Enzyme-based synthesis of single molecule RNA FISH probes

ABSTRACTSingle molecule RNA fluorescence in situ hybridization (smRNA FISH) allows the quantitative analysis of gene expression in single cells. The technique relies on the use of pools of end-labeled fluorescent oligonucleotides to detect specific cellular RNA sequences. These fluorescent probes are currently chemically synthesized. Here I describe a novel technique based on the use of routine molecular biology enzymes to generate smRNA FISH probes without the need for chemical synthesis of pools of oligonucleotides. The protocol comprises 3 main steps: purification of phagemid-derived single stranded DNA molecules comprising a segment complementary to a target RNA sequence; fragmentation of these molecules by limited DNase I digestion; and end-labeling of the resulting oligonucleotides with terminal deoxynucleotide transferase and fluorescent dideoxynucleotides. smRNA FISH probes that are obtained using the technique presented here are shown to perform as well as conventional probes. The main advantages of the method are the low cost of probes and the flexibility it affords in the choice of labels. Enzyme-based synthesis of probes should further increase the popularity of smRNA FISH as a tool to investigate gene expression at the cellular or subcellular level.

Autophagy confers resistance to lipopolysaccharide-induced mouse hepatocyte injury

During sepsis, bacterial products, particularly LPS, trigger injury in organs such as the liver. This common condition remains largely untreatable, in part due to a lack of understanding of how high concentrations of LPS cause cellular injury. In the liver, the lysosomal degradative pathway of autophagy performs essential hepatoprotective functions and is induced by LPS. We, therefore, examined whether hepatocyte autophagy protects against liver injury from septic levels of LPS. Mice with an inducible hepatocyte-specific knockout of the critical autophagy gene Atg7 were examined for their sensitivity to high-dose LPS. Increased liver injury occurred in knockout mice, as determined by significantly increased serum alanine aminotransferase levels, histological evidence of liver injury, terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick end-labeling, and effector caspase-3 and -7 activation. Hepatic inflammation and proinflammatory cytokine induction were unaffected by the decrease in hepatocyte autophagy. Although knockout mice had normal NF-κB signaling, hepatic levels of Akt1 and Akt2 phosphorylation in response to LPS were decreased. Cultured hepatocytes from knockout mice displayed a generalized defect in Akt signaling in response to multiple stimuli, including LPS, TNF, and IL-1β. Akt activation mediates hepatocyte resistance to TNF cytotoxicity, and anti-TNF antibodies significantly decreased LPS-induced liver injury in knockout mice, indicating that the loss of autophagy sensitized to TNF-dependent liver damage. Hepatocyte autophagy, therefore, protects against LPS-induced liver injury. Conditions such as aging and steatosis that impair hepatic autophagy may predispose to poor outcomes from sepsis through this mechanism.

Therapeutic administration of IL-11 exhibits the postconditioning effects against ischemia-reperfusion injury via STAT3 in the heart

Activation of cardiac STAT3 by IL-6 cytokine family contributes to cardioprotection. Previously, we demonstrated that IL-11, an IL-6 cytokine family, has the therapeutic potential to prevent adverse cardiac remodeling after myocardial infarction; however, it remains to be elucidated whether IL-11 exhibits postconditioning effects. To address the possibility that IL-11 treatment improves clinical outcome of recanalization therapy against acute myocardial infarction, we examined its postconditioning effects on ischemia/reperfusion (I/R) injury. C57BL/6 mice were exposed to ischemia (30 min) and reperfusion (24 h), and IL-11 was intravenously administered at the start of reperfusion. I/R injury mediated the activation of STAT3, which was enhanced by IL-11 administration. IL-11 treatment reduced I/R injury, analyzed by triphenyl tetrazolium chloride staining [PBS, 46.7 ± 14.4%; IL-11 (20 μg/kg), 28.6 ± 7.5% in the ratio of infarct to risk area]. Moreover, echocardiographic and hemodynamic analyses clarified that IL-11 treatment preserved cardiac function after I/R. Terminal deoxynucleotide transferase-mediated dUTP nick-end labeling staining revealed that IL-11 reduced the frequency of apoptotic cardiomyocytes after I/R. Interestingly, IL-11 reduced superoxide production assessed by in situ dihydroethidium fluorescence analysis, accompanied by the increased expression of metallothionein 1 and 2, reactive oxygen species (ROS) scavengers. Importantly, with the use of cardiac-specific STAT3 conditional knockout (STAT3 CKO) mice, it was revealed that cardiac-specific ablation of STAT3 abrogated IL-11-mediated attenuation of I/R injury. Finally, IL-11 failed to suppress the ROS production after I/R in STAT3 CKO mice. IL-11 administration exhibits the postconditioning effects through cardiac STAT3 activation, suggesting that IL-11 has the clinical therapeutic potential to prevent I/R injury in heart.

Assessment of the viability of embryos stored in liquid nitrogen produced commercially using culture medium as a complementary test for stereoscopic microscopy

SummaryThe objective of the present study was to evaluate the viability of frozen embryos obtained from various private farmers in a culture medium for 4 h. Forty-seven embryos were used that had been previously graded as good and fair. These embryos were evaluated using stereoscopic microscopy by experienced clinicians prior to freezing. Embryos were divided in two groups: the non-cultured group, made up of six good quality embryos, and five fair; and the cultured group that consisted of 20 good quality embryos and 16 fair. Fifty-four per cent of the good quality embryos achieved a favourable development during culture whereas just 42% of embryos determined to be fair were observed to have adequate development. This evaluation was undertaken by serial photographs obtained at the onset of culture and 4 h later. This finding was corroborated by a more specific technique: terminal deoxynucleotide transferase dUTP nick end labelling–bromodeoxyuridine (TUNEL–BrdU). These results are indicative of the necessity of tight quality controls for commercially produced frozen embryos, as once thawed it is unlikely that clinicians will examine them to determine their physiological status prior to transfer.

Cadherin Engagement Protects Human β-Cells from Apoptosis

The aim of this study was to assess the expression of different types of cadherins in human islets and their role in human β-cell apoptosis. Expression of E-, N-, and P-cadherins was studied by immunofluorescence on pancreas sections and islet cells, and by Western blotting on protein extracts of isolated islets and islet cells. The effects of specific cadherins on cell adhesion and apoptosis were studied using chimeric proteins containing functional E-, N-, or P-cadherin ectodomains fused to Fc fragment of Ig (E-cad/Fc, N-cad/Fc, and P-cad/Fc) and immobilized on glass substrate. β-Cells were identified by immunofluorescence for insulin and apoptotic cells by terminal deoxynucleotide transferase-mediated 2′-deoxyuridine, 5′-triphosphate nick-end labeling. By immunofluorescence, we showed that E- and N-, and not P-, cadherins were expressed at the surface of islet cells. By triple staining, we showed that E-cadherin was expressed at similar extent in β- and α-cells, whereas N-cadherin was preferentially expressed in β-cells. These results were confirmed by Western blot analysis using protein extracts from fluorescence-activated cell sorting-sorted β- and non-β-cells. Adhesion tests showed that the affinity of islet cells for E-cad/Fc and N-cad/Fc and not for P-cad/Fc was increased compared with control. By terminal deoxynucleotide transferase-mediated 2′-deoxyuridine, 5′-triphosphate nick-end labeling, we showed that the percentage of apoptotic cells was lower in aggregated β-cells compared with single β-cells and that attachment to E-cad/Fc and N-cad/Fc and not to P-cad/Fc decreased apoptosis of single β-cells compared with control. Our results show that at least E- and N-cadherins are expressed at the surface of human β-cells and that these adhesion molecules are involved in the maintenance of β-cell viability.

A human postnatal lymphoid progenitor capable of circulating and seeding the thymus

Identification of a thymus-seeding progenitor originating from human bone marrow (BM) constitutes a key milestone in understanding the mechanisms of T cell development and provides new potential for correcting T cell deficiencies. We report the characterization of a novel lymphoid-restricted subset, which is part of the lineage-negative CD34+CD10+ progenitor population and which is distinct from B cell–committed precursors (in view of the absence of CD24 expression). We demonstrate that these Lin−CD34+CD10+CD24− progenitors have a very low myeloid potential but can generate B, T, and natural killer lymphocytes and coexpress recombination activating gene 1, terminal deoxynucleotide transferase, PAX5, interleukin 7 receptor α, and CD3ε. These progenitors are present in the cord blood and in the BM but can also be found in the blood throughout life. Moreover, they belong to the most immature thymocyte population. Collectively, these findings unravel the existence of a postnatal lymphoid-polarized population that is capable of migrating from the BM to the thymus.

Importance of Uterine Cell Death, Renewal, and Their Hormonal Regulation in Hamsters that Show Progesterone-Dependent Implantation

This study was initiated to investigate the significance of uterine cell death and proliferation during the estrous cycle and early pregnancy and their correlation with sex steroids in hamsters where blastocyst implantation occurs in only progesterone-primed uteri. The results obtained in hamsters were also compared with mice where blastocyst implantation occurs in progesterone-primed uteri if estrogen is provided. Apoptotic cells in the uterus were detected by using terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) technique. Uterine cell proliferation was determined by 5-bromo-2′-deoxyuridine labeling followed by immunohistochemistry and methyl-tritiated [3H]thymidine labeling. Active caspase-3, an executor protein of cell death, expression was assayed by immunohistochemistry/immunofluorescence. Our results demonstrate that epithelial proliferation on the second day after mating marks the initiation of pregnancy-related uterine changes in both species despite their differences in hormonal requirements. Hamsters and mice showed subtle differences in uterine proliferative and apoptotic patterns during early pregnancy and in response to steroids. There existed almost a direct correlation between apoptosis and caspase-3 expression, suggesting uterine cell death mostly involves the caspase pathway. Consistent with these findings, we showed, for the first time, that execution of uterine epithelial cell apoptosis by caspase-3 is important for blastocyst implantation because a caspsase-3 inhibitor N-acetyl-DEVD-CHO when instilled inside the uterine lumen on d 3 of pregnancy inhibits implantation in hamsters and mice. The overall results indicate that uterine cell apoptosis and proliferation patterns are highly ordered cell-specific phenomena that play an important role in maintaining the sexual cycle and pregnancy-associated uterine changes.

Sperm DNA damage is related to field fertility of semen from young Norwegian Red bulls

Flow cytometry was utilised for the first time to independently measure five sperm parameters of individual spermatozoa of bull ejaculates to differentiate between outcome successes after artificial insemination (AI). These parameters included plasma membrane and acrosome integrity, mitochondrial functionality and DNA damage measured by sperm chromatin structure assay (SCSA) and terminal deoxynucleotide transferase-mediated dUTP nick end labelling (TUNEL) assays. For each parameter, results of 142 ejaculates (30 bulls) were ranked into three groups according to their flow cytometric measures: (1) ejaculates with the 25% lowest measures; (2) the 50% middle measures; and (3) the 25% highest measures. In total, 20 272 first-service inseminations (18 × 106 spermatozoa per AI dose) were performed, where fertility was defined as non-return within 60 days after first insemination. While plasma membrane and acrosome integrity, and mitochondrial functionality were not significantly related to fertility, data from SCSA and TUNEL assays were significantly associated with fertility. Ejaculates in SCSA group 1 had higher odds of AI success (1.07, 95% CI = 1.02–1.12), whereas those in group 3 had lower odds of AI success (0.94, 95% CI = 0.89–0.99), compared with the average odds of all three groups. Ejaculates in group 2 did not have significantly higher odds of AI success compared with the average odds. For TUNEL-positive spermatozoa, the odds of AI success was higher in group 1 compared with the average odds (1.10, 95% CI = 1.02–1.13), whereas odds of AI success in groups 2 and 3 were not significant compared with the average odds. In conclusion, despite the high number of spermatozoa per AI dose from high-quality bulls, both SCSA and TUNEL assays were valuable measures in this study for evaluating sperm quality in relation to fertility after AI.

Morphology of the Fetal Rat Testis Preserved in Different Fixatives

Histopathological examination of the testes of exposed fetuses and neonates is important in assessing the developmental effects of environmental toxins, including sex hormone modulators. Modified Davidson’s fluid (mDF) has been suggested as a superior substitute for Bouin’s fluid for fixation of adult animal testes. We compared the morphology of fetal rat testes stained with hematoxylin and eosin (H&E) or immunochemically after fixation in 10% neutral buffered formalin (NBF), Bouin’s fluid, or mDF. Fixation in mDF resulted in more sharply defined nuclear detail and better preservation of cellular cytoplasm on H&E-stained sections of rat testes on gestation day 19. Use of Bouin’s fluid did not allow satisfactory detection of apoptotic cells by fluorescent terminal deoxynucleotide transferase-mediated deoxy-UTP nick labeling. Staining with the immunoperoxidase system and the conventional chromogen diaminobenzidine tetrahydrochloride to visualize 5-bromo-2-deoxyuridine-positive cells demonstrated that the number of positive nuclei and intensity of staining were similar with all 3 fixatives. Immunostaining for cytoskeletal protein vimentin was more intense and provided better details of the Sertoli cell cytoplasm with formalin fixation than with mDF. Our study demonstrates that fixation in mDF provided better morphologic detail in the fetal rat testis compared with 10% NBF and Bouin’s fluid and illustrates the importance of establishing the correct fixation conditions for each immunostaining protocol.

Age-Related Olfactory Dysfunction: Cellular and Molecular Characterization in the Rat

Background Olfactory receptor neurons (ORNs) undergo apoptosis at a baseline rate even in the absence of obvious disease. Although the precise triggers of the apoptotic cascade are unclear, ORNs are exposed directly to the external environment, making them susceptible to injury. As an adaptive mechanism, mammals have the ability to replace lost ORNs throughout adult life from neuronal precursors within the olfactory epithelium (OE). In humans, this process fails with age as the surface area of the OE and the number of ORNs decline, coupled with a loss of clinical olfactory function. The question addressed in this study is whether this age-related failure of olfactory sensation is a result of a decrease in neuronal proliferation or an increase in ORN cell death. Methods To begin to address this question the ribonuclease protection assay was used to assess expression of apoptosisrelated genes in rat OE as a function of age. Second, the terminal deoxynucleotide transferase end labeling assay was used to assess the percentage of ORNs undergoing apoptosis (apoptotic index) in three groups of animals: young (12 weeks), old (32 months), and bulbectomized rats. Bulbectomy is a standard model for ORN injury associated with a massive increase in ORN apoptosis and serves as a positive control. Results Ribonuclease protection assay data indicate an age-related increase in Bax, Bcl-xL, and procaspase-3 messenger RNA expression in aged compared with young rats. A similar but more pronounced increase in expression of these apoptotic-related genes is seen after bulbectomy. The terminal deoxynucleotide transferase end labeling assay also showed a statistically significant increase in the apoptotic index with both age and bulbectomy. Conclusion Taken together, the current results indicate that aging and injury induce parallel changes in OE. Furthermore, these findings support the hypothesis that age-related olfactory dysfunction is, at least in part, related to an increase in ORN cell death.