Abstract:Objective To screen the transient and stable cell lines with high production of Nectin-like 4 (Necl-4) protein.Methods First, cDNA sequences encoding the extracellular domain of Necls were cloned into the modified vector pAPtag at the N terminus of alkaline phosphatase (AP) for fusion expression. Next, 293ET cells stably expressed Necls-AP fusion protein and secreted it into the culture medium which were detected by the AP activity assay and Western blot analysis. Then, by adding N-glycosylation processing inhibitor kifunensine into the medium, complex glycan was inhibited to generate. The residual glycan of purified protein was removed by endoglycosidase H. Finally, AP protein was removed by using human rhinovirus protease and size exclusion chromatography. The concentration of purified Necl-4 protein was monitored by measuring the absorbance at 280 nm and analyzed by SDS-PAGE.Results The transient and stable cell lines with high production of Necl-4 protein were screened by the color reaction with the AP-tag in the recombinant vector. The soluble and active form of purified Necl-4 protein was obtained after deglycosylation of native N-glycan protein with an expression level of 4 mg/L culture and purity of 95%.Conclusions By using modified AP mammalian protein expression system, we can easily screen the high productive stable cell lines by using AP activity assay. By adding mannosidase inhibitor kifunensine into the medium and cutting purified protein by using endoglycosidase H, we can obtain deglycosylated Necl-4 protein in milligram quantities. Our method might throw a light on the expression and purification of glycoprotein for structural and functional studies.
Keywords:Nectin-like immunoglobulin;membrane protein;fast and easy screening;glycosylation;mammalian cell
Abstract:Objective To investigate the efficacy and safety of rituximab (RTX) in the treatment of idiopathic membranous nephropathy (IMN) with nephrotic syndrome with a systematic review and meta-analysis.Methods PubMed, Embase, Cochrane Library and Clinical Trials (December 2016) were searched to identify researches investigating the treatment of RTX in adult patients with biopsy-proven IMN. Complete remission (CR) or partial remission was regarded as effective therapy, and the cumulated remission rate was calculated.ResultsSeven studies involved 120 patients (73% were men) were included in our systematic review and meta-analysis. All were prospective observation cohort studies or matched-cohort studies, mainly came from two medical centers, and one study was multi-centric (four nephrology units in northern Italy). The creatinine clearance was more than 20 ml/(min·1.73 m2) and persistent proteinuria higher than 3.5 g/d for at least 6 months. All patients received treatment previously [44 (36.7%) had immunosuppressive treatment]. In 12- and 24-month, 56% (95%CI, 0.47-0.65) and 68% (95%CI, 0.41-0.87) patients could reach remission, while 15% (95%CI, 0.09-0.23) and 20% (95%CI, 0.12-0.32) patients could reach CR. The reduction in proteinuria was gradual and obvious, paralleled with upward trend of serum albumin level and decreasing serum cholesterol level. Renal functions were stable. Relapses happened in 24 months were around 8%. RTX related adverse events were mild and were mostly infusion-related reactions.Conclusions RTX treatment in IMN was efficient, well tolerated and safe. More than 60% patients can reach partial remission or CR in 24 months, and relapse is rare. Adverse events of RTX are mostly infusion-related reactions and generally mild.
Abstract:Objective To investigate cerebral structural signatures of the bulbar- and spinal-onset amyotrophic lateral sclerosis (ALS) using voxel-based morphometry on magnetic resonance imaging.Methods The MR structural images of the brain were obtained from 65 ALS patients (15 bulbar-onset, 50 spinal-onset) and 65 normal controls (NC) on a 3.0T MRI system. Gray matter (GM) volume changes were investigated by voxel-based morphometry, and the distribution of the brain regions with volume changes was compared between ALS and normal controls, as well as between bulbar-onset and spinal-onset ALS based on Neuromorphometrics atlas.Results On voxel-level the decreased volume of brain regions in ALS patients was located in the right precentral gyrus (rPrcGy) and right middle frontal gyrus compared with that in NC. The bulbar-onset ALS presented extra-motor cortex atrophy (fronto-temporal pattern), including left medial orbital gyrus, left inferior temporal gyrus and right middle temporal gyrus; the spinal-onset ALS suffered from motor cortex atrophy (rPrcGy dominance) and extra-motor cortex atrophy (fronto-temporal and extra-fronto-temporal pattern) compared with NC. The spinal-onset ALS featured by GM volume loss of left postcentral gyrus and bulbar-onset ALS featured by GM volume loss of left middle temporal gyrus compared with each other.Conclusions The asymmetric GM atrophy of the motor cortex and extra-motor cortex represents the common MRI structural signatures of spinal-onset ALS, and sole extra-motor cortex atrophy represents the structural signatures of bulbar-onset ALS. The present study also demonstrated that the pattern of GM damage is likely to distribute wider in spinal-onset ALS than in bulbar-onset ALS.
Abstract:Objective To investigate the neuroprotective action of astragaloside IV (AS-IV) on spatial learning and memory impairment induced by amyloid-beta 1-42 (Aβ1-42) in rats and elucidate its underlying molecular mechanisms. Methods Adult-male Sprague-Dawley rats (230-250 g) were divided into six groups randomly: control, Aβ1-42, AS-IV, Aβ1-42 plus 5 mg/kg·d AS-IV, Aβ1-42 plus 25 mg/kg·d AS-IV, and Aβ1-42 plus 50 mg/kg·d AS-IV groups. Aβ1-42 were delivered by intracerebroventricular injection under the guidance of a brain stereotaxic apparatus. The Morris water maze test (hidden platform test, probe trials, visible platform test) was performed one week after Aβ1-42 injection to obtain the ability of rat spatial learning and memory. AS-IV (5, 25 and 50 mg/kg·d) was administrated intraperitoneally once per day from the 8th day after Aβ1-42 injection for 5 consecutive days. Average escape latencies, distances for searching for the platform under water and the percentage of total time elapsed and distance swam in the right quadrant after removing platform were determined by behavior software system. The vision and swim speeds of rats were also determined to exclude the effect of these factors on the parameters of learning and memory. After behavioral tests, the rats were sacrificed immediately by decapitation. Hippocampus were collected. The enzyme activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-px) and catalase (CAT) in the hippocampus obtained from different-treated rat brain were measured by following the manufacturer’s instructions. The levels of interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) in tissue lysates were assayed with ELISA. Results The water maze test results indicated that chronic treatments with AS-IV effectively protected the rats from Aβ1-42-induced spatial learning and memory impairment. Furthermore, the activities of SOD, GSH-px and CAT decreased by Aβ1-42 were also restored by AS-IV treatment in the hippocampus of rats. In addition, AS-IV significantly decreased the levels of IL-1β and TNF-α in the hippocampus of Aβ1-42-induced amnesia’s rats.Conclusion Our findings suggest that AS-IV might be a useful chemical in improving the spatial memory and relieving the oxidative stress and neuroinflammation in Alzheimer patients.
Keywords:amyloid-β protein;astragaloside IV;spatial learning and memory;oxidative stress;neuroinflammation
Abstract:Objective To evaluate the reliability of three dimensional spiral fast spin echo pseudo-continuous arterial spin labeling (3D pc-ASL) in measuring cerebral blood flow (CBF) with different post-labeling delay time (PLD) in the resting state and the right finger taping state.Methods 3D pc-ASL and three dimensional T1-weighted fast spoiled gradient recalled echo (3D T1-FSPGR) sequence were applied to eight healthy subjects twice at the same time each day for one week interval. ASL data acquisition was performed with post-labeling delay time (PLD) 1.5 seconds and 2.0 seconds in the resting state and the right finger taping state respectively. CBF mapping was calculated and CBF value of both the gray matter (GM) and white matter (WM) was automatically extracted. The reliability was evaluated using the intraclass correlation coefficient (ICC) and Bland and Altman plot.Results ICC of the GM (0.84) and WM (0.92) was lower at PLD 1.5 seconds than that (GM, 0.88; WM, 0.94) at PLD 2.0 seconds in the resting state, and ICC of GM (0.88) was higher in the right finger taping state than that in the resting state at PLD 1.5 seconds. ICC of the GM and WM was 0.71 and 0.78 for PLD 1.5 seconds and PLD 2.0 seconds in the resting state at the first scan, and ICC of the GM and WM was 0.83 and 0.79 at the second scan, respectively.Conclusion This work demonstrated that 3D pc-ASL might be a reliable imaging technique to measure CBF over the whole brain at different PLD in the resting state or controlled state.
Abstract:Chorea-acanthocytosis (ChAc) is the most common subtype of neuroacanthocytosis syndrome, characterized by the presence of acanthocytes and neurological disorders. It is thought to be caused by VPS13A mutations. Characteristic movement disorders in ChAc is choreiform movements affecting both trunk and extremities and prominent orolingual dyskinesia is pathognomonic. Acanthocytosis in peripheral blood smear, elevated serum creatine kinase and atrophy of heads of caudate nuclei and dilation of the anterior horn of the lateral ventricles in magnetic resonance imaging could assist the diagnosis of ChAc. Botulinum toxin injection is a possible treatment for the typical orofacial dystonia. Deep brain stimulation is a novel surgical treatment modality. Most cases chose globus pallidus internus as target. Patients with dystonia as a major manifestation will benefit more from high-frequency stimulation and those with major findings of chorea and dysarthria are suitable for low-frequency stimulation. More evidence of long-term outcomes is warranted.
Abstract:Fibronectin glomerulopathy is a rare autosomal dominant inherited glomerular disease associated with massive deposition of fibronectin. We recently diagnosed fibronectin glomerulopathy in a 29-year-old woman presenting nephrotic syndrome. Genetic analysis of fibronectin 1 gene showed heterozygosity for the Y973C mutation. However, this mutation was not found in her parents. She had stable renal function but persistent nephrotic proteinuria after one-year follow-up.
Abstract:We herein reported a 27-year-old woman with a right renal mass for two years. She underwent laparoscopic partial nephrectomy. Immunohistochemical examination of the specimen confirmed the diagnosis of solitary fibrous tumor by revealing its positive staining for cluster of differentiation (CD)34, epithelial membrane antigen (EMA), B-cell lymphoma-2 (Bcl-2) and CD99 in the tumor cells. No adjuvant treatment was carried out. The patient was in good health without local recurrence or metastasis during 2 years of follow-up. Laparoscopic partial nephrectomy for renal solitary fibrous tumor is an alternative treatment to radical nephrectomy. It can provide a good outcome. However, further follow-up and more cases of renal solitary fibrous tumor treated with laparoscopic partial nephrectomy are necessary to compare the oncological outcome with radical nephrectomy.