Accumulation of Prion Triggers the Enhanced Glycolysis via Activation of AMKP Pathway in Prion-Infected Rodent and Cell Models.

Mitochondrial dysfunction is one of the hallmarks in the pathophysiology of prion disease and other neurodegenerative diseases. Various metabolic dysfunctions are identified and considered to contribute to the progression of some types of neurodegenerative diseases. In this study, we evaluated the status of glycolysis pathway in prion-infected rodent and cell models. The levels of the key enzymes, hexokinase (HK), phosphofructokinase (PFK), and pyruvate kinase (PK) were significantly increased, accompanying with markedly downregulated mitochondrial complexes. Double-stained IFAs revealed that the increased HK2 and PFK distributed widely in GFAP-, Iba1-, and NeuN-positive cells. We also identified increased levels of AMP-activated protein kinase (AMPK) and the downstream signaling. Changes of AMPK activity in prion-infected cells by the AMPK-specific inhibitor or activator induced the corresponding alterations not only in the downstream signaling, but also the expressions of three key kinases in glycolysis pathway and the mitochondrial complexes. Transient removal or complete clearance of prion propagation in the prion-infected cells partially but significantly reversed the increases of the key enzymes in glycolysis, the upregulation of AMPK signaling pathway, and the decreases of the mitochondrial complexes. Measurements of the cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) showed lower OCR and higher ECAR in prion-infected cell line, which were sufficiently reversed by clearance of prion propagation. Those data indicate a metabolic reprogramming from oxidative phosphorylation to glycolysis in the brains during the progression of prion disease. Accumulation of PrPSc is critical for the switch to glycolysis, largely via activating AMPK pathway.

Spontaneous prion disease in homozygous and heterozygous transgenic mouse models of T188K genetic Creutzfeldt-Jakob disease.

Genetic Creutzfeldt-Jakob disease with T188K mutation (T188K gCJD) is the most frequent genetic prion disease in China. To explore the penetration of T188K mutation and the pathogenesis of T188K gCJD, we constructed 2 lines of transgenic mouse models: homozygous Tg188K+/+ mice containing T188K mutation in 2 alleles of human PRNP background and heterozygous Tg188K+/- mice containing 1 allele of T188K human PRNP and 1 allele of the wild-type mouse PRNP. Spontaneous neurological illnesses were identified in all Tg188K mice at their old ages (750-800 days old). About half of the Tg188K mice died prior to the final observation (930 days old). Extensive spongiosis, PrPSc deposit, and reactive gliosis of astrocytes and microglia are neuropathologically identified, showing time-dependent exacerbation. Proteinase K-resistant PrP was detected in the brain, muscle, and intestine tissues, and positive real-time quaking-induced conversion reactions were elicited by the brain and muscle tissues of Tg188K mice. Those data verify that the constructed Tg188K mice highly mimic the clinicopathology of human T188K gCJD, strongly indicating the pathogenicity of T188K mutated PrP.

Aberrant SENP1-SUMO-Sirt3 Signaling Causes the Disturbances of Mitochondrial Deacetylation and Oxidative Phosphorylation in Prion-Infected Animal and Cell Models.

Post-translational modifications of proteins, such as acetylation and SUMOylation, play important roles in regulation of protein functions and pathophysiology of different diseases including neurodegenerative diseases. Our previous studies have identified aberrant acetylation profiles and reduced deacetylases Sirt3 and Sirt1 in the brains of prion-infected mouse models. In this study, we have found that the levels of acetylated forms of AceCS2 and LCAD, the key enzymes regulating lipid metabolism, CS and IHD2, the key enzymes regulating complete oxidative metabolism, GDH, the key enzyme regulating the oxidative decomposition of glutamate into the tricarboxylic acid (TCA) cycle, and NDUFA9, the essential component in the complex I of respiratory chain activity, were significantly upregulated in the prion-infected animal and cell models, along with the decrease of Sirt3 activity and mitochondrial cytochrome c oxidase activity. Meanwhile, the increases of SUMO1 modifications and SUMO1-Sirt3 and decrease of SENP1 were identified in the brains and the cultured cells with prion infections. Removal of prion propagation in the cultured cells partially, but significantly, reversed the aberrant situations. Moreover, similar abnormal phenomena were also observed in the cultured 293 T cells transiently expressing cytosolic form PrP (Cyto-PrP), including decreased SENP1, increased SUMO1, decreased Sirt3 activity, increased acetylated forms of the key enzymes, and decreased cytochrome c oxidase activity. Attenuation of the accumulation of Cyto-PrP by co-expression of the p62 protein sufficiently diminished those abnormalities. The data here strongly indicate that deposits of prions in brains or accumulations of Cyto-PrP in cells trigger dysregulation of the SENP1-SUMO1-Sirt pathway and subsequently induce aberrant mitochondrial deacetylation and the mitochondrial respiratory chain.

Proteomics profiling for the global and acetylated proteins of papillary thyroid cancers.

BACKGROUND: Papillary thyroid carcinoma (PTC) is the most common endocrine malignancy cancer among the malignancies of thyroid. Despite of wide usages of proteomics in PTC, the profile of acetylated proteins in PTC remains unsettled, which is helpful for understanding the carcinogenesis mechanism and identifying useful biomarkers for PTC. METHODS: The surgically removed specimens of cancer tissues (Ca-T) and adjacent normal tissues (Ca-N) from 10 female patients pathological diagnosed as PTC (TNM stage III) were enrolled in the study. After preparing the pooled extracts of the whole proteins and the acetylated proteins from 10 cases, TMT labeling and LC/MS/MS methods were applied to the assays of global proteomics and acetylated proteomics separately. Bioinformatics analysis, including KEGG, gene ontology (GO) and hierarchical clustering were performed. Some differentially expressed proteins (DEPs) and differentially expressed acetylated proteins (DEAPs) were validated by individual Western blots. RESULTS: Controlled with the normal tissues adjacent to the lesions, 147 out of 1923 identified proteins in tumor tissues were considered as DEPs in global proteomics, including 78 up-regulated and 69 down-regulated ones, while 57 out of 311 identified acetylated proteins in tumor tissues were DEAPs in acetylated proteomics, including 32 up-regulated and 25 down-regulated, respectively. The top 3 up- and down-regulated DEPs were fibronectin 1, KRT1B protein and chitinase-3-like protein 1, as well as keratin, type I cytoskeletal 16, A-gamma globin Osilo variant and Huntingtin interacting protein-1. The top 3 up- and down-regulated DEAPs were ribosomal protein L18a-like protein, alpha-1-acid glycoprotein 2 and eukaryotic peptide chain release factor GTP-binding subunit ERF3A, as well as trefoil factor 3, thyroglobulin and histone H2B. Functional GO annotation and KEGG pathway analysis based on the DEPs and DEAPs showed completely different changing pictures. Contrary to the top 10 up- and -down regulated DEPs, most of which were addressed in PTC and other types of carcinomas, changes of the majority DEAPs were not mentioned in the literatures. CONCLUSIONS: Taken the profiling of the global and acetylated proteomics together will provide more broad view of protein alterations on the carcinogenesis and new direction for selecting biomarker for diagnosis of PTC.

Global Profiles of Acetylated Proteins in Brains of Scrapie Agents 139A- and ME7-Infected Mice Collected at Mid-Early, Mid-Late, and Terminal Stages.

Objective: To describe the global profiles of acetylated proteins in the brains of scrapie agents 139A- and ME7-infected mice collected at mid-early, mid-late, and terminal stages. Methods: The acetylated proteins from the cortex regions of scrapie agent (139A- and ME7)-infected mice collected at mid-early (80 days postinfection, dpi), mid-late (120 dpi), and terminal (180 dpi) stages were extracted, and the global profiles of brain acetylated proteins were assayed with proteomic mass spectrometry. The proteins in the infected mice showing 1.5-fold higher or lower levels than that of age-matched normal controls were considered as differentially expressed acetylated peptides (DEAPs). Results: A total of 118, 42, and 51 DEAPs were found in the brains of 139A-80, 139A-120, and 139A-180 dpi mice, respectively. Meanwhile, 390, 227, and 75 DEAPs were detected in the brains of ME7-80, ME7-120, and ME7-180 dpi mice, respectively. The overwhelming majority of DEAPs in the mid-early stage were down-regulated, and more portions of DEAPs in the mid-late and late stages were up-regulated. Approximately 22.1% (328/1,485) of acetylated peptides mapped to 74 different proteins were mitochondrial associated. Kyoto Encyclopedia of Genes and Genomes pathway analysis identified 39 (80 dpi), 13 (120 dpi), and 10 (180 dpi) significantly changed pathways in 139A-infected mice. Meanwhile, 55, 25, and 18 significantly changed pathways were observed in the 80, 120, and 180 dpi samples of 139A- and ME7-infected mice ( P < 0.05), respectively. Six pathways were commonly involved in all tested samples. Moreover, many steps in the citrate cycle (tricarboxylic acid cycle) were affected, represented by down-regulated acetylation for relevant enzymes in the mid-early stage and up-regulated acetylation in the mid-late and late stages. Conclusion: Our data here illustrated the changes in the global profiles for brain acetylated proteins during prion infection, showing remarkably inhibited acetylation in the early stage and relatively enhanced acetylation in the late stage.

Enhanced M-CSF/CSF1R Signaling Closely Associates with PrPSc Accumulation in the Scrapie-Infected Cell Line and the Brains of Scrapie-Infected Experimental Rodents.

Activation and proliferation of microglia are one of the hallmarks of prion disease and is usually accompanied by increased levels of various cytokines and chemokines. Our previous study demonstrated that the level of brain macrophage colony-stimulating factor (M-CSF) was abnormally elevated during prion infection, but its association with PrPSc is not completely clear. In this study, colocalization of the increased M-CSF with accumulated PrPSc was observed by IHC with serial brain sections. Reliable molecular interaction between total PrP and M-CSF was observed in the brain of 263 K-infected hamsters and in cultured prion-infected cell line. Immunofluorescent assays showed that morphological colocalization of M-CSF with neurons and microglia, but not with astrocytes in brains of scrapie-infected animals. The transcriptional and expressing levels of CSF1R were also significantly increased in prion-infected cell line and mice, and colocalization of CSF1R with neurons and microglia was observed in the brains of prion-infected mouse models. Removal of PrPSc replication by resveratrol in SMB-S15 cells induced limited reductions of cellular levels of M-CSF and CSF1R. In addition, we found that the level of IL-34, another ligand of CSF1R, did not change significantly after prion infection, but its distribution on the cell types in the brains shifted from neurons in healthy mice to the proliferated astrocytes and microglia in scrapie-infected mice. Our data demonstrate activation of M-CSF/IL-34/CSF1R signaling in the microenvironment of prion infection, strongly indicating its vital role in the pathophysiology of prions. It provides solid scientific evidence for the therapeutic potential of inhibiting M-CSF/CSF1R signaling in prion diseases.

Application of α-Syn Real-Time Quaking-Induced Conversion for Brain and Skin Specimens of the Chinese Patients With Parkinson's Disease.

The real-time quaking-induced conversion (RT-QuIC) assay has been developed and used as an in vitro diagnostic tool for Parkinson's disease (PD). In this study, we established α-Syn RT-QuIC using recombinant human α-Syn as the substrate. All 5 brain homogenates of neuropathological PD cases and 13 skin homogenates of clinical PD cases showed positive results, whereas all the samples of negative controls remain negative. Meantime, randomly selected 6 skin samples of PD cases and 6 skin samples of sCJD cases showed negative in opposite prion RT-QuIC and α-Syn RT-QuIC. Our α-Syn RT-QuIC showed dose-dependent manner between the lag times and peak ThT fluorescent values. Additionally, the detecting limitation was about 10-7 dilution for brain tissues and 10-6 for skins. Those data indicate a reliable specificity and good sensitivity of the established α-Syn RT-QuIC in identifying and amplifying the misfolded α-Syn in brain and skin tissues of patients with PD.

Different Aberrant Changes of mGluR5 and Its Downstream Signaling Pathways in the Scrapie-Infected Cell Line and the Brains of Scrapie-Infected Experimental Rodents.

Metabotropic glutamate receptor subtype 5 (mGluR5) is a G-protein-coupled receptor found widely in the central nervous system. It has been involved in the development and progression of some neurodegenerative diseases, but its role in prion diseases is rarely described. In this study, the changes of mGluR5 and its downstream signaling pathways in prion-infected cell line SMB-S15 and the brains of scrapie-infected experimental rodents were evaluated by various methodologies. We found the levels of mGluR5 were significantly increased in a prion-infected cell line SMB-S15 and the cultured cells transiently express an abnormal form PrP (Cyto-PrP). Using immunoprecipitation tests and immunofluorescent assays (IFA), molecular interaction and morphological colocalization between PrP and mGluR5 were observed in the cultured cells. We identified that the (GPCRs)-IP3-IP3R-Ca2+ pathway was activated and the levels of the downstream kinases p38, ERK, and JNK were increased in SMB-S15 cells. After treated with mGluR5 antagonist (MTEP) or the removal of prion replication by resveratrol in SMB-S15 cells, the upregulations of mGluR5 and the downstream kinases were restored in a certain degree. Moreover, increased mGluR5 contributes to the cell damage in prion-infected cells. Contrarily, the levels of mGluR5 in the brains of several scrapie-infected rodent models were decreased at terminal stage. IFA of the brain sections of scrapie-infected rodents demonstrated that the signals of mGluR5 were preferentially colocalized with the NeuN-positive cells, accompanying with severe neuron losses in Nissl staining, which might be a reason for the decrease of mGluR5. Our data indicate the different aberrant alterations of mGluR5 and the downstream signaling pathways during prion infection in vivo and in vitro.

Accumulation of Prion and Abnormal Prion Protein Induces Hyperphosphorylation of α-Synuclein in the Brain Tissues from Prion Diseases and in the Cultured Cells.

Prion disease (PrD) and Parkinson's disease (PD) are neurodegenerative diseases characterized by aggregation of misfolded proteins in brain tissues, including protease-resistant prion protein (PrPSc) in PrD and α-synuclein in PD. In recent years, overlap of these two proteins has attracted increased attention, and cross-seeding of prion proteins by aggregated α-synuclein has been proposed. However, the changes in α-synuclein after prion infection are still unclear. In this study, we showed that α-synuclein expression was significantly decreased in the brains of prion-infected rodent models, in the SMB-S15 cell line, which exhibits persistent prion replication, and in the brains of humans with PrDs. Meanwhile, α-synuclein phosphorylated at serine 129(p(S129)-α-synuclein) was significantly increased in the brains of scrapie-infected mice and prion-infected SMB-S15 cells. The increased p(S129)-α-synuclein colocalized with GFAP- and NeuN-positive cells in the brains of scrapie-infected mice. p(S129)-α-synuclein was also observed in the cytoplasm of SMB-S15 and HEK-293 cells transiently expressing an abnormal form of prion protein (Cyto-PrP). Molecular interactions between PrP and α-synuclein were detected in recombinant proteins, normal and prion-infected brain tissues, and cultured cells. The increased p(S129)-α-synuclein colocalized with PrP signals from prion-infected SMB-S15 and HEK-293 cells expressing Cyto-PrP. Moreover, increased morphological colocalization of p(S129)-α-synuclein with mitochondrial markers was also detected in the two cell types. Our results indicate that prion replication and accumulation in cells and brains induce hyperphosphorylation of α-synuclein, particularly at S129, which may aggravate mitochondrial damage and facilitate α-synuclein aggregation in the central nervous system tissues from PrDs.

Genetic Prion Disease: Insight from the Features and Experience of China National Surveillance for Creutzfeldt-Jakob Disease.

Human genetic prion diseases (gPrDs) are directly associated with mutations and insertions in the PRNP (Prion Protein) gene. We collected and analyzed the data of 218 Chinese gPrD patients identified between Jan 2006 and June 2020. Nineteen different subtypes were identified and gPrDs accounted for 10.9% of all diagnosed PrDs within the same period. Some subtypes of gPrDs showed a degree of geographic association. The age at onset of Chinese gPrDs peaked in the 50-59 year group. Gerstmann-Sträussler-Scheinker syndrome (GSS) and fatal familial insomnia (FFI) cases usually displayed clinical symptoms earlier than genetic Creutzfeldt-Jakob disease (gCJD) patients with point mutations. A family history was more frequently recalled in P105L GSS and D178N FFI patients than T188K and E200K patients. None of the E196A gCJD patients reported a family history. The gCJD cases with point mutations always developed clinical manifestations typical of sporadic CJD (sCJD). EEG examination was not sensitive for gPrDs. sCJD-associated abnormalities on MRI were found in high proportions of GSS and gCJD patients. CSF 14-3-3 positivity was frequently detected in gCJD patients. Increased CSF tau was found in more than half of FFI and T188K gCJD cases, and an even higher proportion of E196A and E200K gCJD patients. 63.6% of P105L GSS cases showed a positive reaction in cerebrospinal fluid RT-QuIC. GSS and FFI cases had longer durations than most subtypes of gCJD. This is one of the largest studies of gPrDs in East Asians, and the illness profile of Chinese gPrDs is clearly distinct. Extremely high proportions of T188K and E196A occur among Chinese gPrDs; these mutations are rarely reported in Caucasians and Japanese.

[Platelet-rich plasma combined with core decompression and bone grafting in the treatment of non traumatic necrosis of femoral head in ARCO stageâ ¡].

OBJECTIVE: To observe the clinical effect of platelet rich plasma (PRP) combined with ß tricalcium phosphate bioceramic bone in the treatment of non traumatic necrosis of the femoral head in ARCO stageⅡ. METHODS: From January 2017 to December 2018, 100 patients (160 hips) with ARCO stageⅡnon traumatic necrosis of the femoral head were divided into PRP group and control group. In PRP group, 50 patients (80 hips), 22 males and 28 females, aged from 18 to 65 (43.47± 7.23) years, with a course of 4 to 18 (15.8±2.9) months, underwent core decompression and bone grafting combined with PRP implantation. There were 50 cases (80 hips) in the control group, including 27 males and 23 females, aged 20 to 63 (45.72± 7.43) years, and the course of disease was 6 to 19 (14.9±3.8) months. Hip X-ay film was followed up after operation. Harris score and VAS score were used to evaluate the curative effect, and the survival rate of hip joint was recorded. RESULTS: All patients had good wound healing, no infection, thrombosis and other complications. All patients were followed up for 12 to 14 (12.0±0.4) months. Twelve months after operation, the image expression of PRP group was better than that of control group(P<0.05). Harris hip score and VAS score of pain at twelve months after operation were 89.98±6.17 and 1.68±1.02 in PRP group and 81.62±5.62 and 2.52±1.13 in control group, respectively. The survival rate of 96.25% in PRP group was significantly higher than 86.25% in control group. The postoperative score of two groups was higher than that before operation(P<0.05), but PRP group was better than control group at any time point statistical significance (P<0.05). CONCLUSION: Platelet-rich plasma(PRP) combined with artificialbone for core decompression and bone grafting can change the situation of simple artificial bone implantation and uncertain curative effect, improve the success rate of this operation, effectively reduce the collapse rate of femoral head necrosis in the early and middle stage, delay or even avoid hip replacement.

Preparation of PrP-specific Polyclonal Antibody via Immunization of PRNP-knockout Mice with Recombinant Human PrP Protein.

OBJECTIVE: The definite diagnosis of human and animal prion diseases depends on the examination of special pathological changes and/or detection of PrP Sc in the brain tissues of suspected cases. Thus, developing methods to obtain PrP antibody with good specificity and sensitivity is fundamental for prion identification. METHODS: We prepared a PrP-specific polyclonal antibody (pAb P54) in a PRNP-knockout mouse model via immunization with recombinant full-length human PrP protein residues 23-231. Thereafter, we verified that pAb in Western blot, immunohistochemistry (IHC), and immunofluorescent (IFA) assays. RESULTS: Western blot illustrated that the newly prepared pAb P54 could react with recombinant PrP protein, normal brain PrP C from healthy rodents and humans, and pathological PrP Sc in the brains of experimental rodents infected with scrapie and humans infected with different types of prion diseases. The electrophoretic patterns of brain PrP C and PrP Sc observed after their reaction with pAb P54 were nearly identical to those produced by commercial PrP monoclonal antibodies. Three glycosylated PrP molecules in the brain homogenates were clearly demonstrated in the reactions of these molecules with pAb P54. IHC assay revealed apparent PrP deposits in the GdnCl-treated brain slices of 139A-infected mice and 263K-infected hamsters. IFA tests with pAb P54 also showed clear green signals surrounding blue-stained cell nuclei. CONCLUSION: The newly prepared pAb P54 demonstrated reliable specificity and sensitivity and, thus, may have potential applications not only in studies of prion biology but also in the diagnosis of human and experimental rodent prion diseases.

Generation and characterization of two strains of transgene mice expressing chimeric MiniSOG-MusPrP.

BACKGROUND: Although the presences of scrapie associated fibril in the brain tissues is a ultrastructural hallmark for prion diseases, the exact morphological structure of prion during the progression of the disease is still unclear. The host prion protein (PrP) is encoded by PrP gene (PRNP) locating on the chromosome 20 in human and the chromosome 2 in mouse. Recently, a novel correlative light and electron microscopy with Mini Singlet Oxygen Generator (miniSOG) was generated. MiniSOG, a small protein of 106 amino acids, can absorb blue light and emit green fluorescence that is detectable under the fluorescence microscope. MiniSOG can also partially catalyze the polymerization of DAB to form black stained structures in the presence of osmium tetroxide, which is able to be observed under transmission electron microscope. NEW METHODS: Two kinds of miniSOG-PrP expressing recombinant plasmids were generated. Correlative photooxidation and transmission electron microscope were used to detect these plasmids. The plasmids were microinjected into fertilized FVB/NJ eggs and Tg mice expressing miniSOG-PrP fusion proteins were selected after successive bred withPRNP KO Tg mice. RESULTS: Those two strains of Tg mice, TgSOG23 and Tg231SOG, developed normally and maintained healthy without detectable abnormality after one-year observation. Western blots and immunohistochemical assays with PrP- and miniSOG-specific antibodies confirmed that the chimeric miniSOG-PrP proteins were expressed in the brain tissues of Tg mice. Digital PCR assays proposed that the copy numbers of the inserted external gene in TgSOG23 and Tg231SOG were 2 and 12, respectively. COMPARISON WITH EXISTING METHOD(S): Compared with GFP tag miniSOG is significantly smaller, which makes it easy be operated experimentally and possibly has less influence on the biological function of the labeled protein. Additionally, GFP tag is an ideal marker for immunofluorescent assays, but may not be suitable for ultrastructural assays for prion morphology. CONCLUSION: Those Tg mice may supply novel and useful experimental animals for further study on the potential morphological structure formation and deposits of prion in the brain tissues during prion infection.

Enhanced Mitophagy Activity in Prion-Infected Cultured Cells and Prion-Infected Experimental Mice via a Pink1/Parkin-Dependent Mitophagy Pathway.

Mitophagy is an important process for removing damaged mitochondria in cells, the dysfunction of which has been directly linked to an increasing number of neurodegenerative disorders. However, the details of mitophagy in prion diseases still need to be deeply explored. In this study, we identified more autophagosomes and large swelling mitochondria structures in the prion-infected cultured cell line SMB-S15 by transmission electron microscopy, accompanying the molecular evidence of activated autophagic flux. Western blots illustrated that the levels of Pink1 and Parkin, particularly in the mitochondrial fraction, were increased in SMB-S15 cells, whereas the levels of mitochondrial membrane proteins TIMM44, TOMM20, and TIMM23 were decreased. The amount of whole polyubiquitinated proteins decreased, but that of phosphor-polyubiquitinated proteins increased in SMB-S15 cells. The level of MFN2 in SMB-S15 cells were down-regulated, but its polyubiquitinated form was up-regulated. Knockdown of the expressions of Pink1 and Parkin by the individual SiRNAs in SMB-S15 cells reduced autophagic activity but did not seem to influence the expressions of TOMM20 and TIMM23. Moreover, we also demonstrated that the brain levels of Pink1 and Parkin in the mice infected with scrapie strains 139A and ME7 were remarkably increased at the terminal stage of the disease by Western blot and immunohistochemical (IHC) assays. Immunofluorescent assays revealed that Pink1 signals widely colocalized with GAFP-, Iba1-, and NeuN-positive cells in the brains of scrapie-infected mice. IHC assays with serial sections of the brain tissues infected with agents 139A and ME7 showed more Pink1- and Parkin-positive cells located at the areas with more PrPSc deposit. These results suggest an activated mitophagy in prion-infected cells and prion-infected experimental mice, probably via an enhanced Pink-Parkin pathway.

Association of serum adiponectin level with cystatin C in male patients with obstructive sleep apnea syndrome.

PURPOSE: Obstructive sleep apnea syndrome (OSAS) was suggested to exert an effect on renal function. However, the specific mechanism was still unknown. We try to find the association among OSAS, adiponectin, and cystatin C and the effect of adiponectin on renal function in OSAS patients. METHODS: Seventeen healthy men and seventy-three men which only had OSAS were included in the end. Apnea-hypopnea index (AHI), oxygen desaturation index (ODI), the percentage of total sleep time spent with SpO2 < 90% (T90%), lowest O2 saturation (LaSO2), Epworth Sleepiness Scale (ESS) score, serum adiponectin, and high-sensitive C-reactive protein (hsCRP) were detected in all subjects, and renal function was evaluated with creatinine, cystatin C, and estimated glomerular filtration rate (eGFR). RESULTS: Demographic data, creatinine, and eGFR did not differ among the studied groups. Decreased serum adiponectin levels were associated with severe OSAS. OSAS patients had a higher hsCRP and cystatin C than those without OSAS. Serum adiponectin levels had a negative association with cystatin C. After adjusted for confounders, adiponectin, hsCRP, and ODI had a significant prediction on the cystatin C (ß = - 0.218, p = 0.011; ß = 0.226, p = 0.037; and ß = 0.231, p = 0.029). CONCLUSIONS: Decreased serum adiponectin was associated with increased cystatin C in male OSAS patients. These results suggest that serum adiponectin might be a regulatory factor for renal function in OSAS.

Engineering Responses to Amino Acid Substitutions in the VP0- and VP3-Coding Regions of PanAsia-1 Strains of Foot-and-Mouth Disease Virus Serotype O.

The presence of sequence divergence through adaptive mutations in the major capsid protein VP1, and also in VP0 (VP4 and VP2) and VP3, of foot-and-mouth disease virus (FMDV) is relevant to a broad range of viral characteristics. To explore the potential role of isolate-specific residues in the VP0 and VP3 coding regions of PanAsia-1 strains in genetic and phenotypic properties of FMDV, a series of recombinant full-length genomic clones were constructed using Cathay topotype infectious cDNA as the original backbone. The deleterious and compensatory effects of individual amino acid substitutions at positions 4008 and 3060 and in several different domains of VP2 illustrated that the chain-based spatial interaction patterns of VP1, VP2, and VP3 (VP1-3), as well as between the internal VP4 and the three external capsid proteins of FMDV, might contribute to the assembly of eventually viable viruses. The Y2079H site-directed mutants dramatically induced a decrease in plaque size on BHK-21 cells and viral pathogenicity in suckling mice. Remarkably, the 2079H-encoding viruses displayed a moderate increase in acid sensitivity correlated with NH4Cl resistance compared to the Y2079-encoding viruses. Interestingly, none of all the 16 rescued viruses were able to infect heparan sulfate-expressing CHO-K1 cells. However, viral infection in BHK-21 cells was facilitated by utilizing non-integrin-dependent, heparin-sensitive receptor(s) and replacements of four uncharged amino acids at position 3174 in VP3 of FMDV had no apparent influence on heparin affinity. These results provide particular insights into the correlation of evolutionary biology with genetic diversity in adapting populations of FMDV.IMPORTANCE The sequence variation within the capsid proteins occurs frequently in the infection of susceptible tissue cultures, reflecting the high levels of genetic diversity of FMDV. A systematic study for the functional significance of isolate-specific residues in VP0 and VP3 of FMDV PanAsia-1 strains suggested that the interaction of amino acid side chains between the N terminus of VP4 and several potential domains of VP1-3 had cascading effects on the viability and developmental characteristics of progeny viruses. Y2079H in VP0 of the indicated FMDVs could affect plaque size and pathogenicity, as well as acid sensitivity correlated with NH4Cl resistance, whereas there was no inevitable correlation in viral plaque and acid-sensitive phenotypes. The high affinity of non-integrin-dependent FMDVs for heparin might be explained by the differences in structures of heparan sulfate proteoglycans on the surfaces of different cell lines. These results may contribute to our understanding of the distinct phenotypic properties of FMDV in vitro and in vivo.

The Slt2-MAPK pathway is involved in the mechanism by which target of rapamycin regulates cell wall components in Ganoderma lucidum.

The fungal cell wall is very important for cell growth and survival during stress, and the target of rapamycin (TOR) pathway plays a major role in regulating cell growth in response to environmental cues. Ganoderma lucidum is an important edible and medicinal fungus, and the function of TOR in this organism remains unclear. As shown in the present study, the TOR pathway regulates cell wall integrity (CWI) in G. lucidum. Inhibition of TOR signaling by RNA interference (RNAi) or rapamycin treatment reduced the growth of G. lucidum mycelia, increased contents of the cell wall components chitin and ß-1,3-glucan, and increased cell wall thickness. Furthermore, inhibition of TOR signaling enhanced the relative level of phosphorylated Slt2, a member of the MAPK cascade involved in CWI signaling. Moreover, when treated with rapamycin, significantly lower chitin and ß-1,3-glucan contents were observed in Slt2-silenced strains than in WT strains, indicating that TOR regulates the synthesis of these cell wall components through the Slt2-MAPK pathway. These results indicate a potential relationship between TOR signaling and CWI signaling. Additionally, participation of Slt2-MAPK in TOR-mediated regulation of cell wall component production has not previously been reported in a microorganism.

Accelerated crystallization of magnetic 4A-zeolite synthesized from red mud for application in removal of mixed heavy metal ions.

To cope with the increasing environmental issues of red mud, an integrated technological route for its comprehensive utilization was developed through the extraction of valuable components and the synthesis of magnetic 4A-zeolite. To accelerate the crystallization process of the synthesized 4A-zeolite, sodium chloride (NaCl) was innovatively employed under hydrothermal treatment. The effects of various parameters, including mass ratio of red mud/NaOH, alkali fusion temperature, alkali fusion time and molar ratio of NaCl/Al2O3, were systematically investigated. The results showed that approximately 81.0% Al, 76.1% Si and 95.8% Fe were utilized from red mud using alkali fusion and acid leaching methods. The optimal conditions of the alkali fusion process were determined as: mass ratio of red mud/NaOH = 1/2, alkali fusion temperature of 800 °C, and time of 90 min. Furthermore, when the molar ratio of NaCl/Al2O3 was kept at 1.5, the crystallization time reduced from 240 min to 150 min, and particle size distributions narrowed from 20-100 µm to 1-10 µm. The practical applications in removal of mixed heavy metal ions (Zn2+, Cu2+, Cd2+, Ni2+, and Pb2+) from wastewater indicated that the as-synthesized magnetic 4A-zeolite is a promising candidate for heavy metals adsorption.

Bioactivity and structure-activity relationship of cinnamic acid derivatives and its heteroaromatic ring analogues as potential high-efficient acaricides against Psoroptes cuniculi.

A series of cinnamic acid derivatives and its heteroaromatic ring analogues were synthesized and evaluated for acaricidal activity in vitro against Psoroptes cuniculi, a mange mite. Among them, eight compounds showed the higher activity with median lethal concentrations (LC50) of 0.36-1.07mM (60.4-192.1µg/mL) and great potential for the development of novel acaricidal agent. Compound 40 showed both the lowest LC50 value of 0.36mM (60.4µg/mL) and the smallest median lethal time (LT50) of 2.6h at 4.5mM, comparable with ivermectin [LC50=0.28mM (247.4µg/mL), LT50=8.9h], an acaricidal drug standard. SAR analysis showed that the carbonyl group is crucial for the activity. The type and chain length of the alkoxy in the ester moiety and the steric hindrance near the ester group significantly influence the activity. The esters were more active than the corresponding thiol esters, amides, ketones or acids. Replacement of the phenyl group of cinnamic esters with α-pyridyl or α-furanyl significantly increase the activity. Thus, a series of cinnamic esters and its heteroaromatic ring analogues with excellent acaricidal activity emerged.

14-3-3 proteins are involved in growth, hyphal branching, ganoderic acid biosynthesis, and response to abiotic stress in Ganoderma lucidum.

Ganoderma lucidum, which contains many pharmacologically active compounds, is regarded as a traditional medicinal fungus. Nevertheless, the scarcity of basic research limits the commercial value and utilization of G. lucidum. As a class of highly conserved, phosphopeptide-binding proteins present in all eukaryotes, 14-3-3 proteins play vital roles in controlling multiple physiological processes, including signal transduction, primary metabolism, and stress responses. However, knowledge of the roles of 14-3-3 proteins in Basidiomycetes is sparse. In this article, two homologs of 14-3-3 proteins, encoded by the two distinct genes GlBmh1 and GlBmh2, were distinguished in G. lucidum. We found that GlBmh1 and GlBmh2 were expressed at various developmental stages, including in vegetative mycelium cultivated on solid medium and in primordia and fruiting bodies. Moreover, we constructed GlBmh1 single-silenced strains, GlBmh2 single-silenced strains, and 14-3-3 double-silenced mutants for further study. When GlBmh1 and GlBmh2 were inhibited by RNA interference, the growth rate of mycelia was decreased, and the distance between the aerial hyphal branches was reduced; responses to various abiotic stresses such as oxidants and cell wall and osmotic stressors were also changed. Furthermore, the contents of secondary metabolite ganoderic acids (GAs) were increased after GlBmh1 and GlBmh2 were simultaneously silenced. Taken together, we provide evidence that implicates potential roles for the two 14-3-3 proteins in affecting growth and GA biosynthesis, thereby providing new insights into the basic functions of 14-3-3 proteins in G. lucidum.