Increased Specific Labeling of INS-1 Pancreatic Beta-Cell by Using RIP-Driven Cre Mutants with Reduced Activity

Ectopically expressed Cre recombinase in extrapancreatic tissues in RIP-Cre mice has been well documented. The objective of this study was to find a simple solution that allows for improved beta-cell specific targeting. To this end, the RIP-Cre and reporter CMV-loxP-DsRed-loxP-EGFP expression cassettes were configurated into a one-plasmid and two-plasmid systems, which labeled approximately 80% insulin-positive INS-1 cells after 48 h transfection. However, off-target labeling was robustly found in more than 15% insulin-negative Ad293 cells. When an IRES element was inserted in front of Cre to reduce the translation efficiency, the ratio of recombination between INS-1 and Ad293 cells increased 3-4-fold. Further, a series of Cre mutants were generated by site-directed mutagenesis. When one of the mutants, Cre(H289P) in both configurations, was used in the experiment, the percentage of recombination dropped to background levels in a number of insulin-negative cell lines, but decreased only slightly in INS-1 cells. Consistently, DNA substrate digestion assay showed that the enzymatic activity of Cre(H289P) was reduced by 30-fold as compared to that of wild-type. In this study, we reported the generation of constructs containing RIP and Cre mutants, which enabled enhanced beta-cell specific labeling in vitro. These tools could be invaluable for beta-cell targeting and to the study of islet development.     

Erratum to: Endogenous TGFβ1 Plays a Crucial Role in Functional Recovery After Traumatic Brain Injury Associated with Smad3 Signal in Rats

Neurochemical Research -     

Cognitive Changes during Prolonged Stay at High Altitude and Its Correlation with C-Reactive Protein

Hypersensitive C-reaction protein (hsCRP) may be a risk factor for cognitive impairment resulting from Alzheimer’s disease (AD), stroke, and vascular dementia. This study explored the correlation of peripheral blood hsCRP level with cognitive decline due to high altitude exposure. The study was conducted on 100 male military participants who had never been to high altitude. Cerebral oxygen saturation monitoring, event related potentials (P300, N200) detection, and neurocognitive assessment was performed and total hsCRP, interleukin-6 (IL-6), and homocysteine was estimated at 500m altitude, 3650m altitude, 3day, 1, and 3 month post arriving at the base camp (4400m), and 1 month after coming back to the 500m altitude. High altitude increased brain oxygen saturation, prolonged P300 and N200 latencies, injured cognitive functions, and raised plasma hsCRP levels. But they all recovered in varying degrees at 1 and 3 month post arriving at the base camp (4400m). P300 latencies and hsCRP levels were strongly correlated to cognitive performances. These results suggested that cognitive deterioration occurred during the acute period of exposure to high altitude and may recover probably owning to acclimatization after extended stay at high altitude. Plasma hsCRP is inversely correlated to neurological cognition and it may be a potential biomarker for the prediction of high altitude induced cognitive dysfunction.     

Apical Scaffolding Protein NHERF2 Modulates the Localization of Alternatively Spliced Plasma Membrane Ca2+ Pump 2B Variants in Polarized Epithelial Cells

The membrane localization of the plasma membrane Ca²⁺-ATPase isoform 2 (PMCA2) in polarized cells is determined by alternative splicing; the PMCA2w/b splice variant shows apical localization, whereas the PMCA2z/b and PMCA2x/b variants are mostly basolateral. We previously reported that PMCA2b interacts with the PDZ protein Na⁺/H⁺ exchanger regulatory factor 2 (NHERF2), but the role of this interaction for the specific membrane localization of PMCA2 is not known. Here we show that co-expression of NHERF2 greatly enhanced the apical localization of GFP-tagged PMCA2w/b in polarized Madin-Darby canine kidney cells. GFP-PMCA2z/b was also redirected to the apical membrane by NHERF2, whereas GFP-PMCA2x/b remained exclusively basolateral. In the presence of NHERF2, GFP-PMCA2w/b co-localized with the actin-binding protein ezrin even after disruption of the actin cytoskeleton by cytochalasin D or latrunculin B. Surface biotinylation and fluorescence recovery after photobleaching experiments demonstrated that NHERF2-mediated anchorage to the actin cytoskeleton reduced internalization and lateral mobility of the pump. Our results show that the specific interaction with NHERF2 enhances the apical concentration of PMCA2w/b by anchoring the pump to the apical membrane cytoskeleton. The data also suggest that the x/b splice form of PMCA2 contains a dominant lateral targeting signal, whereas the targeting and localization of the z/b form are more flexible and not fully determined by intrinsic sequence features.     

Identification of compound heterozygous DNAH11 variants in a Han-Chinese family with primary ciliary dyskinesia

Primary ciliary dyskinesia (PCD) is a group of genetically and clinically heterogeneous disorders with motile cilia dysfunction. It is clinically characterized by oto-sino-pulmonary diseases and subfertility, and half of the patients have situs inversus (Kartagener syndrome). To identify the genetic cause in a Han-Chinese pedigree, whole-exome sequencing was conducted in the 37-year-old proband, and then, Sanger sequencing was performed on available family members. Minigene splicing assay was applied to verify the impact of the splice-site variant. Compound heterozygous variants including a splice-site variant (c.1974-1G>C, rs1359107415) and a missense variant (c.7787G>A, p.(Arg2596Gln), rs780492669), in the dynein axonemal heavy chain 11 gene (DNAH11) were identified and confirmed as the disease-associated variants of this lineage. The minigene expression in vitro revealed that the c.1974-1G>C variant could cause skipping over exon 12, predicted to result in a truncated protein. This discovery may enlarge the DNAH11 variant spectrum of PCD, promote accurate genetic counselling and contribute to PCD diagnosis.     

Involvement of Kv1.3 and p38 MAPK signaling in HIV-1 glycoprotein 120-induced microglia neurotoxicity

Inflammatory responses mediated by activated microglia play a pivotal role in the pathogenesis of human immunodeficiency virus type 1 (HIV-1)-associated neurocognitive disorders. Studies on identification of specific targets to control microglia activation and resultant neurotoxic activity are imperative. Increasing evidence indicate that voltage-gated K⁺ (K_v) channels are involved in the regulation of microglia functionality. In this study, we investigated K_v1.3 channels in the regulation of neurotoxic activity mediated by HIV-1 glycoprotein 120 (gp120)-stimulated rat microglia. Our results showed treatment of microglia with gp120 increased the expression levels of K_v1.3 mRNA and protein. In parallel, whole-cell patch-clamp studies revealed that gp120 enhanced microglia K_v1.3 current, which was blocked by margatoxin, a K_v1.3 blocker. The association of gp120 enhancement of K_v1.3 current with microglia neurotoxicity was demonstrated by experimental results that blocking microglia K_v1.3 attenuated gp120-associated microglia production of neurotoxins and neurotoxicity. Knockdown of K_v1.3 gene by transfection of microglia with K_v1.3-siRNA abrogated gp120-associated microglia neurotoxic activity. Further investigation unraveled an involvement of p38 MAPK in gp120 enhancement of microglia K_v1.3 expression and resultant neurotoxic activity. These results suggest not only a role K_v1.3 may have in gp120-associated microglia neurotoxic activity, but also a potential target for the development of therapeutic strategies.     

Isoliquiritigenin prevents hyperglycemia-induced renal injuries by inhibiting inflammation and oxidative stress via SIRT1-dependent mechanism

Diabetic nephropathy (DN) as a global health concern is closely related to inflammation and oxidation. Isoliquiritigenin (ISL), a natural flavonoid compound, has been demonstrated to inhibit inflammation in macrophages. Herein, we investigated the effect of ISL in protecting against the injury in STZ-induced type 1 DN and in high glucose-induced NRK-52E cells. In this study, it was revealed that the administration of ISL not only ameliorated renal fibrosis and apoptosis, but also induced the deterioration of renal function in diabetic mice. Mediated by MAPKs and Nrf-2 signaling pathways, respectively, upstream inflammatory response and oxidative stress were neutralized by ISL in vitro and in vivo. Moreover, as further revealed by the results of molecular docking, sirtuin 1 (SIRT1) binds to ISL directly, and the involvement of SIRT1 in ISL-mediated renoprotective effects was confirmed by studies using in vitro models of SIRT1 overexpression and knockdown. In summary, by reducing inflammation and oxidative stress, ISL has a significant pharmacological effect on the deterioration of DN. The benefits of ISL are associated with the direct binding to SIRT1, the inhibition of MAPK activation, and the induction of Nrf-2 signaling, suggesting the potential of ISL for DN treatment.Subject terms: Pharmacology, Molecular biology