Identification of Mineralocorticoid Receptors, Aldosterone, and Its Processing Enzyme CYP11B2 on Parasympathetic and Sympathetic Neurons in Rat Intracardiac Ganglia

Recent interest has focused on the mineralocorticoid receptor (MR) and its impact on the myocardium and the performance of the heart. However, there is a lack of evidence about MR expression and its endogenous ligand aldosterone synthesis with specific regard to the intrinsic cardiac nervous system. Therefore, we looked for evidence of MR and aldosterone in sympathetic and parasympathetic neurons of intracardiac ganglia. Tissue samples from rat heart atria were subjected to conventional reverse-transcriptase polymerase chain reaction (PCR), Western blot, and double immunofluorescence confocal analysis of MR, corticosterone-inactivating enzyme 11β-hydroxysteroid-dehydrogenase-2 (11β-HSD2), aldosterone, and its processing enzyme CYP11B2 together with the neuronal markers vesicular acetylcholine transporter (VAChT) and tyrosine hydroxylase (TH). Our results demonstrated MR, 11β-HSD2, and CYP11B2 specific mRNA and protein bands in rat heart atria. Double immunofluorescence labeling revealed coexpression of MR immunoreactivity with VAChT in large diameter parasympathetic principal neurons. In addition, MR immunoreactivity was identified in TH-immunoreactive small intensely fluorescent (SIF) cells and in nearby VAChT- and TH-immunoreactive nerve terminals. Interestingly, the aldosterone and its synthesizing enzyme CYP11B2 and 11β-HSD2 colocalized in MR– immunoreactive neurons of intracardiac ganglia. Overall, this study provides first evidence for the existence of not only local expression of MR, but also of 11β-HSD2 and aldosterone with its processing enzyme CYP11B2 in the neurons of the cardiac autonomic nervous system, suggesting a possible modulatory role of the mineralocorticoid system on the endogenous neuronal activity on heart performance.

Changes in Homogalacturonan Metabolism in Banana Peel during Fruit Development and Ripening

Though numerous studies have focused on the cell wall disassembly of bananas during the ripening process, the modification of homogalacturonan (HG) during fruit development remains exclusive. To better understand the role of HGs in controlling banana fruit growth and ripening, RNA-Seq, qPCR, immunofluorescence labeling, and biochemical methods were employed to reveal their dynamic changes in banana peels during these processes. Most HG-modifying genes in banana peels showed a decline in expression during fruit development. Four polygalacturonase and three pectin acetylesterases showing higher expression levels at later developmental stages than earlier ones might be related to fruit expansion. Six out of the 10 top genes in the Core Enrichment Gene Set were HG degradation genes, and all were upregulated after softening, paralleled to the significant increase in HG degradation enzyme activities, decline in peel firmness, and the epitope levels of 2F4, CCRC-M38, JIM7, and LM18 antibodies. Most differentially expressed alpha-1,4-galacturonosyltransferases were upregulated by ethylene treatment, suggesting active HG biosynthesis during the fruit softening process. The epitope level of the CCRC-M38 antibody was positively correlated to the firmness of banana peel during fruit development and ripening. These results have provided new insights into the role of cell wall HGs in fruit development and ripening.

Effects of Chemogenetic Inhibition of D1 or D2 Receptor-Containing Neurons of the Substantia Nigra and Striatum in Mice With Tourette Syndrome

As tourette syndrome (TS) is a common neurobehavioral disorder, the primary symptoms of which include behavioral stereotypies. Dysfunction of the substantia nigra–striatum network could be the main pathogenesis of TS, which is closely associated with dopamine (DA) and its receptors. TS is often resistant to conventional treatments. Therefore, it is necessary to investigate the neurobiological mechanisms underlying its pathogenesis. In this study, we investigated whether chemogenetic activation or inhibition of dopaminergic D1 receptor (D1R)- or D2 receptor (D2R)-containing neurons in the substantia nigra pars compacta (SNpc) or dorsal striatum (dSTR) affected the stereotyped behavior and motor functions of TS mice. Intraperitoneal injection of 3,3′-iminodipropionitrile (IDPN) was used to induce TS in mice. Stereotyped behavior test and open-field, rotarod, and grip strength tests were performed to evaluate stereotyped behavior and motor functions, respectively. Immunofluorescence labeling was used to detect the co-labeling of virus fluorescence and D1R or D2R. We found that chemogenetic inhibition of D1R- or D2R-containing neurons in the SNpc and dSTR alleviated behavioral stereotypies and motor functions in TS mice. Chemogenetic activation of D1R-containing neurons in the dSTR aggravated behavioral stereotypies and motor functions in vehicle-treated mice, but neither was aggravated in TS mice. In conclusion, chemogenetic inhibition of D1R- or D2R-containing neurons in the SNpc and dSTR alleviated behavioral stereotypies of TS, providing a new treatment target for TS. Moreover, the activation of D1R-containing neurons in the dSTR may contribute to the pathogenesis of TS, which can be chosen as a more precise target for treatment.

Changes in Epigenetic Patterns Related to DNA Replication in Vicia faba Root Meristem Cells under Cadmium-Induced Stress Conditions

Experiments on Vicia faba root meristem cells exposed to 150 µM cadmium chloride (CdCl2) were undertaken to analyse epigenetic changes, mainly with respect to DNA replication stress. Histone modifications examined by means of immunofluorescence labeling included: (1) acetylation of histone H3 on lysine 56 (H3K56Ac), involved in transcription, S phase, and response to DNA damage during DNA biosynthesis; (2) dimethylation of histone H3 on lysine 79 (H3K79Me2), correlated with the replication initiation; (3) phosphorylation of histone H3 on threonine 45 (H3T45Ph), engaged in DNA synthesis and apoptosis. Moreover, immunostaining using specific antibodies against 5-MetC-modified DNA was used to determine the level of DNA methylation. A significant decrease in the level of H3K79Me2, noted in all phases of the CdCl2-treated interphase cell nuclei, was found to correspond with: (1) an increase in the mean number of intranuclear foci of H3K56Ac histones (observed mainly in S-phase), (2) a plethora of nuclear and nucleolar labeling patterns (combined with a general decrease in H3T45Ph), and (3) a decrease in DNA methylation. All these changes correlate well with a general viewpoint that DNA modifications and post-translational histone modifications play an important role in gene expression and plant development under cadmium-induced stress conditions.

Super Resolution Localization Microscopy Enables Quantitative Diagnosis of Multiple Myeloma

Multiple myeloma (MM) is a bone marrow-based malignancy that has a range of consequences resulting from either its direct effects on the bone marrow microenvironment- causing anaemia, more extensive myelosuppression and bone lysis - or its indirect effects on the kidney and other organ systems. Over the past several years, the introduction of autologous stem cell transplantation, immunomodulatory drugs, proteasome inhibitors, histone deacetylase inhibitors, and monoclonal antibodies has substantially improved survival outcomes. However, MM remains biologically heterogeneous with significant variability among patients in terms of clinical features, response to therapy and overall survival (OS). Several prognostic variables can help predict this variability in outcomes ranging from clinical based systems such as the International Staging System (ISS) to more advanced molecular characterizations of the myeloma PCs by cytogenetics and gene expression profiling. However with the advancement in technology utilized for laboratory testing and the emergence of new treatments for MM, there has been an evolution in the significance of these previously defined prognostic markers with time. Assessment of disease activity and depth of response continues to be a moving target in MM. Super-resolution fluorescence imaging is a major breakthrough in the field of optical imaging in this century. Super-resolution fluorescence imaging has been applied to a variety of biological imaging applications, including membrane, cytoskeletal and cytosolic proteins in fixed and living cells. Molecular motions can be quantified. To establish the detection limit and sensitivity threshold of dSTORM and FC, we used serial dilutions of anti-CD38 antibody to detect expression of CD38 on 8226 cells by Super Resolution localization Microscopy and flow cytometry (FC). Design six different antibody concentrations (300ng/ml, 30ng/ml, 10ng/ml, 3ng/ml, 1ng/ml, 0.3ng/ml) to label MM cells with immunofluorescence, and then detect them by flow cytometry. Similarly, design eight different antibody concentrations (1μg/ml, 300ng/ml, 30ng/ml, 10ng/ml, 3ng/ml, 1ng/ml, 0.3ng/ml, 0.1ng/ml), and perform the same treatment, perform immunofluorescence labeling on MM cells, and then perform super-resolution imaging, and calculate the density of CD38 protein on the surface of MM cells of each concentration. Figure 1A detects the ratio of the number of cells to the total number of cells, it can be seen from Figure 1A that when the antibody concentration is not less than 30ng/ml, almost all MM thinners can detect the signal, but when the antibody concentration is less than at 30ng/ml, only a few or no cells can detect the signal. This shows that when the antibody concentration is lower than 30ng/ml, the sensitivity of flow cytometry is no longer sufficient to detect CD38 protein on the surface of MM cells. From Figure 1B, it can be seen that even when the CD38 antibody concentration is 0.3ng/ml, super-resolution imaging can still accurately identify each CD38 antigen molecule on the surface of MM cells, and the statistical CD38 protein density is 8.5864±1.4180/μm2. This shows that the sensitivity of super-resolution is much higher than that of streaming. Disclosures No relevant conflicts of interest to declare.

Identification and response analysis of xyloglucan endotransglycosylase/hydrolases (XTH) family to fluoride and aluminum treatment in Camellia sinensis

Background Xyloglucan endotransglycosylase/hydrolases (XTH) can disrupt and reconnect the xyloglucan chains, modify the cellulose-xyloglucan complex structure in the cell wall to reconstruct the cell wall. Previous studies have reported that XTH plays a key role in the aluminum (Al) tolerance of tea plants (Camellia sinensis), which is a typical plant that accumulates Al and fluoride (F), but its role in F resistance has not been reported. Results Here, 14 CsXTH genes were identified from C. sinensis and named as CsXTH1–14. The phylogenetic analysis revealed that CsXTH members were divided into 3 subclasses, and conserved motif analysis showed that all these members included catalytic active region. Furthermore, the expressions of all CsXTH genes showed tissue-specific and were regulated by Al3+ and F− treatments. CsXTH1, CsXTH4, CsXTH6–8 and CsXTH11–14 were up-regulated under Al3+ treatments; CsXTH1–10 and CsXTH12–14 responded to different concentrations of F− treatments. The content of xyloglucan oligosaccharide determined by immunofluorescence labeling increased to the highest level at low concentrations of Al3+ or F− treatments (0.4 mM Al3+ or 8 mg/L F−), accompanying by the activity of XET (Xyloglucan endotransglucosylase) peaked. Conclusion In conclusion, CsXTH activities were regulated by Al or F via controlling the expressions of CsXTH genes and the content of xyloglucan oligosaccharide in C. sinensis roots was affected by Al or F, which might finally influence the elongation of roots and the growth of plants.

Peripheral Nerve Impairment in a Mouse Model of Alzheimer’s Disease

Sarcopenia, a geriatric syndrome involving loss of muscle mass and strength, is often associated with the early phases of Alzheimer’s disease (AD). Pathological hallmarks of AD including amyloid β (Aβ) aggregates which can be found in peripheral tissues such as skeletal muscle. However, not much is currently known about their possible involvement in sarcopenia. We investigated neuronal innervation in skeletal muscle of Tg2576 mice, a genetic model for Aβ accumulation. We examined cholinergic innervation of skeletal muscle in adult Tg2576 and wild type mice by immunofluorescence labeling of tibialis anterior (TA) muscle sections using antibodies raised against neurofilament light chain (NFL) and acetylcholine (ACh) synthesizing enzyme choline acetyltransferase (ChAT). Combining this histological approach with real time quantification of mRNA levels of nicotinic acetylcholine receptors, we demonstrated that in the TA of Tg2576 mice, neuronal innervation is significantly reduced and synaptic area is smaller and displays less ChAT content when compared to wild type mice. Our study provides the first evidence of reduced cholinergic innervation of skeletal muscle in a mouse model of Aβ accumulation. This evidence sustains the possibility that sarcopenia in AD originates from Aβ-mediated cholinergic loss.

Abstract P409: GSK-3β Deficiency In Cardiomyocyte Induces Cardiac Progenitor Cell Proliferation In The Ischemic Heart

The cardiomyocytes are terminally differentiated cells and ischemia-induced cardiomyopathy is an irreparable loss. Novel strategies are needed to induce resident cardiac progenitor cells (CPCs) proliferation in situ to enhance the possibility of cardiac regeneration. Here we sought to identify a potential role for glycogen synthase kinase-3β (GSK-3β), a critical regulator of cell proliferation and differentiation, in CPCs proliferation in the ischemic heart. Cardiomyocyte-specific conditional GSK-3β knockout (cKO) and littermate control mice were recruited and challenged with myocardial infarction (MI). The cardiac function was assessed using trans-thoracic M-mode echocardiography. The level of CPC proliferation in the ischemic cKO hearts was determined at 2, 4 and 8 weeks post-MI through immunofluorescence labeling of stem cell marker c-Kit. To confirm the lineage of identified c-Kit -positive cells (KPCs) in the heart, a hematopoietic lineage marker was stained along with c-Kit. The cardiac left ventricular chamber dimension (LVID) and contractile functions were comparable until 2 weeks post-MI. The cKO mice displayed significantly preserved LV chamber [LVIDd(mm); 5.01±0.67 vs.6.09±0.65, p =0.01] and contractile function [LVEF(%); 31.98±8.52 vs. 18.06±7.11., p =0.01] in comparison to control mice at 4 week post-MI. Consistent with protective phenotypes, an increased percentage of KPC was observed in the cKO hearts at 4 and 6-weeks post-MI which was accompanied by an increased level of cardiomyocyte proliferation. Further analysis revealed that the observed increased number of KPCs in the ischemic cKO hearts were mostly from a cardiac lineage as the majority of identified KPCs were negative for the hematopoietic marker, CD45. In conclusion, our findings strongly suggest that GSK-3β inhibits CPCc proliferation post-ischemia, and loss of GSK-3β in cardiomyocytes promotes resident CPCc proliferation potentially through paracrine mechanisms.

The effect of androgen on wool follicles and keratin production in Hetian sheep

To investigate the optimal androgen concentration for culturing Hetian sheep wool follicle and to detect effects of androgen concentration on wool follicle cell proliferation and apoptosis using immunofluorescence labeling and real-time quantitative fluorescence determinations of wool keratin-associated protein gene expression levels. Wool follicles were isolated by microdissection and wool follicles and skin pieces were cultured in various concentrations of dihydrotestosterone (DHT) in culture medium. Next, daily lengthwise growth measurements of wool follicles were obtained using a microscopic micrometer. Cultured Hetian wool follicles were stained using the SACPIC method to reveal wool follicle structure, while sheep skin slices were used to observe cell proliferation by immunostaining and cell apoptosis using the TUNEL method. At the molecular biological level, keratin-associated protein (Kap) gene expression was studied using wool follicles cultured for various numbers of days in vitro. Effects of androgen concentrations on Hetian wool follicle growth and development were experimentally studied. EdU proliferation assays revealed that androgen promoted cell proliferation within wool follicle dermal papillae. TUNEL apoptosis detection demonstrated that androgen treatment could delay cell apoptosis. Quantitative reverse transcription polymerase chain reaction (qPCR) results demonstrated that gene expression level patterns of Hetian mountain sheep super-high sulfur protein. Kap1.1, KIF1.2, Kap2.12 and Kap4.2 gene expression level of the mountainous experimental group was significantly higher than plains Hetian sheep. An androgen concentration of 100 nM can promote the growth of Hetian wool follicle cells in vitro, resulting in overexpression of some genes of the Kap family.

A Comprehensive Approach for the Diagnosis of Primary Ciliary Dyskinesia—Experiences from the First 100 Patients of the PCD-UNIBE Diagnostic Center

Primary ciliary dyskinesia (PCD) is a rare genetic disease characterized by dyskinetic cilia. Respiratory symptoms usually start at birth. The lack of diagnostic gold standard tests is challenging, as PCD diagnostics requires different methods with high expertise. We founded PCD-UNIBE as the first comprehensive PCD diagnostic center in Switzerland. Our diagnostic approach includes nasal brushing and cell culture with analysis of ciliary motility via high-speed-videomicroscopy (HSVM) and immunofluorescence labeling (IF) of structural proteins. Selected patients undergo electron microscopy (TEM) of ciliary ultrastructure and genetics. We report here on the first 100 patients assessed by PCD-UNIBE. All patients received HSVM fresh, IF, and cell culture (success rate of 90%). We repeated the HSVM with cell cultures and conducted TEM in 30 patients and genetics in 31 patients. Results from cell cultures were much clearer compared to fresh samples. For 80 patients, we found no evidence of PCD, 17 were diagnosed with PCD, two remained inconclusive, and one case is ongoing. HSVM was diagnostic in 12, IF in 14, TEM in five and genetics in 11 cases. None of the methods was able to diagnose all 17 PCD cases, highlighting that a comprehensive approach is essential for an accurate diagnosis of PCD.