Fresh Isolation Research Articles

Quantification of Microglial Engulfment of Synaptic Material Using Flow Cytometry.

Microglia play a pivotal role in synaptic refinement in the brain. Analysis of microglial engulfment of synapses is essential for comprehending this process; however, currently available methods for identifying microglial engulfment of synapses, such as immunohistochemistry (IHC) and imaging, are laborious and time-intensive. To address this challenge, herein we present in vitro and in vivo* assays that allow fast and high-throughput quantification of microglial engulfment of synapses using flow cytometry. In the in vivo* approach, we performed intracellular vGLUT1 staining following fresh cell isolation from adult mouse brains to quantify engulfment of vGLUT1+ synapses by microglia. In the in vitro synaptosome engulfment assay, we used freshly isolated cells from the adult mouse brain to quantify the engulfment of pHrodoRed-labeled synaptosomes by microglia. These protocols together provide a time-efficient approach to quantifying microglial engulfment of synapses and represent promising alternatives to labor-intensive image analysis-based methods. By streamlining the analysis, these assays can contribute to a better understanding of the role of microglia in synaptic refinement in different disease models.

The Effect of Short- and Long-Term Cryopreservation on Chicken Primordial Germ Cells

Primordial germ cells (PGCs) are the precursors of functional gametes and the only cell type capable of transmitting genetic and epigenetic information from generation to generation. These cells offer valuable starting material for cell-based genetic engineering and genetic preservation, as well as epigenetic studies. While chicken PGCs have demonstrated resilience in maintaining their germness characteristics during both culturing and cryopreservation, their handling remains a complex challenge requiring further refinement. Herein, the study aimed to compare the effects of different conditions (freezing-thawing and in vitro cultivation) on the expression of PGC-specific marker genes. Embryonic blood containing circulating PGCs was isolated from purebred Green-legged Partridgelike chicken embryos at 14–16 Hamburger–Hamilton (HH) embryonic development stage. The blood was pooled separately for males and females following sex determination. The conditions applied to the blood containing PGCs were as follows: (1) fresh isolation; (2) cryopreservation for a short term (2 days); and (3) in vitro culture (3 months) with long-term cryopreservation of purified PGCs (~2 years). To characterize PGCs, RNA isolation was carried out, followed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) to assess the expression levels of specific germ cell markers (SSEA1, CVH, and DAZL), as well as pluripotency markers (OCT4 and NANOG). The investigated genes exhibited consistent expression among PGCs maintained under diverse conditions, with no discernible differences observed between males and females. Notably, the analyzed markers demonstrated higher expression levels in PGCs when subjected to freezing than in their freshly isolated counterparts.

Bicarbonate signalling via G protein-coupled receptor regulates ischaemia-reperfusion injury

Homoeostatic regulation of the acid–base balance is essential for cellular functional integrity. However, little is known about the molecular mechanism through which the acid–base balance regulates cellular responses. Here, we report that bicarbonate ions activate a G protein-coupled receptor (GPCR), i.e., GPR30, which leads to Gq-coupled calcium responses. Gpr30-Venus knock-in mice reveal predominant expression of GPR30 in brain mural cells. Primary culture and fresh isolation of brain mural cells demonstrate bicarbonate-induced, GPR30-dependent calcium responses. GPR30-deficient male mice are protected against ischemia-reperfusion injury by a rapid blood flow recovery. Collectively, we identify a bicarbonate-sensing GPCR in brain mural cells that regulates blood flow and ischemia–reperfusion injury. Our results provide a perspective on the modulation of GPR30 signalling in the development of innovative therapies for ischaemic stroke. Moreover, our findings provide perspectives on acid/base sensing GPCRs, concomitantly modulating cellular responses depending on fluctuating ion concentrations under the acid–base homoeostasis.

Establishment and functional characterization of immortalized rabbit dermal papilla cell lines

Hair follicle (HF) undergo periodic growth and development in mammals, which regulated by dermal papilla cells (DPCs) are reported to play an important role in HF morphogenesis and development. However, primary DPCs have low proliferative activity, age quickly, and fresh cell isolation is both time-consuming and laborious. In this study, we introduced the SV40 large T antigen (SV40T) into dissociated early passage rabbit vibrissae DPCs with lentiviral vectors and established seven immortalized DPC lines (R-1, R-2, R-3, R-4, R-5, R-6 and R-7). These cell lines displayed early passage morphology and high alkaline phosphatase activity. RT-PCR and immunofluorescence staining showed that all the immortalized cell lines expressed the DPC markers (α-SMA, IGF1, ALPL, FGF2, BMP2 and TGFβ2), but α-SMA was only expressed well in R-3, R-4, and R-7. Furthermore, it was found that R-7 was the only line to survive beyond 50 passages. Compared to melanoma cells, R-7 did not undergo malignant transformation. Karyotyping and cell growth viability analysis illustrated that the R-7 cell line preserved the basic characteristics of primary DPCs. The R-7 DPCs established have potential application for future hair research. The study provides the theoretical basis in the cell research of HF growth and development.

Refined premature chromosome condensation (G0-PCC) with cryo-preserved mitotic cells for rapid radiation biodosimetry

Mitotic cell fusion induced Premature Chromosome Condensation (G0-PCC) assay in human lymphocytes allows rapid detection of cytogenetic damage in interphase stage, within few hours after blood collection. Hence, it is the most suitable method for rapid and high dose biodosimetry. Mitotic cells, used for G0-PCC could be either freshly isolated or previously cryo-preserved. However, under emergency scenarios, only cryo-preserved cells can be relied upon, fresh isolation will only delay the process by 18–24 h. Impact of cryopreservation on mitotic cells and their efficacy to induce PCC are not reported. In the present study, we investigated effect of cryopreservation on mitotic cells and refined the parameters for G0-PCC. More than 95% of the cells were recoverable after 4 months of cryopreservation, within 20 min recovery at 37 °C, without significant change in the mitotic index or viability. Recovered mitotic cells have shown mitotic index of 89 ± 4% and viability of 90 ± 4%, similar to that of freshly isolated cells. Decrease in metaphases was observed within 40 min after recovery as the mitotic cells progressed through cell cycle and reduced to 21% at 1 h. Nevertheless, in presence of Colcemid, the cells progressed slowly and considerably high metaphase index (60%) persisted up to ~ 2 h. The recovered cells efficiently fused with lymphocytes and induced PCC. Average PCC index varied from 10 to 20%, which did not change with cryopreservation duration. Post fusion incubation duration of 2 h was found to be optimum for proper chromosome condensation. In conclusion, use of cryo-preserved mitotic cells is the most practical approach for rapid biodosimetry. The cells can be recovered quickly and efficiently without alteration in viability or mitotic index. Recovered cells are fully competent to induce G0-PCC.

A novel approach to study markers of dopamine signaling in peripheral immune cells

Human monocytes express known markers of dopamine synthesis, storage and clearance, including dopamine transporter (DAT), tyrosine hydroxylase (TH), all subtypes of dopamine receptors and vesicular monoamine transporter 2 (VMAT2). Immunohistochemical and immunofluorescent methodologies have traditionally been employed to determine DAT and TH expression in the CNS, their detection in the blood and specifically in the peripheral monocytes has not been studied by flow cytometry. Flow cytometry assays are widely used in medicine and in basic, preclinical or clinical research to quantify physical and chemical characteristics of target cell populations. Here, we have established a highly sensitive and reproducible flow cytometry panel to detect and quantify DAT and TH expression in freshly isolated or cryopreserved human peripheral monocytes. In healthy humans (n = 41 biological replicates), we show baseline DAT and TH expressing monocytes constitute ~12% of the peripheral blood mononuclear cell (PBMC) fraction when examined in fresh isolation from whole blood. Using an identical flow cytometry panel, we found that cryopreservation of PBMCs using multiple techniques resulted in altered PBMC populations as compared to fresh isolation and relative to one another. Among these, we identified an optimum cryopreservation method for detecting TH and DAT in cryopreserved PBMCs. Our data provide a sensitive and reproducible approach to examine dopamine signaling in peripheral human immune cells. This approach can be applied to study peripheral dopamine signaling under healthy and potentially under disease conditions. The use of dopamine signaling could also be explored as a technique to monitor therapeutic interventions particularly those targeting DAT and TH in the periphery.

ENVIRONMENTAL ASPECTS OF LARGE-TONNAGE PRODUCTION LIQUID WASTE STORAGE INTO THE UNDERGROUND STORAGE

The article presents the schemes of two fundamentally different ways of storing liquid waste. The technologies of large-tonnage production liquid waste storage are analyzed. The advantages and disadvantages of storage methods for liquid waste are identified. The limited suitable reservoirs number during deep storage in mandatory requirements terms for the absence of fresh water and isolation from them, the presence of regional waterproof tires (screens), which are low-permeable or permafrost, the presence of the necessary reservoir and the mining allotment boundaries, because when filling the reservoir, it is necessary to observe the restriction of the underground pollution distribution halo within the mining allotment boundaries. The actual sizes of underground pollution halos in reservoirs depend on the injected liquid waste volume and on the their distribution time. Liquid waste migration continues after the end of their injection into the well. It is possible that the halo of the liquid waste storage after conservation and liquidation of the injection well will exceed the boundaries of mining with drawal of the subsoil, and hazardous components of liquid waste will fall into aquifers of the upper hydrodynamic zone suitable for economic use. In order to eliminate the liquid waste possible migration beyond the boundaries of the subsoil mining allotment during the liquidation of an injection well, it is necessary to take measures to localize the contaminated area. Based on the analysis of the liquid waste storage applied methods, it has been established that deep storage in reservoirs is the most promising both in terms of ecosystem safety and the possibility of storing liquid waste of all hazard classes. Special attention after the conservation and liquidation of the injection well should be paid to the storage halo limitation. To exclude the liquid waste components migration into suitable for economic use aquifers of the upper hydrodynamic zone, a strategy is proposed for localizing the contaminated area by creating insulating screens.

Comparison between three different protocols for isolation and culture of mouse bone marrow derived mesenchymal stem cells

Bone marrow derived mesenchymal stem cells (BM-MSCs) represent a promising source for cell therapy, As they can differentiate into bone, cartilage, fat, tendon and many other organ progenitor cells. Although the culture of MSCs has been studied for over 30 years. The identification of standard protocol for isolation and characterization have yet to be developed. A comparison study was made on three different isolation techniques, which include: direct plating, red blood lysis buffer strategies, and density gradient (DG) method (using two different gradient media: Ficoll and Percoll) to find the optimal isolation and culture condition for adequate amount of MSCs for clinical use.The results demonstrate that direct plating of whole bone marrow (BM) suspension provides a suitable alternative protocol for isolation of BM-MSCs with minimal requirements, which mainly based on the frequent medium change in primary culture. The BM crud cell suspension were isolated by aspiration from albino mice and cultured in fresh complete isolation media (CIM) RPMI-1640/10%FBS. Adherent cells from BM cells suspension were MSCs which then expanded by complete expansion media (CEM) α-MEM/ 10% FBS. By concluding, direct plating of whole bone marrow represent the alternative method for isolating of mesenchymal stem cells from small specimen of bone marrow. By concluding, the present study was designed to investigate the optimal technique for the isolation of BM-MSCs for use in tissue engineering and regenerative medicine.

Confirmation of Tuntun Angin (Elaeocarpus floribundus) Taxonomic Status Using matK and ITS Sequences

<p>Tuntun angin is one of important floodplain plants in and around Kajuik Lake located in Riau Province, Indonesia. Morphological identification shows that the scientific name of this plant is <em>Elaeocarpus floribundus. </em>The study aimed to confirm the taxonomic status of tuntun angin using <em>matK</em> and nuclear intergenic spacer (ITS) sequences. The methods included fresh leaf DNA isolation, polymerase chain reaction, electrophoresis, sequencing, and data analysis using BLASTn program and MEGA software version 6.06 programs. The results showed that the <em>matK</em> sequence (519 bp) of tuntun angin had highest similarity to <em>E. floribundus</em> <em>matK</em> sequence that was available in GenBank. It was supported by the high max score (937), low E-value (0.0), high identity value (100%), and high query cover (100%). However, the ITS sequence of tuntun angin did not show similarity to <em>E. floribundus </em>ITS sequence because there was no database of the sequence in GenBank. This study was able to confirm the taxonomic status of tuntun angin as <em>E. floribundus</em> using <em>matK</em> sequence and also showed that morphological and molecular identification techniques were complementary to each other. Moreover, this study enriched the DNA sequence database of <em>E. floribundus </em>in GenBank which will be useful for this species’ molecular identification.</p><p><strong>How to Cite</strong></p><p>Roslim, D. I., Khumairoh, S. & Herman, H. (2016). Confirmation of Tuntun Angin (<em>Elaeocarpus floribundus</em>) Taxonomic Status Using <em>matK</em> and ITS Sequences. <em>Biosaintifika: Journal of Biology & Biology Education</em>, 8(3), 393-400. </p>

Preservation and enumeration of endothelial progenitor and endothelial cells from peripheral blood for clinical trials.

Endothelial progenitor cells (EPCs) are markers of vascular repair. Increased numbers of circulating endothelial cells (ECs) are associated with endothelial damage. We enumerated EPC-EC by using Enrichment kit with addition of anti-human CD146-PE/Cy7 from peripheral blood mononuclear cell (PBMC) isolated either by red blood cell (RBC) lysing solution or by Ficoll centrifugation, and from fresh and preserved samples. PBMCs were quantified by flow cytometry. RBC lysis yielded higher percentage of PBMC (p = 0.0242) and higher numbers of PBMC/ml (p = 0.0039) than Ficoll. Absolute numbers of CD34(+)CD133(+)VEGFR2(+) and CD146(+)CD34(+)VEGFR2(+) were higher (p = 0.0117 for both), when isolated by RBC lysis than by Ficoll, when no difference in other subsets was found. Cryopreservation at -160°C and -80°C and short-term preservation at room temperature decreased EPC-EC. Our data support use of fresh samples and isolation of PBMC from human blood by RBC lysis for enumeration of EPC and EC.

Time Course of Mitophagy‐Related Signals in Skeletal Muscle Following a Single Bout of Aerobic Exercise in Mice

This study aimed to investigate mitophagy‐related signals in skeletal muscle following a single bout of aerobic exercise in mice. C57BL6 mice were submitted to a single bout of treadmill aerobic exercise for 80 minutes at 60% of maximal speed achieved in a graded treadmill test. Skeletal muscles were harvested at rest, immediately after the bout of aerobic exercise and after 3, 6, 12 and 24h of recovery. One hindlimb was used for fresh isolation of skeletal muscle mitochondrial‐enriched fraction (MF), and contralateral muscles were immediately frozen. Immunoblotting for LC3, p62 and Bnip3 were performed either in quadriceps muscles or MF. qPCR were performed to evaluate mRNA levels of LC3, p62 and Bnip3 in quadriceps muscles. No protein was differentially expressed in total lysate of quadriceps muscle. However, LC3‐II levels were significantly increased in MF immediately after the bout of aerobic exercise (72.5%) and after 12h of recovery (63.3%), whereas p62 protein levels were increased at 6 (30%) and 12h (62.5%) of recovery. Bnip3 protein levels displayed a trend toward to increase (p=0.06) after 3h of recovery (36.4%). No difference was found in mRNA levels of LC3, p62 and Bnip3 at any time point. Collectively, these results suggest that mitophagy signaling is increased during aerobic exercise and decreases after its cessation. Interestingly, the retake of mitophagy signaling after 12h of recovery suggests that mitophagy plays an important role in skeletal muscle mitochondrial remodeling to acute aerobic exercise stimulus, independently of transcriptional regulation. Thus, these results provide new insights into aerobic exercise‐induced mitophagy and mitochondrial remodeling.Financial support: FAPESP 2013/21065‐3

Brucellosis seroprevalence in Bali cattle with reproductive failure in South Sulawesi and Brucella abortus biovar 1 genotypes in the Eastern Indonesian archipelago

BackgroundBrucellosis is a major cause of infertility and reproductive failure in livestock. While cattle in the Eastern Indonesian archipelago suffers from reproductive problems information on bovine brucellosis in the region is fragmentary. The control of brucellosis requires a major and prolonged effort and confirmation of the infection by isolation with detailed knowledge of the spread of the infection is essential when planning a control program.ResultsSerological investigation of Brucella infection in beef cattle tended under extensive farming conditions revealed a high seroprevalence (19.3%; 95% CI, 17–22) in the compliment fixation tests. The results of a rapid and simple field test correlated well with the Rose Bengal test (kappa, 0.917) and indicated an acceptable sensitivity (87.5%) and specificity (98.1%) compared with the complement fixation test. Reproductive failure was reported for 39.0% of the cows with a loss of calves due to abortion or early death amounting to 19.3%. Past reproductive failure did not, however, correlate with seropositivity in the complement fixation test (RP = 1.21; P = 0.847). B. abortus biovar 1 was freshly isolated from the hygromas of two cows and together with thirty banked isolates collected since 1990 from different parts of Sulawesi and Timor eight related genotypes could be distinguished with one genotype being identical to that of an isolate (BfR91) from Switzerland. The Indonesian genotypes formed together with BfR91 and one African and one North American isolate a distinct branch on the B. abortus biovar 1 dendogram.ConclusionsBovine brucellosis appears to be widespread in the Eastern Indonesian archipelago and calls for urgent intervention. The fresh isolation of the pathogen together with the observed high seroprevalence demonstrates the presence and frequent exposure of cattle in the area to the pathogen. The application of a rapid and simple field test for brucellosis could be very useful for the quick screening of cattle at the pen side.

Effect of Cryopreservation on Canine and Human Activated Nucleus Pulposus Cells: A Feasibility Study for Cell Therapy of the Intervertebral Disc

It has been shown that coculture of bone marrow–derived stromal cells (BMSCs) with intervertebral disc (IVD) nucleus pulposus (NP) cells significantly activates the biological characteristics of NP cells in animal models and in humans. We therefore predicted that activated NP cells would be a useful graft source for cellular transplantation therapy in the treatment of degenerative IVDs. However, the activation protocol is based on fresh isolation and activation of NP cells, which limits the timing of clinical application. Cell transplantation therapy could be offered to more patients than is now possible if activated NP cells could be transplanted as and when required by the condition of the patient. No study has investigated the effect of cryopreservation on NP cells after enzymatic isolation. We investigated the effects of cryopreservation of canine and human NP cells in both cell and tissue form before coculture with autologous BMSCs. Cell viability, proliferation, glycosaminoglycan production, aggrecan transcriptional activity, colony generation, and gene expression profile of the cells after cryopreservation and subsequent coculture were analyzed. The influence of cryopreservation on cell chromosomal abnormalities and tumorigenesis was also studied. The results showed that there were no clear differences between the noncryopreserved and cryopreserved cells in terms of cell viability, proliferation capacity, and capacity to synthesize extracellular matrix. Furthermore, the cells showed no apparent chromosomal abnormalities or tumorigenic ability and exhibited similar patterns of gene expression. These findings suggest that by using cryopreservation, it may be possible to transplant activated NP cells upon request for patients' needs.

Life in the Open Air: Place as a Therapeutic and Preventative Instrument in Australia's Early Open-air Tuberculosis Sanatoria

As the open-air sanatorium movement gained popularity during the nineteenth century, the therapeutic benefits of place were promoted in the crusade against tuberculosis by both medical professionals and architects. Fresh air, isolation, hygiene, discipline and education in a non-urban environment at high altitude were seen to be the keys to successful open-air treatment at sanatoria around the world. However, open-air sanatoria in Australia were not only therapeutic places, they were instruments of prevention. This article examines Australian tuberculosis sanatoria designed on the open-air treatment principles from 1895 to 1910. It focuses predominantly on Kalyra and Nunyara, both at Belair, South Australia, and the Queen Victoria Home for Consumptives at Wentworth Falls, New South Wales. In doing so, it explores the contribution of the buildings and landscape to the machinery of health as both an instrument of cure and prevention.

Differences in the Properties and Mirna Expression Profiles between Side Populations from Hepatic Cancer Cells and Normal Liver Cells

AimsBecause hepatic cancer stem cells (HCSCs) are believed to derive from the conversion of hepatic normal stem cells (HNSCs), the identification of the differences that distinguish HCSCs from HNSCs is important.MethodsThe HCC model was established in F344 rats by DEN induction. Using FACS analysis, side population cells from HCC (SP-HCCs) were isolated from the epithelial-like cells of HCC tissues, and the side population cells from normal liver (SP-NLCs) were isolated from syngeneic normal liver cells. The expression of stem cell markers was detected in both freshly isolated and amplified subpopulations. After induction with HGF, the differentiation of each subpopulation was analyzed by detection of early and late liver markers. In vivo, the biological characteristics of SP-HCCs and SP-NLCs were analyzed by repairing injured livers or forming tumors in nude mice. In addition, the expression of miRNAs was examined in both populations by miRNA array and QRT-PCR.ResultsSP-NLCs and SP-HCCs were 4.30±0.011% and 2.100±0.010% of the whole population, respectively. Both SP-NLCs and SP-HCCs displayed greater expression of stem cell markers (CD133 and EpCAM) than NSP-NLCs and NSP-HCCs, respectively (P<0.01), both after fresh isolation and amplification. Upon HGF induction, SP-NLCs generated many ALB positive cells and few CK-7 positive cells, but NSP-NLCs could generate only ALB positive cells. In contrast, SP-HCCs gave rise to only AFP positive cells. As few as 5×105 SP-NLCs were capable of repairing liver injury, while the same number of NSP-NLCs could not repair the liver. Furthermore, only 1×104 SP-HCCs were necessary to initiate a tumor, while NSP-HCCs could not form a tumor. Compared to SP-NLCs, 68 up-regulated and 10 down-regulated miRNAs were present in SP-HCCs (P<0.01).ConclusionBased on the decisive roles of some miRNAs in the genesis of HCSCs, miRNAs may contribute to the different characteristics that distinguish SP-HCCs from SP-NLCs.

Origin and hierarchy of basal lamina-forming and -non-forming myogenic cells in mouse skeletal muscle in relation to adhesive capacity and Pax7 expression in vitro

As a novel approach to distinguish skeletal myogenic cell populations, basal lamina (BL) formation of myogenic cells was examined in the mouse compensatory enlarged plantaris muscles in vivo and in fiber-bundle cultures in vitro. MyoD(+) myogenic cells located inside the regenerative muscle fiber BL were laminin(-) but interstitial MyoD(+) cells were laminin(+). This was also confirmed by electron microscopy as structural BL formation. Similar trends were observed in the fiber-bundle cultures including satellite cells and interstitial myogenic cells and laminin(+) myogenic cells predominantly showed non-adhesive (non-Ad) behavior with Pax7(-), whereas laminin(-) cells were adhesive (Ad) with Pax7(+). Moreover, non-Ad/laminin(+) and Ad/laminin(-) myotubes were also observed and the former type showed spontaneous contractions, while the latter type did not. The origin and hierarchy of Ad/Pax7(+)/laminin(-) and non-Ad/Pax7(-)/laminin(+) myogenic cells were also examined using skeletal muscle interstitium-derived CD34(+)/45(-) (Sk-34) and CD34(-)/45(-) (Sk-DN) multipotent stem cells, which were composed of non-committed myogenic cells with a few (<1%) Pax7(+) cells in the Sk-DN cells at fresh isolation. Both cell types were separated by Ad/non-Ad capacity in repetitive culture. As expected, both Ad/Pax7(+)/laminin(-) and non-Ad/Pax7(-)/laminin(+) myogenic cells consistently appeared in the Ad and non-Ad cell culture. However, Ad/Pax7(+)/laminin(-) cells were repeatedly detected in the non-Ad cell culture, while the opposite phenomenon did not occur. This indicates that the source of non-Ad/ Pax7(-)/laminin(+) myogenic cells was present in the Sk-34 and Sk-DN stem cells and they were able to produce Ad/ Pax7(+)/ laminin(-) myogenic cells during myogenesis as primary myoblasts and situated hierarchically upstream of the latter cells.

Rab32 Modulates Apoptosis Onset and Mitochondria-associated Membrane (MAM) Properties

The mitochondria-associated membrane (MAM) has emerged as an endoplasmic reticulum (ER) signaling hub that accommodates ER chaperones, including the lectin calnexin. At the MAM, these chaperones control ER homeostasis but also play a role in the onset of ER stress-mediated apoptosis, likely through the modulation of ER calcium signaling. These opposing roles of MAM-localized chaperones suggest the existence of mechanisms that regulate the composition and the properties of ER membrane domains. Our results now show that the GTPase Rab32 localizes to the ER and mitochondria, and we identify this protein as a regulator of MAM properties. Consistent with such a role, Rab32 modulates ER calcium handling and disrupts the specific enrichment of calnexin on the MAM, while not affecting the ER distribution of protein-disulfide isomerase and mitofusin-2. Furthermore, Rab32 determines the targeting of PKA to mitochondrial and ER membranes and through its overexpression or inactivation increases the phosphorylation of Bad and of Drp1. Through a combination of its functions as a PKA-anchoring protein and a regulator of MAM properties, the activity and expression level of Rab32 determine the speed of apoptosis onset.

β-Catenin/T-cell Factor Signaling Is Activated during Lung Injury and Promotes the Survival and Migration of Alveolar Epithelial Cells

The Wnt/beta-catenin signaling cascade activates genes that allow cells to adopt particular identities throughout development. In adult self-renewing tissues like intestine and blood, activation of the Wnt pathway maintains a progenitor phenotype, whereas forced inhibition of this pathway promotes differentiation. In the lung alveolus, type 2 epithelial cells (AT2) have been described as progenitors for the type 1 cell (AT1), but whether AT2 progenitors use the same signaling mechanisms to control differentiation as rapidly renewing tissues is not known. We show that adult AT2 cells do not exhibit constitutive beta-catenin signaling in vivo, using the AXIN2(+/LacZ) reporter mouse, or after fresh isolation of an enriched population of AT2 cells. Rather, this pathway is activated in lungs subjected to bleomycin-induced injury, as well as upon placement of AT2 cells in culture. Forced inhibition of beta-catenin/T-cell factor signaling in AT2 cultures leads to increased cell death. Cells that survive show reduced migration after wounding and reduced expression of AT1 cell markers (T1alpha and RAGE). These results suggest that AT2 cells may function as facultative progenitors, where activation of Wnt/beta-catenin signaling during lung injury promotes alveolar epithelial survival, migration, and differentiation toward an AT1-like phenotype.

Limits of CD133 as a marker of glioma self‐renewing cells

In human gliomas, self-renewing and tumor-initiating cells are characterized by the expression marker CD133. Although, widely used, the validity of CD133 is debated as recent data show that CD133(+) and CD133(-) cells share similar stemness and tumorigenic properties. To clarify this "CD133 controversy", we reexamined the methods of purification and the stem behavior of both CD133 compartments in fresh gliomas and gliomasphere cultures. Using human anti-CD133-coupled microbeads and magnetic activated cell sorting, we observed a nonspecific sorting of glioma cells irrespective of their CD133 expression. In contrast, when purified by fluorescence activating cell sorting, a specific expression and enrichment of CD133 was successfully observed in fresh human gliomas and gliomasphere cultures. However, neither the expression of stemness genes nor the long-term self-renewal capacities of CD133(+) and CD133(-) cells were significantly different, even after fresh isolation. Altogether, our data show that purification of CD133(+) glioma cells using hCD133-microbeads presents a lack of specificity and demonstrate that the use of CD133 as a unique glioma stem cell marker is likely not sufficient to tag the whole self-renewing tumor cell reservoir.

Fresh isolation and identification of the rat liver Kupffer cells suitable to patch clamp technique

To establish a method for isolation of rat liver Kupffer cells suitable for patch clamp technique (PCT).Liver Kupffer cells were isolated from Sprague-Dawley rats by in situ perfusion via the portal vein with Ca2+/Mg2+-free Krebs buffer followed by collagenase IV digestion. Then Percoll Isopyknic gradient centrifugation was performed. Kupffer cells were purified after selective adhesion. The cells were identified by endocytosis (ink and latex beads) experiments in vivo and in vitro. The electric current in Kupffer cells was recorded by whole-cell patch-clamp recording technique.The isolated Kupffer cells showed polymorphism with typical polygon-like and star-like shapes. A total of 2.5-3.5×106 cells were collected per gram of rat liver. The purity and adhesion rate were 90% and 39.4%, respectively. The activity is over 90%. The isolated and purified rat Kupffer cells were easy to be used in whole-cell PCT, and the store-operated Ca2+ current was successfully recorded in the cells.The Kupffer cells suitable for PCT were successfully isolated from rats by enzyme digestion combined with gradient centrifugation and adhesion purification.