Netrin-1 and its receptor DCC modulate survival and death of dopamine neurons and Parkinson's disease features.

The netrin-1/DCC ligand/receptor pair has key roles in central nervous system (CNS) development, mediating axonal, and neuronal navigation. Although expression of netrin-1 and DCC is maintained in the adult brain, little is known about their role in mature neurons. Notably, netrin-1 is highly expressed in the adult substantia nigra, leading us to investigate a role of the netrin-1/DCC pair in adult nigral neuron fate. Here, we show that silencing netrin-1 in the adult substantia nigra of mice induces DCC cleavage and a significant loss of dopamine neurons, resulting in motor deficits. Because loss of adult dopamine neurons and motor impairments are features of Parkinson's disease (PD), we studied the potential impact of netrin-1 in different animal models of PD. We demonstrate that both overexpression of netrin-1 and brain administration of recombinant netrin-1 are neuroprotective and neurorestorative in mouse and rat models of PD. Of interest, we observed that netrin-1 levels are significantly reduced in PD patient brain samples. These results highlight the key role of netrin-1 in adult dopamine neuron fate, and the therapeutic potential of targeting netrin-1 signaling in PD.

Manual compression technique improves the success rate in the treatment of thrombosed aneurysmal arteriovenous fistula: A single-center experience.

OBJECTIVE: Endovascular intervention for thrombosed aneurysmal arteriovenous fistula (AVF) is still a challenge. Manual compression technique (MCT)-assisted angioplasty may be helpful, but there is no evidence or data to support it. METHODS: From January 2018 to May 2021, patients with thrombosed aneurysmal AVFs were retrospectively enrolled. The patients were separated into the MCT group or the traditional group according to the procedure received. Technical failure, clinical failure, 90-day patency, and safety were analyzed. RESULTS: A total of 159 cases (64 ± 12 years old, 60% male) were enrolled, of which 87 cases received MCT and 72 underwent traditional angioplasty. No technical failure was observed in the MCT group, while five technical failures were observed in the traditional group (0% vs. 7%, p = 0.02). There were no differences in the clinical failure rate (3% vs. 7%, p = 0.30), 90-day patency rate, or procedure time between the MCT group and the traditional group. There was no symptomatic pulmonary embolism or other complication in the two groups. CONCLUSION: MCT is a low-cost, less invasive, and safe procedure for thrombosed aneurysmal AVF, and it achieves a higher technical success rate than traditional angioplasty.

The cytoprotective protein MANF promotes neuronal survival independently from its role as a GRP78 cofactor.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER)-stress-regulated protein exhibiting cytoprotective properties through a poorly understood mechanism in various in vitro and in vivo models of neuronal and non-neuronal damage. Although initially characterized as a secreted neurotrophic factor for midbrain dopamine neurons, MANF has recently gained more interest for its intracellular role in regulating the ER homeostasis, including serving as a cofactor of the chaperone glucose-regulated protein 78 (GRP78). We aimed for a better understanding of the neuroprotective mechanisms of MANF. Here we show for the first time that MANF promotes the survival of ER-stressed neurons in vitro as a general unfolded protein response (UPR) regulator, affecting several UPR pathways simultaneously. Interestingly, MANF does not affect naïve neurons. We hypothesize that MANF regulates UPR signaling toward a mode more compatible with neuronal survival. Screening of MANF interacting proteins from two mammalian cell lines revealed a conserved interactome of 15 proteins including several ER chaperones such as GRP78, GRP170, protein disulfide isomerase family A member 1, and protein disulfide isomerase family A member 6. Further characterization confirmed previously published finding that MANF is a cofactor of GRP78 interacting with its nucleotide binding domain. Using microscale thermophoresis and nuclear magnetic resonance spectroscopy, we discovered that MANF is an ATP binding protein and that ATP blocks the MANF-GRP78 interaction. Interestingly, functional analysis of the antiapoptotic properties of MANF mutants in cultured neurons revealed divergent roles of MANF as a GRP78 cofactor and as an antiapoptotic regulator of UPR. We conclude that the co-factor type interaction with GRP78 is dispensable for the survival-promoting activity of MANF in neurons.

CDNF Interacts with ER Chaperones and Requires UPR Sensors to Promote Neuronal Survival.

Cerebral dopamine neurotrophic factor (CDNF) is a neurotrophic factor that has beneficial effects on dopamine neurons in both in vitro and in vivo models of Parkinson's disease (PD). CDNF was recently tested in phase I-II clinical trials for the treatment of PD, but the mechanisms underlying its neuroprotective properties are still poorly understood, although studies have suggested its role in the regulation of endoplasmic reticulum (ER) homeostasis and the unfolded protein response (UPR). The aim of this study was to investigate the mechanism of action of CDNF through analyzing the involvement of UPR signaling in its anti-apoptotic function. We used tunicamycin to induce ER stress in mice in vivo and used cultured primary neurons and found that CDNF expression is regulated by ER stress in vivo and that the involvement of UPR pathways is important for the neuroprotective function of CDNF. Moreover, we used AP-MS and BiFC to perform the first interactome screening for CDNF and report novel binding partners of CDNF. These findings allowed us to hypothesize that CDNF protects neurons from ER-stress-inducing agents by modulating UPR signaling towards cell survival outcomes.

Theoretical Study of a Two-Photon Fluorescent Probe Based on Nile Red Derivatives with Controllable Fluorescence Wavelength and Water Solubility.

Hypochloric acid (HOCl) plays a vital role in the natural defense system, but abnormal levels of it can cause cell damage, accelerated human aging, and various diseases. It is of great significance to develop new probes for detecting HOCl in biosystems nondestructively and noninvasively. The purpose of this work is to explore new chemical modification strategies of two-photon excitation fluorescence (TPEF) probes to improve the poor water solubility and low efficiency in imaging applications. Nil-OH-6 has a two-photon absorption cross-section value as high as 243 GM and attains a good quantum yield of 0.49. In addition, the modification of terminal groups with different azetidine-heterospirocycles or N,N-dialkyl fused amino groups to Nile Red can effectively improve the fluorescence efficiency as well as increase the solubility to some extent. This study provides some strategies to simultaneously improve the fluorescence performance and solubility of these two-photon probes and, hence, reliable guidance and a foundation for the subsequent synthesis of TPEF probes based on Nile Red.

The dependence receptor TrkC triggers mitochondria-dependent apoptosis upon Cobra-1 recruitment.

The neurotrophin receptor TrkC was recently identified as a dependence receptor, and, as such, it triggers apoptosis in the absence of its ligand, NT-3. The molecular mechanism for apoptotic engagement involves the double cleavage of the receptor's intracellular domain, leading to the formation of a proapoptotic "killer" fragment (TrkC KF). Here, we show that TrkC KF interacts with Cobra1, a putative cofactor of BRCA1, and that Cobra1 is required for TrkC-induced apoptosis. We also show that, in the developing chick neural tube, NT-3 silencing is associated with neuroepithelial cell death that is rescued by Cobra1 silencing. Cobra1 shuttles TrkC KF to the mitochondria, where it promotes Bax activation, cytochrome c release, and apoptosome-dependent apoptosis. Thus, we propose that, in the absence of NT-3, the proteolytic cleavage of TrkC leads to the release of a killer fragment that triggers mitochondria-dependent apoptosis via the recruitment of Cobra1.

Small-Molecule Inhibitors of the RNA M6A Demethylases FTO Potently Support the Survival of Dopamine Neurons.

The fat mass and obesity-associated protein (FTO), an RNA N6-methyladenosine (m6A) demethylase, is an important regulator of central nervous system development, neuronal signaling and disease. We present here the target-tailored development and biological characterization of small-molecule inhibitors of FTO. The active compounds were identified using high-throughput molecular docking and molecular dynamics screening of the ZINC compound library. In FTO binding and activity-inhibition assays the two best inhibitors demonstrated Kd = 185 nM; IC50 = 1.46 µM (compound 2) and Kd = 337 nM; IC50 = 28.9 µM (compound 3). Importantly, the treatment of mouse midbrain dopaminergic neurons with the compounds promoted cellular survival and rescued them from growth factor deprivation induced apoptosis already at nanomolar concentrations. Moreover, both the best inhibitors demonstrated good blood-brain-barrier penetration in the model system, 31.7% and 30.8%, respectively. The FTO inhibitors demonstrated increased potency as compared to our recently developed ALKBH5 m6A demethylase inhibitors in protecting dopamine neurons. Inhibition of m6A RNA demethylation by small-molecule drugs, as presented here, has therapeutic potential and provides tools for the identification of disease-modifying m6A RNAs in neurogenesis and neuroregeneration. Further refinement of the lead compounds identified in this study can also lead to unprecedented breakthroughs in the treatment of neurodegenerative diseases.

Exploring factors influencing the retention of nurses in a religious hospital in Taiwan: a cross-sectional quantitative study.

BACKGROUND: Long-term deficits in the nursing labor force and high turnover rates are common in the Taiwanese medical industry. Little research has investigated the psychological factors associated with the retention of nursing staff. However, in practice, religious hospitals often provide nursing staff with education in medicine or the medical humanities to enhance their psychological satisfaction. The objective of this study was to explore factors influencing nursing staff retention in their work in relation to different levels of needs. A further objective was to investigate whether medical humanities education was associated with the retention of nursing staff. METHODS: This study used self-administrated questionnaires to survey nurses working in northern areas of Taiwan. The questionnaire design was based on the six levels of Maslow's hierarchy of needs. Participation was voluntary, and the participants signed informed consent documents. Self-administrated questionnaires were distributed to a total of 759 participants, and 729 questionnaires were returned (response rate 96.04%). Logistic regression analysis was used to estimate the impact of seniority on nurses' reported intention to stay after adjustment for nurse characteristics (gender and age). RESULTS: In the Pearson correlation analysis, nurses' willingness to stay was moderately correlated with "physical needs", "safety needs", "love and belonging needs", and "esteem needs" (r = 0.559, P < 0.001; r = 0.533, P < 0.001; r = 0.393, P < 0.001; and r = 0.476, P < 0.001, respectively). Furthermore, nurses' willingness to stay was highly correlated with "self-actualization needs", "beyond self-actualization needs" and "medical humanities education-relevant needs" (r = 0.707, P < 0.001; r = 0.728, P < 0.001; and r = 0.678, P < 0.001, respectively). We found that the odds ratios (ORs) of retention of nursing staff with less than 1 year (OR = 4.511, P = 0.002) or 1-3 years (OR = 3.248, P = 0.003) of work experience were significantly higher than that of those with 5-10 years of work experience. CONCLUSIONS: With regard to medical humanities education, we recommend adjusting training, as the compulsory activities included in the official programs are inadequate, and adjusting the number of required hours of medical humanities education. Tailoring different educational programs to different groups (especially nurses who have worked 3-5 years or 5-10 years in the case study hospital) might improve acceptance by nursing staff.

[Selection of q RT-PCR reference genes for Amomum tsaoko seeds during dormancy release].

Freshly collected seeds of Amomum tsaoko demonstrate obvious dormancy. Therefore, the selection of stable reference genes during seed dormancy release is very important for the subsequent functional research of related genes. In this study, ten commonly used reference genes(GAPDH, 40S, actin, tubulin, EIF4A-9, EIF2α, UBC, UBCE2, 60S, and UBQ) were selected as candidates for quantitative Real-time polymerase chain reaction(qRT-PCR) of the embryo samples of A. tsaoko at different dormancy release stages. Three kinds of software(BestKeeper, geNorm, and Normfinder) and the Delta CT method were used to evaluate the expression stability of the candidate reference genes, and the RefFinder online tool was employed to integrate the results and generate a comprehensive ranking. The results showed that the expression levels of the ten candidate reference genes differed greatly in different embryo samples. GAPDH and UBC had high expression levels, as manifested by the small Ct values. GeNorm identified 40S and UBCE2 as the most stable genes. NormFinder ranked EIF2α as the most stable gene and UBC as the least stable gene. UBCE2 was found to be the most stable gene and actin the least stable one by BestKeeper. Delta CT analysis suggested that the expression of 40S was most stable. UBCE2 was recommended as the most stably expressed gene by RefFinder. Thus, UBCE2 is the ideal reference gene for qRT-PCR analysis of A. tsaoko seeds at different dormancy release stages. The results may lay a foundation for analyzing the expression of related genes during seed dormancy release of A. tsaoko.

[Genetic diversity and antibacterial activity of endophytic fungi from Zanthoxylum nitidum].

In order to reveal the distribution and population characteristics of endophytic fungi from Zanthoxylum nitidum and the antibacterial potential,this study performed molecular identification and analyzed the genetic diversity and antibacterial activity of endophytic fungi from Z. nitidum in Guangxi. Through culture and molecular identification,35 strains,belonging to 15 genera,12 families,10 orders,4 classes,and 2 phyla,were isolated from various tissues of Z. nitidum,of which Colletotrichum and Fusarium were the dominant genera,respectively accounting for 20% of total strains. The diversity of endophytic fungi was significantly different among roots,stems,and leaves,as manifested by the significantly higher Shannon index( H') in stems( 1. 678) than in roots( 0. 882 1) and leaves( 0. 515 4). The antimicrobial activity analysis showed that 14. 28% of endophytic fungi inhibited at least one indicator pathogen. Among them,Fusarium sp. ZN-34 and Fusarium sp. ZN-26 separately demonstrated the strongest inhibitory effect on Escherichia coli and Staphylococcus aureus. In general,Fusarium sp. ZN-26 and Phialemoniopsis plurioloculosa ZN-35 were advantageous in suppressing the two bacteria owing to the broad spectrum and strong efficacy. In summary,Z. nitidum in Guangxi boasts rich endophytic fungi with the majority showing strong antibacterial activity,which can be used as candidates for the extraction and separation of basic antibacterial substances and the development of natural antibacterial agents.

[Effects of exogenous IBA and fungal elicitor on growth of in vitro roots culture of Dysosma versipellis and production of podophyllotoxin].

Using the White as basic medium, the effects of the exogenous IBA and endophytic fungal elicitor on the growth of in vitro roots cultures of Dysosma versipellis and production of podophyllotoxin were investigated in this study. The results showed that the IBA and the endophytic fungus Zasmidium syzygii elicitor could increase the content of podophyllotoxin of in vitro roots of D. versipellis after 3 weeks. The White medium added with 3 mg·L~(-1) IBA induced the highest increase of podophyllotoxin(1 830.86 µg·g~(-1)), which was 2.07 folds greater than the control, and followed by 1.5 mg·L~(-1) IBA, fungal elicitor, 1 mg·L~(-1) IBA, 0.5 mg·L~(-1) IBA and 4.5 mg·L~(-1) IBA, which was 1.82, 1.71, 1.63, 1.43 and 1.1 folds greater than the control, respectively. The results also showed that the growth of roots was certain positively correlated with the change of IBA concentration. Therefore, 3 mg·L~(-1) IBA was the most suitable for the production of podophyllotoxin in the in vitro roots of D. versipellis, and the stimulating effect of Z. syzygii fungal elicitor was between 1.5 mg·L~(-1) and 1 mg·L~(-1) IBA, which was a potential natural elicitor to induce the accumulation of podophyllotoxin in future production.

[Inhibitory effect of endophytic fungi from Dysosma versipellis on HIV-1 IN-LEDGF/p75 interaction].

To determine the inhibitory effect of endophytic fungi from Dysosma versipellis on HIV-1 IN-LEDGF/p75 interaction,the protein-protein interaction between human immunodeficiency virus type 1( HIV-1) integrase and lens epithelial growth factor p75 protein( LEDGF/p75) was used as a target. The homogeneous time-resolved fluorescence( HTRF) technique was used in the inhibitory activity assay. The results showed that eight endophytic fungi with anti-IN-LEDGF/p75 interaction activity were screened out from fifty-three strains with different morphological characteristic. Among them,106 strain showed strong inhibitory activity against HIV-1 IN-LEDGF/p75 interaction with IC50 value of 5. 23 mg·L-1,and was identified as a potential novel species of Magnaporthaceae family by the analyses of ITS-rDNA,LSU and RPB2 sequences data. This study demonstrated that potential natural active ingredients against the HIV-1 IN-LEDGF/p75 interaction exist in the endophytic fungi of D. versipellis. These results may provide available candidate strain resources for the research and development of new anti-acquired immunodeficiency syndrome drugs.

Theoretical design and investigation of 1,8-naphthalimide-based two-photon fluorescent probes for detecting cytochrome P450 1A with separated fluorescence signal.

As a type of enzyme with a terminal oxygen, the CYP1A subfamily possesses the ability to catalyze the reactions of many environmental toxins, endogenous substrates and clinical drugs. The development of efficient methods for the rapid and real-time detection of CYP1A enzyme activity in complex biological systems is of considerable significance for identifying potential abnormalities in these cancer-related enzymes. With this goal, we firstly provided a series of 1,8-naphthalimide-based two-photon fluorescent chromophores with large two-photon absorption (TPA) cross-sections (500-7000 GM) and remarkable changes in fluorescence spectra upon recognizing the CYP1A enzyme from its theoretical aspect. Moreover, we have thoroughly studied the effects of cyclic acceptor (dichlorobenzene and benzothiadiazole) and donor (fluorene and carbazole) groups on the one-photon absorption (OPA), TPA, and fluorescence properties of CYP1A enzyme probes and the corresponding reaction products. The connection of a heterocycle as the donor group to a 1,8-naphthalimide-based molecule to form a D-π-A-π-D-type electronic structure can effectively cause red shifts in the absorption and emission wavelengths to facilitate bioimaging in the near infrared (NIR) region, which is attributed to the lower transition energy, larger transition dipole moment and amount of transferred charge. Docking analysis suggests that the two-photon fluorescent probes NCMN-3 and NCMN-5 that were designed will guarantee and achieve excellent selectivity for the CYP1A enzyme.

Effect of External Electric Field on Substrate Transport of a Secondary Active Transporter.

Substrate transport across a membrane accomplished by a secondary active transporter (SAT) is essential to the normal physiological function of living cells. In the present research, a series of all-atom molecular dynamics (MD) simulations under different electric field (EF) strengths was performed to investigate the effect of an external EF on the substrate transport of an SAT. The results show that EF both affects the interaction between substrate and related protein's residues by changing their conformations and tunes the timeline of the transport event, which collectively reduces the height of energy barrier for substrate transport and results in the appearance of two intermediate conformations under the existence of an external EF. Our work spotlights the crucial influence of external EFs on the substrate transport of SATs and could provide a more penetrating understanding of the substrate transport mechanism of SATs.

[Analysis phylogenetic relationship of Gynostemma (Cucurbitaceae)].

The sequences of ITS, matK, rbcL and psbA-trnH of 9 Gynostemma species or variety including 38 samples were compared and analyzed by molecular phylogeny method. Hemsleya macrosperma was designated as outgroup. The MP and NJ phylogenetic tree of Gynostemma was built based on ITS sequence, the results of PAUP phylogenetic analysis showed the following results: (1) The eight individuals of G. pentaphyllum var. pentaphyllum were not supported as monophyletic in the strict consensus trees and NJ trees. (2) It is suspected whether G. longipes and G. laxum should be classified as the independent species. (3)The classification of subgenus units of Gynostemma plants is supported.

[Determination of yogliptin and its metabolite in Wistar rat plasma by liquid chromatography-tandem mass spectrometry].

A rapid, sensitive and simple liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed for the simultaneous determination of yogliptin and its metabolite in Wistar rat plasma. Linagliptin and dexamethasone were chosen as the internal standards of yogliptin and its metabolite, (R)-8-(3-hydroxypiperidine- -yl)-7-(but-2-yn-1-yl)-1-((5-fluorobenzo[d]thiazol-2-yl)methyl)-3-methyl- H-purine-2, 6 (3H, 7H)-dione, respectively. After a simple protein precipitation using acetonitrile as the precipitating solvent, both analytes and ISs were separated on a Grace Altima HP C18 column (2.1 mm x 50 mm, 5 microm) with gradient elution using methanol (containing 0.1% formic acid, 4 mmol x L(-1) ammonium acetate)-0.1% formic acid (containing 4 mmol x L(-1) ammonium acetate) as the mobile phase. A chromatographic total run time of 4.4 min was achieved. Mass spectrometric detection was conducted with electrospray ionization under positive-ion and multiple-reaction monitoring modes. Linear calibration curves for yogliptin and its metabolite were over the concentration range of 0.5 to 500 ng x mL(-1) with a lower limit of quantification of 0.5 ng x mL(-1). The intra- and inter- assay precisions were all below 14%, the accuracies were all in standard ranges. The method was used to determine the concentration of yogliptin and M1 in Wistar rat plasma after a single oral administration of yogliptin (27 mg x kg(-1)). The method was proved to be selective, sensitive and suitable for pharmacokinetic study of yogliptin and M1 in Wistar rat plasma.

[Analysis of critical genes expression of chlorogenic acid and luteolin biosyntheses in Lonicera confusa].

This study analysed the tissue specific expression of critical genes involved in chlorogenic acid and luteolin biosynthesis, for exploiting the molecular mechanism of components biosynthesis in Lonicera confusa. Expression of PAL, 4CL, C4H, CHS, CHI, FNS and HQT gene families of chlorogenic acid and luteolin biosynthesis-related genes in buds and leaves of L. confusa were analyed by Real-time PCR. Expressions of PAL1, C4H1, 4CL1, CHS1, CHI3 and HQT2 in buds were lower than that in leaves, and expressions of PAL3, 4CL2, CHI2 and FNS2 in buds were higher than that in leaves. The results indicated that that PAL3 and 4CL2 may be associated with accumulation of chlorogenic acid, and the expression patterns of PAL1, CHS1, CHI3 and HQT2 in buds and leaves of L. confusa were different with L. japonica. This study provided some theoretical basis for the further research on genetic mechanism of active components differences in L. confusa and L. japonica.

[Microscopic observation on mycorrhiza of rare herb Dysosma versipellis].

Endophytic fungi played an important role in the growth of its host plant. To investigate the mycorrhizal characteristics and the distribution of fungi in the root, an endangered wild plant-Dysosma versipellis was collected and observed by electron microscope. The results showed that the host was closely associated with endophytic fungi. The fungi were mainly distributed in the epidermis and cortex. The aseptate and septate fungi with swollen hyphae were observed in some cell of the cortex. The result provides a reference for the study of mycorrhizal structure of Dysosma genus and the interaction between the fungi and its host.

MANF regulates neuronal survival and UPR through its ER-located receptor IRE1α.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER)-located protein with cytoprotective effects in neurons and pancreatic ß cells in vitro and in models of neurodegeneration and diabetes in vivo. However, the exact mode of MANF action has remained elusive. Here, we show that MANF directly interacts with the ER transmembrane unfolded protein response (UPR) sensor IRE1α, and we identify the binding interface between MANF and IRE1α. The expression of wild-type MANF, but not its IRE1α binding-deficient mutant, attenuates UPR signaling by decreasing IRE1α oligomerization; phosphorylation; splicing of Xbp1, Atf6, and Txnip levels; and protecting neurons from ER stress-induced death. MANF-IRE1α interaction and not MANF-BiP interaction is crucial for MANF pro-survival activity in neurons in vitro and is required to protect dopamine neurons in an animal model of Parkinson's disease. Our data show IRE1α as an intracellular receptor for MANF and regulator of neuronal survival.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) has a unique mechanism to rescue apoptotic neurons.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) protects neurons and repairs the Parkinson disease-like symptoms in a rat 6-hydroxydopamine model. We show a three-dimensional solution structure of human MANF that differs drastically from other neurotrophic factors. Remarkably, the C-terminal domain of MANF (C-MANF) is homologous to the SAP domain of Ku70, a well known inhibitor of proapoptotic Bax (Bcl-2-associated X protein). Cellular studies confirm that MANF and C-MANF protect neurons intracellularly as efficiently as Ku70.