[Molecular mechanisms of quinolone resistance in non-typhoidal Salmonella].

By conducting retrospective analysis, this study aim to investigate the resistance mechanism of quinolones in non-typhoidal Salmonella (NTS). A total of 105 strains of NTS isolated from clinical specimens from the Fifth Affiliated Hospital of Southern Medical University from May 2020 to February 2021 were used as research objects. VITEK2 Compact automatic identification drug sensitivity analysis system and serological test were used to identify the strains. The sensitivity of the strains to ciprofloxacin, levofloxacin and nalidixic acid was detected by AGAR dilution method. The whole genome of 105 strains of NTS was sequenced. Abricate and other softwares were used to analyze drug-resistant genes, including plasmid-mediated quinolone resistance gene (PMQR) and Quinolone resistance determination region (QRDR). Serotypes and ST types were analyzed using SISTR and MLST, and phylogenetic trees were constructed. The results showed that the NTS isolated in this region were mainly ST34 Salmonella typhimurium (53.3%). The drug sensitivity results showed that the drug resistance rates of NTS to ciprofloxacin, levofloxacin and nalidixic acid were 30.4%, 1.9% and 22.0%, respectively, and the intermediate rates of ciprofloxacin and levofloxacin were 27.6% and 54.2%.A total of 46 (74.2%) of the 62 quinolone non-susceptible strains carried the PMQR gene, mainly qnrS1 (80.4%), followed by aac(6')-Ib-cr(15.2%); there were 14 NTS and 8 NTS had gyrA and parC gene mutations, respectively. The gyrA was mutations at the amino acid position 87, Asp87Tyr, Asp87Asn, Asp87Gly, and Thr57Ser mutations were detected in parC. In conclusion, this study found that NTS had relatively high resistance to quinolones, carrying qnrS1 gene mainly resulted in decreased sensitivity of NTS to ciprofloxacin and levofloxacin, and gyrA:87 mutation mainly resulted in NTS resistance to Nalidixic acid; Salmonella typhimurium in clinical isolates showed clonal transmission and required further epidemiological surveillance.

Clinical effectiveness of sodium fluorescein-guided microsurgery in patients with high-grade gliomas.

OBJECTIVE: The aim of this study was to assess the clinical effectiveness of sodium fluorescein-guided microsurgery in patients with high-grade gliomas. PATIENTS AND METHODS: 120 patients with high-grade gliomas who were hospitalized in our Neurosurgery Department from January 2018 to January 2021 were selected and then divided into a control and a study group using the random number table method, with 60 cases in each group. To compare the clinical efficacy of patients in both groups, neuronavigation microsurgery was used in the control group and neuronavigation microsurgery combined with sodium fluorescein-guided microsurgery was used in the study group. RESULTS: The Gross Total Resection Rate (GTRR) of the study group was significantly higher than that of the control group. There was no significant difference in intraoperative bleeding loss or hospital stay between the two groups, and the study group had a much shorter operation time than the control group. The Karnofsky Performance Score (KPS) and the National Institutes of Health Stroke Scale (NIHSS) scores did not significantly differ between the two groups prior to surgery but declined significantly in the study group compared to the control group following treatment. In terms of adverse effects, there was no significant difference between the two groups. In the control group, the median progression-free survival (PFS) was 7.5 months, and the median overall survival (OS) was 9.6 months, whereas in the study group, the median PFS was 9.5 months, and the median OS was 11.5 months. PFS did not significantly differ between the two groups (HR=1.389, 95% CI=0.926-2.085, p=0.079); however, OS was significantly higher in the study group compared to the control group (HR=1.758, 95% CI=1.119-2.762, p=0.013). CONCLUSIONS: Fluorescein-guided microsurgery can dramatically improve total resection rate, postoperative neurological functional status, and overall survival with higher efficacy and safety in patients with high-grade gliomas.

[D-dimer contributes to the diagnosis and prognosis in hepatitis B-related acute-on-chronic liver failure].

Objective: To investigate the diagnostic and prognostic value of D-dimer level in patients with hepatitis B-related acute-on-chronic liver failure (HBV-ACLF). Methods: A total of 142 cases diagnosed with ACLF were randomly selected as research objects in the open cohort using the Chinese Group on the Study of Severe Hepatitis B-ACLF (COSSH-ACLF). Plasma D-dimer levels were compared between patients with ACLF and non-ACLF and patients with different ACLF grades. Survival and death group D-dimer levels were compared with the end points of 28 days and 90 days, respectively. The correlation between D-dimer and other laboratory indicators and prognostic scores were investigated. Area under receiver operating characteristic curve (AUROC) was used to evaluate the D-dimer value for predicting the prognosis of ACLF patients. 125 external ACLF cases were used for validation. A Student t test or Mann-Whitney U test was used to compare continuous measurement data between two groups. Kruskal-Wallis test was used to compare continuous measurement data between multiple groups. Results: Plasma D-dimer levels in the ACLF [2 588.5 (1 142.8, 5 472.8) µg/L] ] and non-ACLF group [1 385.5 (612.0, 3 840.3) µg/L] had a significant difference (P<0.001). ACLF-3 patients had significantly higher D-dimer levels than ACLF-1/2 patients (ACLF-3 vs. ACLF-1, P<0.001; ACLF-3 vs. ACLF-2, P<0.05). Patients who died at 28/90 days had significantly higher D-dimer levels than those whom survived (P<0.001). There was a significant positive correlation between D-dimer level with prothrombin time (PT), international normalized ratio (INR), high-density lipoprotein C, as well as various prognostic scores (COSSH-ACLFs, CLIF-C ACLFs, CLIF-OFs, MELDs). AUROC of D-dimer in predicting the prognosis of ACLF patients at 28 days and 90 days was 0.751 (95% CI: 0.649-0.852) and 0.787 (95% CI: 0.695-0.878), respectively, which did not differ significantly compared with the predictive ability of other scores (P<0.05), and similar results were confirmed by an external validation group of 125 cases. Conclusion: D-dimer level is significantly higher in patients with ACLF, so it is an independent predictor of prognosis at 28 and 90 days.

[Depression symtoms and related factors of fire fighters].

Objective: To analysis the status of the mental health and related factors of fire fighters in Tianjin Binhai New District, and to provide suggestions for their psychological health protection. Methods: 399 fire fighters in Tianjin Binhai New District were selected as study subjects in Jan to April 2019. Depression symptoms were measured by the depression module of the Patient Health Questionnaire (PHQ-9) . The Chinese version of Efrort Reword Imbalance (ERI) Questionnnaire were used to investigate and evaluate their occupational stress. Chi-Square test was used to analysis Categorical data. Binary logistic regression model was used to analysis the ralated factors of depression. Results: Among the 399 fire fighters, 71.1% (280/394) were found high level of depression symptom. The detection rates of depression symptoms in the related influceing factors ERI、station、disease、life pressure、eating habits and sleep disorder occupational stress were difierent (P<0.05) . Sleep disorder, life pressure and ERI occupational stress were risk factors for depressive symptoms (OR=1.921, 95% CI=1.002-3.682; OR=2.852, 95% CI=1.561-5.212; OR=2.367, 95% CI=1.163-4.818, P<0.05) . Conclusion: The rate of depression of fire fighters is relatively higher. Government should pay attention to and take measures to improve the psychological condition of fire fighters.

[CD44 regulates biological behavior and Ras signaling pathway in nasopharyngeal carcinoma stem cells].

Objective: To study the biological behavior of nasopharyngeal carcinoma stem cells and to explore the activation of Ras signaling pathway regulated by CD44. Methods: CNE2-SC and 5-8F-SC were nasopharyngeal carcinoma stem cells and obtained by serum-free suspension culture. Cell counting kit-8 (CCK-8) assay, colony formation assay, Transwell migration assay, cell adhesion array were used to investigate the growth, proliferation, migration and adhesion of nasopharyngeal carcinoma stem cells. Western blot test was used to detect the expressions of Ras signaling pathway related proteins and siRNA-mediated interference was used to determine the activation of Ras signaling pathway regulated by CD44. Results: The growth rates of CNE2-SC and 5-8F-SC cells were significantly lower than those of nasopharyngeal carcinoma cells at 24, 48 and 72 hours after inoculation (P<0.05). After 14 days of implantation, the colony formation rates of CNE2-SC (44.5±1.9)% and 5-8F-SC (47.4±1.8)% were higher than those of CNE2 (34.9±1.5)% and 5-8F (37.2±1.7)%, respectively(P<0.01). The migration cell number of CNE2-SC was (87.6±7.8), 3.97 times higher than that of CNE2 (P<0.01). The migration cell number of 5-8F-SC was (67.2±5.7), 3.07 times higher than 5-8F (P<0.01). The adhesion rates of CNE2-SC and CNE2 cells were (42.1±7.6)% and (8.9±2.0)%, respectively at 3 hours after inoculation and were (82.4±5.0)% and (12.1±2.2)% at 6 hours after inoculation, respectively. The adhesion rate of CNE2-SC cells was higher than that of CNE2 cells (all P<0.01). The adhesion rates of 5-8F-SC and 5-8F cells were (53.6±6.1)% and (7.3±1.5)% at 3 hours after inoculation, and (90.7±3.6)% and (11.0±1.2)% at 6 hours after inoculation, respectively. The adhesion rate of 5-8F-SC cells was higher than that of 5-8F cells (P<0.01). The expression levels of CD44, Ras and N-cadherin were significantly higher, while phosphatase and tensin homolog deleted on chromosome 10 (PTEN), E-cadherin in nasopharyngeal carcinoma stem cells were lower than those of the nasopharyngeal carcinoma cells. Furthermore, the levels of phosphorylated mitogen extracellular kinase1/2 (p-MEK1/2) and phosphorylated extracellular signal-regulated protein kinase1/2 (p-ERK1/2)were significantly increased in nasopharyngeal carcinoma stem cells (P<0.01). Correlation analysis showed that the protein expression levels of CD44 was highly positively correlated with RAS in nasopharyngeal carcinoma stem cells(r=0.985, P=0.002; r=0.962, P=0.038). Deletion of CD44 in CNE2-SC decreased the expression levels of HER-2, Ras and p-ERK1/2, p-Akt and phosphorylated protein kinase C-δ(p-PKCδ) (P<0.01). Conclusions: Despite compare to the nasopharyngeal carcinoma cell, nasopharyngeal carcinoma stem cells grows at a relatively slow rate, the capacities of clone formation, migration, adhesion are promoted. This may be related to the CD44-regulated abnormal activation of Ras signaling pathway.

[Hemodynamic response in cirrhotic patients with transjugular intrahepatic portosystemic shunt].

Objective: To discuss the effects of transjugular intrahepatic portosystemic shunt (TIPS) procedure on hemodynamics in cirrhotic patients. Methods: A total of 23 cirrhotic patients for TIPS insertion were enrolled from January 2018 to October 2018. Serum N-terminal pro-B-type natriuretic peptide (NT-proBNP), transthoracic echocardiography and non-invasive cardiac output measurement based on impedance cardiogram were carried out before and 24h, 1 month, 6 months after TIPS in order to observe cardiac function and hemodynamic changes after TIPS. Results: Significant increases in right atrial area [(17.2±4.0) cm(2) vs. (15.0±3.4) cm(2), P<0.05], right ventricular area [(15.1±3.8) cm(2) vs. (13.7±3.5) cm(2), P<0.05] and left ventricular volume [(97.4±21.5) ml vs. (91.1±22.7) ml, P<0.05] were observed 24 h after TIPS. These changes were accompanied with significant reduction in collapsible index of inferior vena cava [(20.7± 8.1)% vs. (28.6±11.3)%, P<0.01] and elevation in pulmonary arterial systolic pressure [(36.0±8.4) mmHg (1 mmHg=0.133 kPa) vs. (31.8±5.4) mmHg, P<0.01]. There also existed significantly elevated serum NT-proBNP [(551.2±325.1) ng/L vs. (124.2±94.4) ng/L, P<0.01], cardiac output [(5.82±0.96) L/min vs. (5.12±1.28) L/min, P<0.01], cardiac index [(3.47±0.64) L·min(-1)·m(-2) vs. (3.05±0.78) L·min(-1)·m(-2), P<0.01], early diastolic filling rate [(59.0±14.3)% vs. (54.5±11.0)%, P<0.05], and reduced systemic vascular resistance index (SVRi) [(1 798.4±357.3) dyne·s·cm(-5)·m(-2) vs. (2 195.7±508.7) dyne·s·cm(-5)·m(-2), P<0.01] 24 h after TIPS. At the end of 6-month follow-up, all these parameters, but not SVRi, returned towards baseline values. Moreover, peak early to late diastolic tissue velocity ratio at the level of lateral mitral annulus (E'/A') was significantly higher at the end of 6-month follow-up than that at baseline (1.06±0.32 vs. 0.90±0.45, P<0.05). Neither the right ventricular fractional area changes nor the left ventricular ejection fractions during the follow-up period were different from those at baseline (P>0.05). Conclusion: Cirrhotic patients who had no cardiovascular pathologies had adequate adaptation and good compensation ability to reach a new hemodynamic homeostasis for the increased volume load after TIPS insertion.

[Distribution characteristics and correlation analysis of antibody detection value in myasthenia gravis].

Objective: To determine the factors affecting distribution and magnitude of antibody detection value in myasthenia gravis (MG). Methods: A total of 406 MG patients diagnosed at Department of Neurology, Peking University First Hospital from May 2015 to November 2017 were included.All of them exhibited muscle fatigue with decreased response in repetitive nerve stimulation test. There were 200 males and 206 females whose ages ranged from 2 to 85 years old. According to clinical classification of MG recommended by Myasthenia Gravis Foundation of America (MGFA), patients assigned to class I to class V included 200,140, 46, 15 and 5 cases, respectively. There were 33 cases of thymic hyperplasia and 63 cases of thymoma confirmed by radiological or pathological findings. Quantile plots and quantile regression model were used to determine the effects of age, gender and MGFA classification, thymus disease on acetylcholine receptors (AChR)antibody, acetylcholinesterase (AChE) antibody, Titin antibody, ryanodine receptor (RyR) antibody and muscle-specific tyrosine kinase (MuSK) antibody detection values detected by enzyme-linked immunosorbent assay (ELISA). Results: MGFA classification had effects on distribution of AChR antibody level. There was a positive correlation between age and AChR antibody level(P<0.05). Negative correlation was found between age and AChE, Titin and RyR antibody level (P<0.05). No significant correlation was shown between any factors and MuSK antibody level(P≥0.05). MGFA classification had a positive correlation with AChR antibody level (P<0.05) and no correlation with other antibody levels (P>0.05). Gender and thymus disease had no correlation with any tested antibody levels (P>0.05). Conclusion: MGFA classification has significant effects on distribution of AChR antibody level. Age and MGFA classification have positive correlation with AChR antibody level.

Mouse Models for Studying Oral Cancer: Impact in the Era of Cancer Immunotherapy.

Model systems for oral cancer research have progressed from tumor epithelial cell cultures to in vivo systems that mimic oral cancer genetics, pathological characteristics, and tumor-stroma interactions of oral cancer patients. In the era of cancer immunotherapy, it is imperative to use model systems to test oral cancer prevention and therapeutic interventions in the presence of an immune system and to discover mechanisms of stromal contributions to oral cancer carcinogenesis. Here, we review in vivo mouse model systems commonly used for studying oral cancer and discuss the impact these models are having in advancing basic mechanisms, chemoprevention, and therapeutic intervention of oral cancer while highlighting recent discoveries concerning the role of immune cells in oral cancer. Improvements to in vivo model systems that highly recapitulate human oral cancer hold the key to identifying features of oral cancer initiation, progression, and invasion as well as molecular and cellular targets for prevention, therapeutic response, and immunotherapy development.

[Evaluation of a high-intensity fluorescent fluorophage method for diagnosis of drug-resistance in tuberculosis].

Objective: To evaluate the high-intensity green fluorescent protein fluorophage Φ(2)GFP10 method for drug susceptibility testing of tuberculosis for isoniazid(INH), rifampin(RIF), and streptomycin(SM). Methods: A total of 128 clinical M. tuberculosis strains were isolated from patients with suspected drug-resistant tuberculosis visiting Beijing Chest Hospital (Beijing, China) from April to June 2014.All of the isolates were tested by the phage assay, while conventional drug susceptibility tests were performing on Lwenstein-Jensen culture medium as reference. Results: The sensitivities of Φ(2)GFP10 assay for INH, RIF, and SM resistance detection were 100.0%, 98.1%(53/54), and 92.6%(50/54), respectively, while their specificities were 84.8%(56/66), 91.9%(68/74), and 91.9%(68/74), respectively. The agreement between the phage assay and the conventional assay for detecting INH, RIF, and SM resistance was 0.92, 0.95 and 0.92, respectively. The Φ(2) GFP10-phage assay could be done within 2 days for RIF and SM, and 3 days for INH. Conclusions: The Φ(2)GFP10-phage method for drug susceptibility test is very sensitive and specific. The method has the potential to be a valuable, rapid and economical screening method for detecting drug-resistant tuberculosis.

The reliability of the Cutaneous Dermatomyositis Disease Area and Severity Index (CDASI) among dermatologists, rheumatologists and neurologists.

BACKGROUND: Previous studies have shown that skin disease in dermatomyositis (DM) is best assessed using the Cutaneous Dermatomyositis Disease Area and Severity Index (CDASI). Although the CDASI has been validated for use by dermatologists, it has not been validated for use by other physicians such as rheumatologists and neurologists, who also manage patients with DM and assess skin activity in clinical trials. OBJECTIVES: To assess the reliability of the CDASI among dermatologists, rheumatologists and neurologists. METHODS: Fifteen patients with cutaneous DM were assessed using the CDASI and the Physician Global Assessment (PGA) by five dermatologists, five rheumatologists and five neurologists. RESULTS: The mean CDASI activity scores for dermatologists, rheumatologists and neurologists were 21·0, 21·8 and 20·8, respectively. These mean scores were not different among the specialists. The CDASI damage score means for dermatologists, rheumatologists and neurologists were 5·3, 7·0 and 4·8, respectively. The mean scores between dermatologists and rheumatologists were significantly different, but the means between dermatologists and neurologists were not. The intraclass correlation coefficients (ICCs) for interrater reliability for CDASI activity and damage were good to excellent for dermatologists and rheumatologists, and moderate to excellent for neurologists. The ICCs for intrarater reliability for CDASI activity and damage were excellent for dermatologists and rheumatologists and moderate to excellent for neurologists. The PGA displayed lower interrater and intrarater reliability relative to the CDASI. CONCLUSIONS: Our results confirm the reliability of the CDASI when used by dermatologists and rheumatologists. The data for its use by neurologists were not as robust.

Public Perception of Cadaver Organ Donation in Hunan Province, China.

OBJECTIVE: Our aim was to (1) survey public' perception and attitudes toward organ donation and (2) analyze the relationship between knowledge, attitudes, and willingness to donate. METHODS: We developed a questionnaire, and conducted the survey with stratified random sampling. Overall, 600 residents, aged ≥18 who resided in Hunan, and 600 undergraduates from 3 universities in Hunan were surveyed randomly. For this study, 1085 valid questionnaires were completed, with a response rate of 90.4%. RESULTS: Of the 1085 participants, 581 (53.5%) were students, 504 (46.5%) were residents, and 519 (47.8%) were male and 566 (52.2%) female. The mean accuracy rate was 71.96%, and the students' mean accuracy rate was slightly higher than that of the resident population (73.06% vs 70.68%, respectively). The results showed that 82.2% of public support organ donation, and 53.5% were willing to donate their organs after death. Students scored higher than the residents (88% vs 75.6% and 55.6% vs 51.2%). Nearly 1.8% felt that organ donation was against their religion, 14.9% thought it was important to ensure the integrity of the body, 71.7% agreed that organ donation allowed a positive outcome after a person's death, and 61.5% agreed that organ donation represented a continuation of life, to help families cope with grief. Age and gender were related to attitudes. Public knowledge of organ donation and their attitudes were correlated positively (r = 0.666). CONCLUSIONS: Public knowledge of organ donation is poor, biased, and incomplete, and based on television, movies, and communication networks. Positive attitudes toward donation displayed in the surveys were not matched by actual organ donation.

[Clinical value of contrast-enhanced ultrasound in evaluating portal hypertension in patients with decompensated liver cirrhosis].

OBJECTIVE: To investigate the correlation between contrast-enhanced ultrasound parameters and hepatic venous pressure gradient (HVPG), and to develop a new noninvasive method for the evaluation of portal hypertension in patients with decompensated liver cirrhosis. METHODS: One-hundred patients with decompensated liver cirrhosis were examined by contrast-enhanced ultrasound, and the dynamic images were collected for offline analysis. The contrast arrival time was obtained in the hepatic artery (HA), portal vein (PV), and hepatic vein (HV), and HA-HV transit time (HA-HVTT) and PV-HV transit time (PV-HVTT) were calculated. At the same time, HVPG was measured within 24 hours after contrast-enhanced ultrasound, Pearson correlation analysis was performed between each parameter and HVPG, and the receiver operating characteristic (ROC) curve was also used for analysis. RESULTS: HV arrival time (HVAT), HA-HVTT, and PV-HVTT were negatively correlated with HVPG (r = -0.385, -0.409, and -0.572, respectively). The area under the ROC curve (AUROC) was 0.903 for PV-HVTT < 2.5 s in judging HVPG≥ l0 mmHg in patients with decompensated liver cirrhosis, and the sensitivity and specificity were 74.4% and 89.5%, respectively. The AUROC was 0.861 for PV-HVTT < 1.5 s in judging HVPG≥l6 mmHg in these patients, and the sensitivity and specificity were 80.4% and 81.5%, respectively. CONCLUSIONS: HVAT and intrahepatic transit time demonstrate negative linear correlations with HVPG in patients with decompensated liver cirrhosis, and among all parameters, PV-HVTT shows the strongest correlation with HVPG and can be used to determine and predict the severity of portal hypertension.

Gene cloning of porcine adiponectin gene from adipose tissue and construction of its eukaryotic expression vector.

To clone adiponectin (ADPN) gene from Shaziling porcine adipocyte and construct its eukaryotic expression vector, total RNA was extracted from subcutaneous fatty tissue. One pair of specific primers was designed by Primer 5.0 software according to the sequence of ADPN gene of porcine available in GenBank. The ADPN gene was amplified by PCR from cDNA and cloned into pMD18-T vector to construct recombinant clonal vector pMD-ADPN, sequenced and analysed. A recombinant expression plasmid pPICZaA-ADPN was constructed by subcloning the cloned ADPN gene into the linearized pPICZaA vector. Then, the plasmid pPICZaA-ADPN was expressed in Pichia pastoris (GS115) by electrotransformation. Western blot and Bradford analysis were used to determine the target protein induced by methanol. Results showed that the genome size of ADPN was 732 bp and encoded 244 amino acid, the nucleotide sequence of ADPN shared 100% identity with that of porcine available in GenBank. Western blot and Bradford analysis showed that the recombinant ADPN was expressed in GS115 correctly and has certain immune activity. The expression level of ADPN was 28.5 µg/ml. In conclusion, the recombinant ADPN could express in eukaryotic expression vector pPICZaA-ADPN constructed in this study effectively.

Phoenixin: a novel peptide in rodent sensory ganglia.

Phoenixin-14 amide, herein referred to as phoenixin, is a newly identified peptide from the rat brain. Using a previously characterized rabbit polyclonal antiserum against phoenixin, enzyme-immunoassay detected a high level (>4.5 ng/g tissue) of phoenixin-immunoreactivity (irPNX) in the rat spinal cords. Immunohistochemical studies revealed irPNX in networks of cell processes in the superficial dorsal horn, spinal trigeminal tract and nucleus of the solitary tract; and in a population of dorsal root, trigeminal and nodose ganglion cells. The pattern of distribution of irPNX in the superficial layers of the dorsal horn was similar to that of substance P immunoreactivity (irSP). Double-labeling the dorsal root ganglion sections showed that irPNX and irSP express in different populations of ganglion cells. In awake mice, intrathecal injection of phoenixin (1 or 5 µg) did not significantly affect the tail-flick latency as compared to that in animals injected with artificial cerebrospinal fluid (aCSF). Intrathecal administration of phoenixin (0.5, 1.25 or 2.5 µg) significantly reduced the number of writhes elicited by intraperitoneal injection of acetic acid (0.6%, 0.3 ml/30 g) as compared to that in mice injected with aCSF. While not affecting the tail-flick latency, phoenixin antiserum (1:100) injected intrathecally 10 min prior to the intraperitoneal injection of acetic acid significantly increased the number of writhes as compared to mice pre-treated with normal rabbit serum. Intrathecal injection of non-amidated phoenixin (2.5 µg) did not significantly alter the number of writhes evoked by acetic acid. Our result shows that phoenixin is expressed in sensory neurons of the dorsal root, nodose and trigeminal ganglia, the amidated peptide is bioactive, and exogenously administered phoenixin may preferentially suppress visceral as opposed to thermal pain.

Irisin-immunoreactivity in neural and non-neural cells of the rodent.

Irisin is a recently identified myokine secreted from the muscle in response to exercise. In the rats and mice, immunohistochemical studies with an antiserum against irisin peptide fragment (42-112), revealed that irisin-immunoreactivity (irIRN) was detected in three types of cells; namely, skeletal muscle cells, cardiomyocytes, and Purkinje cells of the cerebellum. Tissue sections processed with irisin antiserum pre-absorbed with the irisin peptide (42-112) (1 µg/ml) showed no immunoreactivity. Cerebellar Purkinje cells were also immunolabeled with an antiserum against fibronectin type II domain containing 5 (FNDC5), the precursor protein of irisin. Double-labeling of cerebellar sections with irisin antiserum and glutamate decarboxylase (GAD) antibody showed that nearly all irIRN Purkinje cells were GAD-positive. Injection of the fluorescence tracer Fluorogold into the vestibular nucleus of the rat medulla retrogradely labeled a population of Purkinje cells, some of which were also irIRN. Our results provide the first evidence of expression of irIRN in the rodent skeletal and cardiac muscle, and in the brain where it is present in GAD-positive Purkinje cells of the cerebellum. Our findings together with reports by others led us to hypothesize a novel neural pathway, which originates from cerebellum Purkinje cells, via several intermediary synapses in the medulla and spinal cord, and regulates adipocyte metabolism.

C-peptide of preproinsulin-like peptide 7: localization in the rat brain and activity in vitro.

With the use of a rabbit polyclonal antiserum against a conserved region (54-118) of C-peptide of human preproinsulin-like peptide 7, referred to herein as C-INSL7, neurons expressing C-INSL7-immunoreactivity (irC-INSL7) were detected in the pontine nucleus incertus, the lateral or ventrolateral periaqueductal gray, dorsal raphe nuclei and dorsal substantia nigra. Immunoreactive fibers were present in numerous forebrain areas, with a high density in the septum, hypothalamus and thalamus. Pre-absorption of C-INSL7 antiserum with the peptide C-INSL7 (1 microg/ml), but not the insulin-like peptide 7 (INSL7; 1 microg/ml), also known as relaxin 3, abolished the immunoreactivity. Optical imaging with a voltage-sensitive dye bis-[1,3-dibutylbarbituric acid] trimethineoxonol (DiSBAC4(3)) showed that C-INSL7 (100 nM) depolarized or hyperpolarized a small population of cultured rat hypothalamic neurons studied. Ratiometric imaging studies with calcium-sensitive dye fura-2 showed that C-INSL7 (10-1000 nM) produced a dose-dependent increase in cytosolic calcium concentrations [Ca2+]i in cultured hypothalamic neurons with two distinct patterns: (1) a sustained elevation lasting for minutes; and (2) a fast, transitory rise followed by oscillations. In a Ca2+-free Hanks' solution, C-INSL7 again elicited two types of calcium transients: (1) a fast, transitory increase not followed by a plateau phase, and (2) a transitory rise followed by oscillations. INSL7 (100 nM) elicited a depolarization or hyperpolarization in a small population of hypothalamic neurons, and an increase of [Ca2+]i with two patterns that were dissimilar from that of C-INSL7. [125I]C-INSL7 bindings to rat brain membranes were inhibited by C-INSL7 in a dose-dependent manner; the Kd and Bmax. values were 17.7 +/- 8.2 nM and 45.4 +/- 20.5 fmol/mg protein. INSL7 did not inhibit [125I]C-INSL7 binding to rat brain membranes, indicating that C-INSL7 and INSL7 bind to distinct binding sites. Collectively, our result raises the possibility that C-INSL7 acts as a signaling molecule independent from INSL7 in the rat CNS.

High-aspect-ratio aluminum borate nanowire bundles supported by sucrose.

High-aspect-ratio and single-crystal aluminum borate (Al(18)B(4)O(33)) nanowire bundles with an ordered orientation were synthesized by using an innovative sucrose-assisted growth process. The process involves the dehydration and polycondensation of aluminum borate-sucrose solution to form a highly viscous precursor. The sucrose plays a crucial role in the growth of the nanowire bundles by supporting as a polymeric substrate and a type of adhesive template. Electron microscopy was used to characterize the high-aspect-ratio nanowire bundles. A possible growth mechanism for the nanowire bundles is proposed.

Characterization of the neurotrophic interaction between nerve growth factor and secreted alpha-amyloid precursor protein.

The expression and secretion of amyloid precursor protein (beta APP) is increased in rat cerebral cortices that have been denervated by subcortical lesions of the nucleus basalis of Meynert. The physiological role of the secreted beta APP in response to this injury has not been established. We have previously shown that secreted beta APP produced by alpha-secretase activity (sAPP(alpha)) potentiates the neuritogenic activity of nerve growth factor (NGF) in vitro on naive PC12 cells. In this investigation, we have further characterized the neurotrophic interaction of NGF and sAPP(alpha) using differentiated PC12 cells and rat primary cortical neurons. NGF required the expression of beta APP to maintain a neuronal phenotype. Reduction of endogenous beta APP expression by introduction of antisense oligonucleotides in the presence of NGF resulted in loss of neurites from differentiated PC12 cells but no apparent cell death. Addition of exogenous sAPP(alpha) (60--200 pM) potentiated the protective activity of NGF in serum-deprived differentiated PC12 cells as determined by retention of neurites and cell viability. In addition, exogenous sAPP(alpha) increased neuron viability in both short-term (3 days) cortical neuron cultures grown in the absence of serum and in long-term (9 days) cultures grown with serum. Disruption of the insulin signaling pathway by reduction of IRS-1 expression inhibited the ability of sAPP(alpha) to potentiate neurotrophic activity. These observations suggest that sAPP(alpha) acts as an injury-induced neurotrophic factor that interacts with NGF to enhance neuronal viability using the insulin signaling pathway.

Death of PC12 cells and hippocampal neurons induced by adenoviral-mediated FAD human amyloid precursor protein gene expression.

We used adenoviral-mediated gene transfer of human amyloid precursor proteins (h-APPs) to evaluate the role of various h-APPs in causing neuronal cell death. We were able to infect PC12 cells with very high efficiency because approximately 90% of the cells were cytochemically positive for beta-galactosidase activity when an adenoviral vector containing LacZ cDNA was used to infect cells. Cells infected with adenovirus containing h-APP cDNA showed high-level transcription and expression of h-APP as measured by reverse transcriptase-polymerase chain reaction and Western immunoblot analyses, respectively. Intracellular and extracellular levels of h-APP were elevated approximately 17-and 24-fold in cultures infected with recombinant adenovirus containing wild-type mutant and 13- and 17-fold with V642F mutant. No elevation in h-APP was seen in cultures infected with antisense h-APP or null adenovirus. H-APP levels were maximal 3 days after infection. Overexpression of V642F mutant h-APP in PC12 cells and hippocampal neurons resulted in about a twofold increase in death compared with overexpression of wild-type h-APP. These results demonstrate the usefulness of recombinant adenoviral mediated gene transfer in cell culture studies and suggest that overexpression of a familial Alzheimer's disease mutant APP may be toxic to neuronal cells.

Effects of PS1 deficiency on membrane protein trafficking in neurons.

We have examined the trafficking and metabolism of the beta-amyloid precursor protein (APP), an APP homolog (APLP1), and TrkB in neurons that lack PS1. We report that PS1-deficient neurons fail to secrete Abeta, and that the rate of appearance of soluble APP derivatives in the conditioned medium is increased. Remarkably, carboxyl-terminal fragments (CTFs) derived from APP and APLP1 accumulate in PS1-deficient neurons. Hence, PS1 plays a role in promoting intramembrane cleavage and/or degradation of membrane-bound CTFs. Moreover, the maturation of TrkB and BDNF-inducible TrkB autophosphorylation is severely compromised in neurons lacking PS1. We conclude that PS1 plays an essential role in modulating trafficking and metabolism of a selected set of membrane and secretory proteins in neurons.