The hypothalamic steroidogenic pathway mediates susceptibility to inflammation-evoked depression in female mice.

BACKGROUND: Depression is two-to-three times more frequent among women. The hypothalamus, a sexually dimorphic area, has been implicated in the pathophysiology of depression. Neuroinflammation-induced hypothalamic dysfunction underlies behaviors associated with depression. The lipopolysaccharide (LPS)-induced mouse model of depression has been well-validated in numerous laboratories, including our own, and is widely used to investigate the relationship between neuroinflammation and depression. However, the sex-specific differences in metabolic alterations underlying depression-associated hypothalamic neuroinflammation remain unknown. METHODS: Here, we employed the LPS-induced mouse model of depression to investigate hypothalamic metabolic changes in both male and female mice using a metabolomics approach. Through bioinformatics analysis, we confirmed the molecular pathways and biological processes associated with the identified metabolites. Furthermore, we employed quantitative real-time PCR, enzyme-linked immunosorbent assay, western blotting, and pharmacological interventions to further elucidate the underlying mechanisms. RESULTS: A total of 124 and 61 differential metabolites (DMs) were detected in male and female mice with depressive-like behavior, respectively, compared to their respective sex-matched control groups. Moreover, a comparison between female and male model mice identified 37 DMs. We capitalized on biochemical clustering and functional enrichment analyses to define the major metabolic changes in these DMs. More than 55% of the DMs clustered into lipids and lipid-like molecules, and an imbalance in lipids metabolism was presented in the hypothalamus. Furthermore, steroidogenic pathway was confirmed as a potential sex-specific pathway in the hypothalamus of female mice with depression. Pregnenolone, an upstream component of the steroid hormone biosynthesis pathway, was downregulated in female mice with depressive-like phenotypes but not in males and had considerable relevance to depressive-like behaviors in females. Moreover, exogenous pregnenolone infusion reversed depressive-like behaviors in female mice with depression. The 5α-reductase type I (SRD5A1), a steroidogenic hub enzyme involved in pregnenolone metabolism, was increased in the hypothalamus of female mice with depression. Its inhibition increased hypothalamic pregnenolone levels and ameliorated depressive-like behaviors in female mice with depression. CONCLUSIONS: Our study findings demonstrate a marked sexual dimorphism at the metabolic level in depression, particularly in hypothalamic steroidogenic metabolism, identifying a potential sex-specific pathway in female mice with depressive-like behaviors.

Routine hematological parameters are associated with short- and long-term prognosis of patients with ischemic stroke.

BACKGROUND: Previous studies indicated that some routine hematological parameters are associated with the prognosis of ischemic stroke (IS), but none of study has evaluated them simultaneously. The aim of this study was to investigate the prognostic value of routine hematological parameters in IS patients. METHODS: Using medical record database, we retrospectively reviewed the patients with IS admitted in Gansu Province Hospital between June 2014 and July 2015. The prognostic value of routine hematological parameters on admission was analyzed using logistic regression model, receiver operating characteristic (ROC) curve analysis and Cox proportional hazards model. RESULTS: Patients with hospital mortality had significantly higher white blood cell (WBC), neutrophil, neutrophil to lymphocyte ratio (NLR), red blood cell distribution width (RDW) and National Institutes of Health Stroke Scale (NIHSS), while their lymphocyte, monocyte, and eosinophil were significantly lower. The area under ROC curve (AUC) for eosinophil, neutrophil, WBC, RDW, NLR, monocyte, and lymphocyte were 0.74 (95% CI, 0.67-0.82), 0.76 (95% CI, 0.67-0.84), 0.72 (95% CI, 0.64-0.81), 0.65 (95% CI, 0.56-0.73), 0.76 (95% CI, 0.68-0.84), 0.67 (95% CI, 0.59-0.76), and 0.75 (95% CI, 0.67-0.83), respectively. In a multivariable logistical regression model, only WBC, NLR, and NIHSS were independently associated with hospital mortality. In a multivariable model, age, NIHSS, RDW, NLR, and eosinophil were independent prognostic factors for all-cause mortality. CONCLUSION: Red blood cell distribution width, NLR and eosinophil are independent prognostic factors for IS.

[Antigenic Characterization of Immunodominant Epitopes of Alpha-8 giardin in Giardia lamblia].

Objective: To identify the dominant epitopes of α-8 giardin in Giardia lamblia, and analyze their immunoreactivity. Methods: Three dominant epitopes G1（7-17 aa）, G2（30-40 aa） and G3（296-306 aa） were predicted for α-8 giardin by bioinformatic analysis using DNAstar, Biosun and CLUSTAL W softwares. Each epitope was conjugated to keyhole limpethemocyanin (KLH) and then used to immunize female Chinchilla rabbits via multi-point（8-10 points） subcutaneous injections on the back（0.5 mg conjugate/rabbit, 100 µl at each point）. Then the rabbits were boosted（0.2 mg conjugate/rabbit） on days 14, 21 and 28 after the first immunization. Blood was collected prior to each immunization, and carotid artery blood（50 ml） was collected on day 7 after the last immunization. The antibody titer for anti-KLH-G1, anti-KLH-G2 and anti-KLH-G3 sera was determined with indirect ELISA. The reactivity of the three anti-sera with recominant giardin (rGiardin) was analyzed with Western blotting. Results: The concentration of the purified conjugates KLH-G1, KLH-G2, and KLH-G3 was 0.66, 0.95 and 0.25 mg/ml， respectively. ELISA showed that the antibody titer for anti-KLH-G1, anti-KLH-G2 and anti-KLH-G3 sera was 1∶12800, 1∶51200, 1∶51200, respectively. SDS-PAGE revealed a specific band at M(r) 36 000 for the three anti-sera. Western blotting showed that the three anti-sera all specifically recognized rGiardin. Conclusion: Immunization with the KLH-G1, KLH-G2, and KLH-G3 conjugates induced production of specific IgG antibody in rabbits, which can recognize rGiardin.

[Prokaryotic Expression and Immunoreactivity Analysis of α-8 Giardin in Giardia lamblia].

OBJECTIVE: To clone and express α8-giardin gene of Giardia lamblia, and analyze its immunoreactivity. METHODS: The open reading frame (ORF) of α8-giardin gene was amplified by PCR. The PCR product was cloned into prokaryotic expression vector pET-30a (+) with restriction enzymes EcoR I and Xho I . The recombinant vector pET30a (+)-α8-giardin was transformed into E. coli BL21 (DE3), and the positive clones were then selected. The constructed pET30 a (+)-α8-giardin was induced with IPTG for expression, and purified through Ni-affinity chromatography. The recombinant protein was examined by SDS-PAGE and Western blotting. RESULTS: The length of α8-giardin gene was 930 bp. PCR and restriction enzyme digestion analysis confirmed the construction of recombinant plasmid pET30a (+)-α8-giardin. SDS-PAGE and Western blotting analysis showed that the recombinant protein rGiardin (about M, 36,000) was expressed in E. coli as inclusion body protein, and reacted positively with anti-His tag antibody and rabbit anti-G. lamblia serum. CONCLUSION: The recombinant plasmid pET30a (+)-α8-giardin is constructed, and the purified rGiardin protein shows immunoreactivity.

[Construction of GCV-specific hammerhead ribozyme recombinant vector of alpha-8 giardin in Giardia lamblia].

OBJECTIVE: To construct a GCV-ribozyme recombinant vectors of alpha-8 giardin in Giardia lamblia. METHODS: The secondary structure of alpha-8 giardin mRNA (GenBank Accession No. AY781323) was analyzed with the RNA draw software. According to the proportion of G:C and principles of designing hammerhead ribozyme, suitable ribozyme cleavage points were chosen. A specific antisense-hammerhead ribozyme (H8) was designed and synthesized. The ribozyme was cloned into Giardia canis virus (GCV) vector to construct a recombinant viral vector-pGCV634/H8/1423. The vector was linearized and transcript into the trophozoites of G. lamblia by electroporation method. The alpha-8 giardin mRNA level of the transfectants and normal trophozoites were analyzed 24 h after electroporation by RT-PCR. RESULTS: The recombinant vector of GCV-specific hammerhead ribozyme of alpha-8 giardin in Giardia lamblia (pGCV634/H8/ 1423) was constructed. RT-PCR assays showed the ribozyme (H8) mRNA can be detected 24h after transfection and alpha-8 giardin mRNA was cleaved effectively by ribozyme (H8) intracellularly. CONCLUSION: pGCV634/H8/1423 can transfect Giardia trophozoites and cleave mRNA of alpha-8 giardin intracellularly.

[Construction of GCV-specific hammerhead ribozyme recombinant vector of pyruvate kinase in Giardia lamblia].

OBJECTIVE: To construct a recombinant vector of Giardia canis virus (GCV)-specific hammerhead ribozyme of pyruvate kinase (PK) in Giardia lamblia. METHODS: Using RNA draw software, the secondary structure of PK gene in Giardia was predicted and the cleavage site of ribozyme was selected. The antisense-specific hammerhead ribozyme (PKH) targeting this site was designed. The DNA encoding the ribozyme was synthesized and the recombinant vector pGCV-PKH was constructed. The extracellular and intracellular cleavage of PK mRNA was carried out with the linear recombinant vector. Trophozoites in each group were observed with fluorescent microscopy at 24th hour after transfection. Real-time PCR was used for relative quantitative analysis of cleavage products. RESULTS: The recombinant vector of GCV-specific hammerhead ribozyme of pyruvate kinase in Giardia lamblia (pGCV-PKH) was constructed. Intracellular cleavage assays showed that the green fluorescence could only be seen in pGCV-GFP transfected trophozoites. The relative content of PK mRNA in pGCV-PKH transfected group was 33.14% of that in normal control group. It could cleave PK mRNA extracellularly and the efficiency was 58.5%. CONCLUSION: The recombinant vector can transfect Giardia trophozoites, and cleave mRNA of PK intracellularly and extracellularly.

Giardia lamblia: intracellular localization of alpha8-giardin.

Alpha8-giardin (α8-giardin) is a member of the multi-gene α-giardin family in the intestinal parasitic protozoan, Giardia lamblia. This gene family shares an ancestry with the annexin super family, whose common characteristic is calcium dependent binding to membranes that contain acidic phospholipids. In the present study, the antigenicity, hydrophilicity, flexibility, surface probability, and secondary structure of α8-giardin amino acids were predicted by bioinformatics applications. A specific anti-peptide antiserum, anti-P3, was used to determine the intracellular location of α8-giardin with confocal immunofluorescence microscopy and immunoelectron microscopy. The results indicated that α8-giardin was located on the plasma membrane and flagella, but not on the ventral disk. Reduction of α8-giardin transcript levels by ribozyme-mediated cleavage decreased trophozoite motility and growth rate, indicating the functional importance of α8-giardin to Giardia trophozoite biology.

Follow-Up on Percutaneous Vertebroplasty Using PMMA in Osteoporotic Patients.

We reviewed the follow-up findings of patients who received percutaneous vertebroplasty, in order to understand the effects of vertebroplasty on following issues: pain relief, height restoration, wedge deformity correction, kyphosis correction, and other radiological findings found in follow up studies, such as new fractures. Generally, the pain relieving effect after vertebroplasty is long lasting except when new fracture occurred, or presence of other problems that caused the back pain. Height restoration and wedge deformity correction last for more than one year. The effect of kyphosis angle correction may vanish one year after procedure due to occurrence of new fracture in adjacent vertebral bodies.

Assessment of CT urography in the diagnosis of urinary tract abnormalities.

BACKGROUND: To evaluate the accuracy of helical computerized tomography urography (CTU) in diagnosing urinary tract disease. METHODS: We collected 102 patients who underwent CTU from March 2001 to September 2002. The clinical symptoms of these patients included: flank pain or hematuria, which were clinically suggestive of urinary system disorders. All patients received CT scan with or without intravenous contrast medium administration, except patients who had allergy history or poor renal function. Reformatted CT urography was performed and the accuracy of imaging diagnosis was evaluated. RESULTS: Of these 102 patients, 40 cases were proved to have urolithiasis, 24 cases were with renal or ureteral tumors, and there were 38 cases of other urinary system disorders (including inflammation, ureteral stricture and congenital anomalies). Precontrast CTU correctly diagnosed 39 of 40 urolithiasis cases (97.5%). Precontrast and contrast-enhanced CTU were performed in the non-urolithiasis group and correctly diagnosed 23 cases of renal or ureteral tumors, 4 cases of congenital disorder (2 duplications, 1 congenital renal agenesis due to VATER syndrome and 1 retrocaval ureter). Five of 34 patients with chronic inflammatory process of urinary tract or ureteral stricture were indistinct from malignancies according to the imaging of CT urography. CONCLUSIONS: CTU is good modality for demonstration of urinary tract disorders. It is better in showing radiolucent stones and non-opacified urinary system. CTU demonstrates more soft tissue information than IVU in the patients with urinary tract tumors. CTU may be an alternative modality for patients with deterioration of renal function or allergic history of contrast medium.