This paper introduces a microtiter-plate scanning instrument for medical laboratory equipment,which is controlled by the embedded PC when moving along the X direction and Y direction on the microtiter-plate plane. The designs of the driving circuit and orienting circuit are included in this paper. This instrument has been used in PKU fluorescence laboratory equipment for the newborn baby.

ObjectiveTo establish the autophagy model of normal human liver cell line 7702 induced by hypoxia and starvation, and to lay a foundation for further studies on the influence of autophagy on liver function. MethodsThe 7702 cells were selected and incubated with 95% air and 5% CO2 at a temperature of 37 ℃(normal control group). The Binder three-gas incubator was used, with a temperature of 37 ℃, a CO2 concentration of 5%, and an O2 concentration of 0.3% to provide a hypoxic environment, and the serum-free DMEM was used to induce starvation. These cells were divided into 6-, 12-, 18-, and 24-hour hypoxia-starvation groups. Western blot was used to measure the protein expression of Beclin 1, Atg5, and LC3 in the normal control group and experimental groups, RT-qPCR was used to measure the mRNA expression of Beclin 1 and Atg5 in each group, and after transfection of LC3 plasmid, immunofluorescence assay was used to observe autophagy in each group. An analysis of variance was used for comparison of continuous data between groups, and the least significant difference t-test was used for further comparison between any two groups; the chi-square test was used for comparison of categorical data between groups. ResultsThe 6-hour hypoxia-starvation groups had higher protein expression of Beclin 1, Atg5, and LC3 than the normal control group or other treated groups. Compared with all the other groups, the 6-hour hypoxia-starvation group showed significantly increased mRNA expression of Beclin 1 and Atg5, as well as significantly greater increases in the mRNA expression of Beclin 1 and Atg5 (all P＜0.05). The hypoxia-starvation groups had significantly lower numbers of autophagosomes than the normal control group, and the 6-hour hypoxia-starvation group had the highest number of autophagosomes (all P＜0.05). ConclusionHypoxia and starvation established by physical methods can successfully induce hepatocyte autophagy, which is the most remarkable at 6 hours of hypoxia and starvation.

OBJECTIVETo investigate the effect of peroxisome proliferator-activated receptors γ (PPARγ) on insulin receptor substrate-4 (IRS-4) gene expression in the brain.

METHODSPrimarily cultured cortical neurons from E17-18 Sprague Dawley rats, after 1 week of plating, were exposed to 10 µmol/L PPARγ agonist rosiglitazone for 0, 1, 4 or 24 h. Adult C57BL/6J mice or conditional brain PPARγ knock-out mice (B-PPARγ-KO, BKO) received an intraperitoneal injection of rosiglitazone in 10% DMSO at 12 mg/kg or injection of the same volume of saline containing 10% DMSO. The effect of rosiglitazone on the survival of the neurons was examined by MTT assay. The expression of IRS-4 mRNA was analyzed by real-time quantitative PCR.

RESULTSThe survival of the cortical neurons showed no significant difference between the agonist groups and the control group. The expression of IRS-4 mRNA was significantly up-regulated in the cortical tissues and neurons of the agonist groups compared with the control groups (P<0.05), but in BKO mice without treatment, IRS-4 mRNA expression was significantly down-regulated (P<0.05).

CONCLUSIONPPARγ can enhance the expression of IRS-4 mRNA in the brain.

Animals ; Cell Survival ; drug effects ; Cells, Cultured ; Cerebral Cortex ; cytology ; metabolism ; Female ; Gene Transfer Techniques ; Insulin Receptor Substrate Proteins ; genetics ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Neurons ; cytology ; metabolism ; PPAR gamma ; agonists ; genetics ; metabolism ; Pregnancy ; RNA, Messenger ; metabolism ; Rats ; Rats, Sprague-Dawley ; Thiazolidinediones ; pharmacology ; Up-Regulation

Objective To identify the pathogenic genes and mutations in a family with Usher syndrome type 2.Methods A three-generation family including 7 individuals was enrolled in this study.There were 2 male patients and 5 unaffected individuals.All participants was underwent related ophthalmologic examination,including best corrected visual acuity,slit-lamp,indirect ophthalmoscopy,electroretinogram (ERG),optical coherence tomography and visual field test.DNA was extracted from 3 ml peripheral venous blood of all participants.A total of 136 hereditary retinal disease target genes were screened and the DNA sequence was performed by Next-generation sequence analysis.Then the suspected mutations compared with databases to identify the suspected mutations,which should be verified with non-affected family members and 100 normal subjects by PCR and Sanger sequence.Results The sequence result showed that 2 patients,the proband and his brother,carried complex heterozygous mutations in the USH2A gene:c.5459T＞C (p.M1820T) in exon 27,c.802G ＞A (p.G268R) in exon 5 and c.1190T＞A (p.I397K) in exon 7.The c.5459T ＞ C and c.1190T ＞A mutations in USH2A have not been reported in the literature and database.Although their mother carried c.5459T＞C (p.M1820T) and c.802G＞A (p.G268R),and their father carried c.1190T＞A (p.I397K) heterozygous mutations,the parents did not present phenotype.These mutations were not detected in other normal family members.The result was supported by co-segregation analysis.Conclusion The heterozygous mutations c.5459T＞C (p.M1820T),c.1190T＞A (p.I397K) and c.802G＞A (p.G268R) in USH2A gene cause Usher syndrome in this family.

Objective To evaluate the clinical improvements after autologous bone marrow mononuclear cells (BMMNCs) percutaneously injected into coronary artery in patients with heart failure due to non-ischemic cardiomyopathy using PET myocardial peffusion/metabolic imaging.Methods From February 2011 to October 2012,40 patients with heart failure due to non-ischemic cardiomyopathy were selected.The test group including 15 patients (13 males,2 females,average age (57.5±14.5) years) received the autologous BMMNCs intracoronary injection on the basis of drug treatment.The other 25 cases (21 males,4 females,average age (58.0±12.0) years) were taken as the control group and only received the drug treatment.All patients were followed up for 24 months,and the myocardial perfusion/metabolism imaging,echocardiography,brain natriuretic peptide (BNP) test,6-minute walking experiment were performed.The data were analyzed by two-sample t test.Results During the follow-up period,the test group had no ventricular arrhythmia and other serious complications,and the patients' symptoms had been improved.There was no change in myocardial perfusion after treatment of autologous BMMNCs,but the myocardial metabolic defect by volume reduced from (43.79± 17.99) cm3 to (28.19±9.27) cm3 (t =3.33,P＜0.01) 24 months after the treatment.The myocardial metabolic defect by volume at the baseline and after 24 months in the control group was (43.30±15.70) cm3,(48.51±15.77) cm3 respectively (t=1.01,P＞0.05).In the test group,the left ventricular end-diastolic diameter decreased from (64.0±8.0) mm to (59.0±7.0) mm 24 months after the treatment (t=2.04,P＜0.05),and the left ventricular ejection fraction was significantly higher than that before treatment:(45.0±4.0) ％ vs (27.0±6.0) ％ (t =10.81,P＜0.01).Conclusion PET myocardial perfusion/metabolic imaging can be used as tools in evaluating the therapeutic effect of autologous BMMNCs in patients with heart failure due to non-ischemic cardiomyopathy.

Objective:To evaluate the effectiveness and safety of intravitreal injection of different doses of aflibercept for polypoidal choroidal vasculopathy (PCV) with serous pigment epithelial detachment (PED) resistant to ranibizumab.Methods:A non-randomized controlled clinical study was conducted.Seventy-three eyes of 73 patients with PCV and serous PED resistant to ranibizumab were enrolled at the First Affiliated Hospital of Zhengzhou University from January 2019 to December 2020.All patients were treated by intravitreal injection of 2 mg or 4 mg aflibercept according to patients' willingness.2 mg aflibercept or 4 mg aflibercept was intravitreally injected monthly for three consecutive months following pro re nata (PRN) regimen in 2 mg aflibercept group (38 eyes) and 4 mg aflibercept group (35 eyes), respectively.PED height and central macular thickness (CMT) were measured by optical coherence tomography, and the best corrected visual acuity (BCVA) was examined with a visual acuity chart and converted to logarithm of the minimum angle of resolution (LogMAR) unit before injection and 1 month, 2, 3, 6 months from the first injection.Intraocular pressure and treatment-related adverse events were recorded.This study adhered to the Declaration of Helsinki and was approved by an Ethics Committee of The First Affiliated Hospital of Zhengzhou University (No.2021-KY-1252).Written informed consent was obtained from each patient prior to entering study cohort.Results:Thirty-three patients (86.84%) in 2 mg aflibercept group and 30 patients (85.71%) in 4 mg aflibercept group finished the treatment and follow-up, respectively. The PED, BCVA and CMT before treatment and at the end of follow-up were (379.24±95.50) and (280.09±120.50)μm, 0.68±0.27 and 0.51±0.19, (393.96±100.81) and (291.70±44.09)μm in 2 mg aflibercept group, respectively, showing statistically significant differences (all at P<0.05).The PED, BCVA and CMT before treatment and at the end of follow-up were (393.07±93.76) and (278.63±145.07)μm, 0.66±0.31 and 0.48±0.22, (377.43±79.61) and (284.67±84.88)μm in 4 mg aflibercept group, respectively, with statistically significant differences (all at P<0.05).The CMT value in 4 mg aflibercept group was significantly lower than that in the 2 mg aflibercept group in one month after injection ( P<0.05).No severe ocular and systemic adverse events were found during the follow-up, such as retinal detachment, endophthalmitis, cataract, and persistent high intraocular pressure. Conclusions:Both 2 mg and 4 mg aflibercept can effectively treat ranibizumab-resistant PCV with serous PED, and improve the anatomical structure of retina and BCVA.4 mg aflibercept can accelerate the recovery of PED and CMT.

Objective To investigate the risk factors of postoperative vitreous hemorrhage after minimal vitrectomy without endotamponade for proliferative diabetic retinopathy (PDR).Methods From June 2015 to June 2017,103 eyes of 103 patients with PDR diagnosed and underwent minimalvitrectomy in Henan Provincial People's Hospital were enrolled in the study.There were 58 males and 45 females,with the average age of 58.37± 10.14 years and diabetes duration of 8.7± 7.2 years.Baseline systemic parameters including sex,age,diabetes duration,hypertension,HbA1c,creatinine,whether received anticoagulants,ocular parameters including whether combined with vitreous hemorrhage,whether finished panretinal photocoagulation (PRP),whether received treatment of anti-VEGF,whether combined with iris neovascularization (NVI),lens status preoperatively,whether hypotony postoperatively and intraoperative parameters including whether disc neovascularization (NVD) bleeding,whether fibrovascular membrane (FVM) residual,laser points,whether combined with cataract phacoemulsification were identified by multivariate logistic regression analysis.Results Twenty-nine of 103 eyes (28.15％) developed PVH in 1 day to 6 months after surgery,with self absorption of 18 eyes and reoperation of 11 eyes.Univariate analysis showed there were significant differences in age (t=2.124,P=0.036),anti-VEGF(x2=7.105,P=0.008),NVD bleeding (x2=10.158,P=0.001) and FVM residual(x2=8.445,P=0.004) between patients with and without postoperative vitreous hemorrhage.Sex (x2=0.021,P=0.884),diabetes duration (t=0.87,P=0.386),hypertension (x2=2.004,P=0.157),HbA1c (t=1.211,P=0.229),creatinine (t=0.851,P=0.397),preoperative oral anticoagulants (x2=0.985,P=0.321),preoperative vitreous hemorrhage (x2=0.369,P=0.544),PRP (X2=1.122,P=0.727),NVI (x2=2.635,P=0.105),lens status (x2=0.172,P=0.679),hypotony postoperatively (x2=1.503,P=0.220),laser points (x2=1.391,P=0.238) and combined phacoemulsification surgery (x2=0.458,P=0.499) were not associated with PVH.Multivariate logistic regression analysis revealed the more PVH appeared in younger (OR=1.065,P=0.009) and NVD bleeding (OR=6.048,P=0.001) patients.Conclusion Younger age and NVD bleeding are the important risk factors for PVH after minimal vitrectomy without endotamponade in PDR.

Objective:To report the BEST1 gene mutations and clinical phenotypes in two pedigrees with Best vitelliform macular dystrophy (BVMD) and autosomal recessive bestrophinopathy (ARB). Methods:A retrospective clinical study. From November 2019 to March 2021, in the Department of Ophthalmology of The First Affiliated Hospital of Zhengzhou University, the BVMD family (4 patients and 6 family members) and the ARB family (2 patients, 2 family members), a total of 6 patients and 8 normal family members were included in the study. Detailed medical history was obtained; best corrected visual acuity, fundus color photography, electrophysiology, optical coherence tomography and fundus autofluorescence examination were performed. The clinical characteristics for all patients in the two families were analyzed. Three milliliter peripheral venous blood of all participants in the family was collected, and the whole genomic DNA was extracted with gene sequencing using next-generation sequencing technology based on targeted capture. Compared with the database to identify the pathogenicity mutation sites, suspected pathogenic mutation sites were selected, then mutations in other members in the family was assayed by Sanger sequencing.Results:In family 1, the proband was demonstrated as typical BVMD, other patients were multifocal vitelliform macular dystrophy. The DNA sequencing result showed that all the 4 patients carried heterozygous missense mutations in exon 3 of BEST1 gene: c.240C>G (p.F80L) (M1) and 2 members carried this mutation, but without clinical phenotype. M1 was a likely-pathogenic mutation reported for the first time. In family 2, the proband and the other patient were diagnosed as ARB. The DNA result showed that the 2 patients carried heterozygous missense mutations in exon 5 and exon 2 of BEST1 gene: c.584C>T (p.A195V) (M2)、c.139C>A (p.R47S) (M3), and a heterozygous frameshift mutation in exon 3 of BEST1 gene: c.235dupT (p.S79Ffs*153) (M4). M2 was a pathogenic mutation reported previously. M3 variant was of undetermined significance. M4 was a first reported pathogenic mutation. Conclusions:The BEST1 gene mutation is the main cause of BVMD and ARB. Different mutation sites have different clinical phenotypes. BVMD and ARB have genetic and clinical heterogeneity.

The spread of COVID-19 has greatly threatened human health and economic growth. Angiotensin-converting enzyme 2 (ACE2) is a receptor for severe acute respiratory syndrome coronavirus 2 (SARS-COV-2). By attaching to ACE2, SARS-COV-2 reduces its expression and induces lung injury. Vitamin D can inhibit the progression of COVID-19 by inhibiting the activity of ROCK pathway, up-regulating ACE2 expression and bio-availability, and slowing down the adverse reactions caused by Ang II accumulation. This study explored a novel mechanism, i.e., vitamin D protects against COVID-19-induced injury by upregulating ACE2 expression. It provides theoretical guidance for the role of Vitamin D in the prevention and treatment of COVID-19.