Orexin-A protects against oxygen-glucose deprivation/reoxygenation-induced cell damage by inhibiting endoplasmic reticulum stress-mediated apoptosis via the Gi and PI3K signaling pathways

The neuropeptide orexin-A (OXA) has a neuroprotective effect, acting as an anti-apoptotic factor in response to multiple stimuli. Apoptosis induced by endoplasmic reticulum stress (ERS) underlies oxygen-glucose deprivation and reoxygenation (OGD/R)-induced cell damage, an in vitro model of ischemia/reperfusion injury. However, that OXA inhibits ERS-induced apoptosis in the OGD/R model has not been reported. In the present study, we investigated the neuroprotective effect of OXA (0.1 μM) on OGD/R-induced damage in the human neuroblastoma cell line SH-SY5Y. After OXA treatment following 4 h oxygen-glucose deprivation (OGD) and then 4 h reoxygenation (R), cell morphology, viability, and apoptosis were analyzed by histology, Cell Counting Kit-8 assay, and flow cytometry, respectively. Western blotting was used to measure expression levels of ERS- and apoptosis-related proteins. To determine signaling pathways involved in OXA-mediated neuroprotection, the Gi pathway inhibitor pertussis toxin (PTX; 100 ng/mL) and PI3K inhibitor LY294002 (LY; 10 μM) were added. In addition, in order to prove the specificity of these characteristics, the OXA antagonist Suvorexant (DORA; Ki of 0.55 nM and 0.35 nM for OX1R and OX2R) was used for intervention. Our results showed that OGD/R induced cell damage, manifested as morphological changes and a significant decrease in viability. Furthermore, Western blotting detected an increase in ERS-related proteins GRP78, p-IRE1α, p-JNK, and Cleaved caspase-12, as well as apoptosis-related proteins Cleaved caspase-3 and Bax, and a decrease in the anti-apoptosis factor Bcl-2. OXA intervention alleviated the degree of cellular damage, and protein expression was also reversed. In addition, the protective effect of OXA was reduced by adding PTX and LY. Meanwhile, after the use of DORA, changes in the expression of related proteins were detected, and it was found that the protective effect of OXA was weakened. Collectively, our results indicate that OXA has a neuroprotective effect on OGD/R-induced cell damage by inhibiting ERS-induced apoptosis through the combined action of Gi and PI3K signaling pathways. These findings help to clarify the mechanism underlying the neuroprotective action of OXA, which should aid the development of further candidate drugs, and provide a new therapeutic direction for the treatment of ischemic stroke

Molecular Characterization and Expression Pattern of Tripartite Motif Protein 39 in Gallus gallus with a Complete PRY/SPRY Domain

Members of tripartite motif (TRIM) proteins in mammals play important roles in multiple cellular processes in the immune system. In the present study we have obtained the chicken TRIM39 with the insertion of a base A at position 1006 bp, compared to the sequence in the NCBI database (Accession No: NM 001006196), which made TRIM39 fulfill the TRIM rule of domain composition with both PRY, and SPRY domains. The open reading frame consisted of 1392 bp encoding 463 amino acid residues. The amino acid sequences of TRIM39 protein in mammals were highly similar (from 91.48% to 99.61%), while chicken TRIM39 had relatively low homology with mammals (from 29.2% to 39.59%). Real time RT-PCR indicated that the mRNA expression level of TRIM39 was the highest in spleen, with a lower expression in liver, brain, and lung, suggesting it might be an important protein participating in the immune system

Transmembrane peptide 4 and 5 of APJ are essential for its heterodimerization with OX1R

Increasing evidence indicates some G protein-coupled receptors function as a heterodimer, which provide a novel target for therapeutics investigation. However, study on the receptor-receptor interaction interface, a potent target on interfering dimer formation, are still limited. Here, using bioluminescence resonance energy transfer (BRET) combined with co-immunoprecipitation (Co-IP), we found a new constitutive GPCR heterodimer, apelin receptor (APJ)-orexin receptor type 1 (OX1R). Both APJ and OX1R co-internalized when constantly subjected to cognate agonist (apelin-13 or orexin-A) specific to either protomer. Combined with BRET and immunostaining, the in vitro synthesized transmembrane peptides (TMs) interfering experiments suggests that TM4 and 5 of APJ act as the interaction interface of the APJ-OX1R heterodimer, and co-internalization could be disrupted by these peptides as well. Our study not only provide new evidence on GPCR heterodimerization, but address a novel heterodimerization interface, which can be severed as a potential pharmacological target

Cell transcriptomic atlas of the non-human primate Macaca fascicularis.

Studying tissue composition and function in non-human primates (NHPs) is crucial to understand the nature of our own species. Here we present a large-scale cell transcriptomic atlas that encompasses over 1 million cells from 45 tissues of the adult NHP Macaca fascicularis. This dataset provides a vast annotated resource to study a species phylogenetically close to humans. To demonstrate the utility of the atlas, we have reconstructed the cell-cell interaction networks that drive Wnt signalling across the body, mapped the distribution of receptors and co-receptors for viruses causing human infectious diseases, and intersected our data with human genetic disease orthologues to establish potential clinical associations. Our M. fascicularis cell atlas constitutes an essential reference for future studies in humans and NHPs.We thank W. Liu and L. Xu from the Huazhen Laboratory Animal Breeding Centre for helping in the collection of monkey tissues, D. Zhu and H. Li from the Bioland Laboratory (Guangzhou Regenerative Medicine and Health Guangdong Laboratory) for technical help, G. Guo and H. Sun from Zhejiang University for providing HCL and MCA gene expression data matrices, G. Dong and C. Liu from BGI Research, and X. Zhang, P. Li and C. Qi from the Guangzhou Institutes of Biomedicine and Health for experimental advice or providing reagents. This work was supported by the Shenzhen Basic Research Project for Excellent Young Scholars (RCYX20200714114644191), Shenzhen Key Laboratory of Single-Cell Omics (ZDSYS20190902093613831), Shenzhen Bay Laboratory (SZBL2019062801012) and Guangdong Provincial Key Laboratory of Genome Read and Write (2017B030301011). In addition, L.L. was supported by the National Natural Science Foundation of China (31900466), Y. Hou was supported by the Natural Science Foundation of Guangdong Province (2018A030313379) and M.A.E. was supported by a Changbai Mountain Scholar award (419020201252), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA16030502), a Chinese Academy of Sciences–Japan Society for the Promotion of Science joint research project (GJHZ2093), the National Natural Science Foundation of China (92068106, U20A2015) and the Guangdong Basic and Applied Basic Research Foundation (2021B1515120075). M.L. was supported by the National Key Research and Development Program of China (2021YFC2600200).S

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Using bottleneck analysis to examine the implementation of standard precautions in hospitals.

BackgroundService providers are often inadequately compliant with standard precaution protocols. This study used bottleneck analysis to identify the weakest link in standard precaution implementation and its associated challenges in hospitals.MethodsBottleneck analysis was conducted in 12 hospitals in Fujian Province, China. In each hospital, a focus group was organized among the key informants to illustrate the sequential steps of standard precaution implementation graphically. The level of difficulty and the specific challenges associated with each step were discussed.ResultsThe sequential activities of standard precaution implementation generally start with making budget for personal protection equipment (PPE), followed by procurement, storage/inventory, in-hospital distribution, in-department distribution, usage/monitoring, and recycling of PPE. Service providers' improper use of PPE was the primary bottleneck. The reasons for improper use of PPE included high workload, time constraints, the sense of wearing PPE would interfere with clinical judgment, and various misconceptions. Making financial planning, recycling, and procurement of PPE were the secondary bottlenecks.ConclusionsBottleneck analysis is useful to illustrate workflow in healthcare systems and pinpoint constraints in standard precaution implementation. Institutional changes, including targeted provider training, adjustment of providers' workloads, and allocation of budget, are suggested strategies to address the identified bottlenecks in standard precaution

Using bottleneck analysis to examine the implementation of standard precautions in hospitals.

BACKGROUND:Service providers are often inadequately compliant with standard precaution protocols. This study used bottleneck analysis to identify the weakest link in standard precaution implementation and its associated challenges in hospitals. METHODS:Bottleneck analysis was conducted in 12 hospitals in Fujian Province, China. In each hospital, a focus group was organized among the key informants to illustrate the sequential steps of standard precaution implementation graphically. The level of difficulty and the specific challenges associated with each step were discussed. RESULTS:The sequential activities of standard precaution implementation generally start with making budget for personal protection equipment (PPE), followed by procurement, storage/inventory, in-hospital distribution, in-department distribution, usage/monitoring, and recycling of PPE. Service providers' improper use of PPE was the primary bottleneck. The reasons for improper use of PPE included high workload, time constraints, the sense of wearing PPE would interfere with clinical judgment, and various misconceptions. Making financial planning, recycling, and procurement of PPE were the secondary bottlenecks. CONCLUSIONS:Bottleneck analysis is useful to illustrate workflow in healthcare systems and pinpoint constraints in standard precaution implementation. Institutional changes, including targeted provider training, adjustment of providers' workloads, and allocation of budget, are suggested strategies to address the identified bottlenecks in standard precaution

New PID Controllers for Step Response of Robot Manipulators with an Uncertain Jacobian Matrix

In framework of PID controllers, there is only three parameters available to time, as a result, performance of the resulting system is always limited As for Cartesian regulation of robot manipulators with uncertain Jacobian matrix, a new scheme of PID controllers is proposed to improve performance for step response. Compare with the traditional PID controllers, a time-dependent integration is employ in this proposed controller to improve performance

Ghrelin through GHSR1a and OX1R heterodimers reveals a Gαs–cAMP-cAMP response element binding protein signaling pathway in vitro

Growth hormone secretagogue receptor 1α (GHSR1a) and Orexin 1 receptor (OX1R) are involved in various important physiological processes, and have many similar characteristics in function and distribution in peripheral tissues and the central nervous system. We explored the possibility of heterodimerization between GHSR1a and OX1R and revealed a signal transduction pathway mechanism. In this study, bioluminescence and fluorescence resonance energy transfer and co-immunoprecipitation (Co-IP) analyses were performed to demonstrate the formation of functional GHSR1a/OX1R heterodimers. This showed that a peptide corresponding to the 5-transmembrane domain of OX1R impaired heterodimer construction. We found that ghrelin stimulated GHSR1a/OX1R heterodimer cells to increase the activation of Gαs protein, compared to the cells that express GHSR1a. Stimulation of GHSR1a/OX1R heterodimers with orexin-A did not alter GPCR interactions with Gα protein subunits. GHSR1a/OX1R heterodimers induced Gαs and downstream signaling pathway activity, including increase of cAMP-response element luciferase reporter activity and cAMP levels. In addition, ghrelin induced a higher proliferation rate in SH-SY5Y cells than in controls. This suggests that ghrelin GHSR1a/OX1R heterodimers promotes an upregulation of a Gαs-cAMP-cAMP-responsive element signaling pathway in vitro and an increase in neuroblastoma cell proliferation

Using conjoint analysis to investigate hospital directors’ preference in adoption of an evidence-based intervention

This study used conjoint analysis, a marketing research technique, to investigate hospital stakeholders' decision-making in adoption of evidence-based interventions (EBI). An efficacious hospital-based stigma-reduction intervention was used as a "product" to study adoption of EBI. Sixty hospital directors in Fujian, China evaluated the likelihood of adopting the EBI in their hospitals by rating across eight hypothetical scenarios with preferred and non-preferred levels of seven attributes, including 1) administrative support, 2) cost, 3) personnel involvement, 4) format, 5) duration, 6) technical support, and 7) priority alignment with the hospital. A hierarchical generalized linear model was fit to the likelihood of intervention adoption for the eight scenarios, with the seven attributes served as independent variables. Monetary cost of intervention implementation (impact score=2.12) had the greatest impact on the directors' reported likelihood of adopting the EBI, followed by duration of the intervention (impact score=0.88), availability of technical support (impact score=0.69), and flexibility of format (impact score=0.36). The impact scores of other attributes were not statistically significant. Conjoint analysis was feasible in modeling hospital directors' decision-making in adoption of EBI. The findings suggested the importance of considering cost, duration, technical support, and flexibility of format in development and dissemination of interventions in healthcare settings