (AP) staining and expression of pluripotency genes (OCT-3/4, NANOG, SOX2) and pluripotency-related antigens (SSEA-4, TRA1-60, TRA1-81), which have been widely used to characterize human embryonic stem cells. In particular, the levels of OCT-3/4 and NANOG expression were significantly increased in the AID-transfected cells when compared with the control and empty vector-transfected cells (p < 0.05). Finally, DNA demethylation in the promoter regions of pluripotency genes (OCT-3/4, NANOG) was significantly increased compared with the control (p < 0.05). These results demonstrate that the induction of the AID gene into bovine differentiated cells improves DNA demethylation and expression of pluripotency genes.

Proposal of Helicobacter canicola sp. nov., previously identified as Helicobacter cinaedi, isolated from canines

During the course of our taxonomic investigation of Helicobacter cinaedi, it was realized that the strains isolated from dogs, which have been identified as H. cinaedi, showed different biochemical traits than did the isolates obtained from humans. None of the three dog isolates could reduce nitrate to nitrite, whereas all of the human H. cinaedi isolates could do so. The dog isolates showed a strong positive alkaline phosphatase reaction and could grow at 42°C, however the human isolates showed negative to very weak responses to those tests. The GyrA protein based phylogenetic analysis showed that the three isolates from dogs formed a slightly distinct cluster from the human isolate cluster. Phylogenetic analysis of the 16S rRNA, 23S rRNA, gyrB, and hsp60 gene sequences further confirmed that the dog isolates differed from the human H. cinaedi isolate cluster. The whole-genome in silico DNA similarities of each isolate based on their full genome sequences revealed that the isolates from dogs shared more than 94.9% ANIb (average nucleotide identity based on BLAST), while 94.0% ANIb were found between the isolates from dogs and the humans, including the H. cinaedi type strain ATCC BAA-847T (=CCUG 18818T). From these data, we propose a new species, ‘H. canicola’ sp. nov., for the isolates from dogs. The type strain is PAGU 1410T (CCUG 33887T=LMG 29580T).

Modified STAP conditions facilitate bivalent fate decision between pluripotency and apoptosis in Jurkat T-lymphocytes

Low extracellular pH (pHe) is not only the result of cancer metabolism, but a factor of anti-cancer drug efficacy and cancer immunity. In this study, the consequences of acidic stress were evaluated by applying STAP protocol on Jurkat T-lymphocytes (2.0 × 106 cells/ml, 25 min in 37 °C). We detected apoptotic process exclusively in pH 3.3 treated cells within 8 h with western blotting (WB). This programmed cell death led to significant drop of cell viability in 72 h measured by MTT assay resulting PI positive population on flow cytometry (FCM) at day 7. Quantified RT-PCR (qRT-PCR) data indicated that all of above mentioned responses are irrelevant to expression of OCT4 gene variants. Interestingly enough, pluripotent cells represented by positive alkaline phosphatase (AP) staining survived acidic stress and consequently proportion of AP positive cells was significantly increased after pH 3.3 treatment (day 7). In general, acidic treatment led to an apoptotic condition for Jurkat T-lymphocytes, which occurred independent of OCT4 induction.

Distribution of Phosphatases, Oxidoreductases and Esterases in Sublingual Salivary Gland of Buffalo During Neonatal Development

The present study was aimed to elucidate the distribution of various enzymes in sublingual salivary gland of neonatal buffalo (n = 10), ranging from 1 to 3 months age, and correlate these enzyme systems with the secretory activity of gland. At 1 month age, a uniform moderate to strong positive diffused granular alkaline phosphatase activity was observed in serous acini and ductal epithelium. A moderate positive glucose-6-phosphatase and succinic dehydrogenase activity was seen close to the basement membrane. At 2 months age, glutamic dehydrogenase showed moderate activity in basal portions of serous acinar cells, while succinic dehydrogenase activity was strong, granular and evenly distributed in basement membrane of serous cells. Weak activity of monoamine oxidase and moderate non-specific esterase activity was seen. At 3 months age, alkaline phosphatase showed strong positive perinuclear activity in acinar cells, while glucose-6-phosphatase showed a moderate to strong activity in acinar cells and in ductal epithelium. Moderate to strong positive non-specific esterase activity was localized in nerve fibres of intralobular and interlobular connective tissues. Glucose-6-phosphate dehydrogenase and nicotinamide adenine dinucleotide diaphorase showed strong positive granular activity in basement membrane of serous acinar cells and ducts. The high basal activities of oxidoreductases were because of basal mitochondria and were associated with high energy production to meet the energy requirement for active and rapid transport of ions and water.

Characteristics of Human Amniotic Epithelial Cells and Bone Marrow Mesenchymal Stem Cells in a Parallel Study: Hanging Drop Colony Formation and Doubling Time

Characteristics of Human Amniotic Epithelial Cells and Bone Marrow Mesenchymal Stem Cells in a Parallel Study: Hanging Drop Colony Formation and Doubling Time Amniotic epithelial cells exhibit pluripotent capacity with some unique properties. They are considered as a good candidate in regenerative medicine. Human bone marrow mesenchymal stromal cells (hBM-MSC) are the gold standard in cell therapy procedures, but whether these cells could be replaced with hAEC remains a debate? We compared the stem cell characteristics of hAEC with hBM-MSC considering isolation, morphology, population doubling time, colony formation on substratum and in suspension, alkaline phosphatase activity and expression of some CD Markers. hAEC were round and cubical but hBM-MSC appeared spindle-shaped in culture. Both groups of cells expressed mesenchymal stem cells markers (CD44, CD90) and they did not express CD34. OCT4 was highly expressed in hAEC but not in hBM-MSC. Their proliferation rate was slower than hBM-MSC. Either of the cell groups could form colonies on substratum as well as suspension with a positive alkaline phosphatase activity. Our results showed that biological properties of human amniotic epithelial cells are to some extent similar to human bone marrow mesenchymal cells.

Phosphatase Activity of Microbial Populations in Different Milk Samples in Relation to Protein and Carbohydrate Content

Cattle milk is a rich source of protein, carbohydrate, vitamins, minerals and all other major and micro nutrients. At a moderate pH, milk is an excellent media for the growth of microbes and thus, intake of raw milk is precarious. In this study, attempt was made for a qualitative study of eight raw milk samples of different varieties of cow and goat milk, collected from Jorhat district of Assam, India, on the basis of nutritional value and microbial population. The highest microbial population was found in the milk collected from cross hybrid variety of cow, whereas microbial contamination was the least in Jersey cow milk. Samples of C1 (Jersey cow) variety showed presence of the highest amount of protein and carbohydrate content as compared to the others. Almost all the milk samples showed positive acid and alkaline phosphatase activity. Maximum acid phosphatase activity was observed in cross hybrid cow milk, whereas local cow milk exhibited the highest alkaline phosphatase activity. Phosphatase activity did not show any co-relationship with microbial population of the milk samples. Similarly, the protein and carbohydrate content of the samples did not have any significant impact on both acid and alkaline phosphatase activity.