Seropositivity rates of water channel protein 4 antibodies compared between a cell-based immunofluorescence assay and an enzyme-linked immunosorbent assay in neuromyelitis optica patients

A total of 66 samples （from 27 cases with neuromyelitis optica, 26 cases with multiple sclerosis, aa 13 cases with optic neuritis） were tested for aquaporin-4 antibody by a cell-based immunofluorescence assay and an enzyme-linked immunosorbent assay. The sensitivities and specificities of the two assays were similar. We further analyzed an additional 68 patients and 93 healthy controls using the enzyme-linked immunosorbent assay. A Kappa test showed good consistency between the two methods in terms of detection of anti-aquaporin-4 antibody in the se of neuromyelitis optica patients. No significant correlations were identified with onset age or disea duration, suggesting that aquaporin-4 antibody is a good marker for neuromyelitis optica. The enzyme-linked immunosorbent assay can be used for quantifying aquaporin-4 antibody concentrations and may be useful to dynamically monitor changes in the levels of aquaporin-4 antibody during disease duration.

Real-time RT-PCR Assay for the detection of Tahyna Virus

A real-time RT-PCR （RT-qPCR） assay for the detection of Tahyna virus was developed to monitor Tahyna virus infection in field-collected vector mosquito samples. The targets selected for the assay were S segment sequences encoding the nucleocapsid protein from the Tahyna virus. Primers and probes were selected in conserved regions by aligning genetic sequences from various Tahyna virus strains available from GenBank. The sensitivity of the RT-qPCR approach was compared to that of a standard plaque assay in BHK cells. RT-qPCR assay can detect 4.8 PFU of titrated Tahyna virus. Assay specificities were determined by testing a battery of arboviruses, including representative strains of Tahyna virus and other arthropod-borne viruses from China. Seven strains of Tahyna virus were confirmed as positive; the other seven species of arboviruses could not be detected by RT-qPCR. Additionally, the assay was used to detect Tahyna viral RNA in pooled mosquito samples. The RT-qPCR assay detected Tahyna virus in a sensitive, specific, and rapid manner; these findings support the use of the assay in viral surveillance.

Real-time RT-PCR Assay for the Detection of Culex flavivirus

Based on the Culex flavivirus (CxFV) E gene sequences in GenBank, CxFV-specific primers and probes were designed for real-time reverse transcription-polymerase chain reaction (RT-qPCR). The specificity test revealed that CxFV could be detected using RT-qPCR with the specific CxFV primers and probes; other species of arboviruses were not detected. The stability test demonstrated a coefficient of variation of <1.5%. A quantitative standard curve for CxFV RT-qPCR was established. Quantitative standard curve analysis revealed that the lower detection limit of the RT-qPCR system is 100 copies/mu L. Moreover, RT-qPCR was used to detect CxFV viral RNA in mosquito pool samples. In conclusion, we established a real-time RT-PCR assay for CxFV detection, and this assay is more sensitive and efficient than general RT-PCR. This technology may be used to monitor changes in the environmental virus levels.

Complete genome sequence of a Rodent Torque teno virus in Hainan Island, China and establishment of a real-time PCR for detecting Rodent Torque teno virus 3

Objective:To perform whole-genome sequencing and phylogenetic analysis of the local endemic strain of Rodent Torque teno virus (RoTTV), RoTTV3-HMU1, found in Rattus norvegicus, Haikou City, Hainan Province, and establish a SYBR Green I based real-time PCR detection assay for RoTTV3.Methods: Based on the high-throughput genome sequencing analysis, specific primers were designed and the whole genome sequence was amplified by PCR and Sanger sequencing. Specific detection primers were designed based on the conserved sequences of RoTTV3. The recombinant plasmid contained the whole genome of RoTTV3-HMU1 was constructed as a standard control. The experimental conditions were optimized and the real-time PCR detection assay of RoTTV3 was established.Results: The genomic sequence of RoTTV carried by Rattus norvegicus in Haikou City was successfully sequenced. Phylogenetic analysis indicated that the virus belongs to the RoTTV3 genotype. In this experiment, the real-time PCR detection method of RoTTV3 was established. The standard curve generated had a wide dynamic range from 1×(102-108) copies/μL, with a linear correlation (R2=1.000). The melting curve analysis using SYBR Green showed only one specific melting peak and no primer-dimmers represented. The detection limit was 100 copies/reaction.Discussion: This study was the first report of the RoTTV in Hainan Islands, and its phylogenetic analysis was of great significance to the origin and evolution of RoTTV. The RoTTV3 real-time PCR detection method established in this experiment has a high sensitivity and good specificity, which lays a technical foundation for the epidemiological investigation of RoTTV3.

Diagnostic value of antigenemia assay for cytomegalovirus gastrointestinal disease in immunocompromised patients

AIM:To investigate the utility of the cytomegalovirus(CMV)antigenemia assay for the diagnosis of CMV gastrointestinal disease(GID). METHODS:One hundred and thirty immunocompromised patients were enrolled in this study.Patients with a history of anti-CMV treatment and who had not undergone examination using the antigenemia assay were excluded.CMV-GID was defined as the detection of large cells with intranuclear inclusions alone or associated with granular cytoplasmic inclusions by biopsy.Biopsy sections were stained with hematoxylin and eosin and immunohistochemically stained with anti-CMV.We evaluated the association between CMV-GID and patient characteristics(symptoms,underlying disease,medication,leukocyte counts,and antigenemia assay).All patients were checked with an human immunodeficiency virus(HIV)antibody test before endoscopic examination.White blood cell(WBC)counts were obtained from medical records within 1 wk of endoscopy.Leukopenia was defined as a total WBC count<5000 cells/mm 3 . For HIV patients,we also checked CD4+counts from medical records. RESULTS:A total of 99 patients were retrospectively selected for analysis.Of the immunocompromised patients,19 had malignant disease,18 had autoimmune disease,19 had disorders of biochemical homeostasis, three had undergone transplantation,and 45 had HIV infection.A total of 50 patients had received immunosuppressive therapy.No patients had inflammatory bowel disease.Fifty-five patients were diagnosed as having CMV-GID.Univariate analysis indicated an association between HIV infection,leukopenia,and positive antigenemia and CMV-GID(P<0.05).Multivariate analysis using logistic regression revealed that HIV infection and positive antigenemia were the only independent factors related to CMV-GID(P<0.01).The sensitivity,specificity,positive predictive value,and negative predictive value of antigenemia for CMV-GID were 65.4%,93.6%, 91.9%,and 71.0%,respectively.In a subgroup analy-sis,patients with leukopenia displayed low sensitivity and high specificity.Minimal differences in accuracy were seen among patients with or without leukopenia. HIV-infected patients displayed low sensitivity and high specificity.Accuracy barely differed between HIV-positive and-negative patients.In HIV-infected patients, CD4 count<50 cells/μL resulted in low sensitivity and high specificity.Differences in accuracy among patients were minor,regardless of CD4 count.In patients who had undergone both quantitative real-time polymerase chain reaction(PCR)and antigenemia assay,real-time PCR was slightly more accurate in terms of sensitivity than the antigenemia assay;however,this difference was not statistically significant(P=0.312). CONCLUSION:If the antigenemia test is positive,endoscopic lesions are acceptable for the diagnosis of CMVGID without biopsy.The accuracy is not affected by HIV infection and leukopenia.Either PCR or the antigenemia assay are valid.

Leishmania tropica: The comparison of two frequently-used methods of parasite load assay in vaccinated mice

Objective:To compare limiting dilution assay and real-time PCR methods in Leishmania tropica parasite load measurement in vaccinated mice.Methods:BALB/c mice were vaccinated by Leishmania tropica soluble Leishmania antigen or recombinant Leishmania tropica stressinducible protein-1 with/without adjuvant.After three vaccinations,mice were challenged by Leishmania tropica promastigotes.Two months after challenge,the draining lymph nodes of mice footpad were removed and parasite load was assayed by limiting dilution assay and real-time PCR methods.Limiting dilution assay was done by diluting tissue samples to extinction in a biphasic medium.For real-time PCR,DNA of the lymph nodes was extracted,equal dilutions of each sample were prepared and hot-start real-time PCR was done using appropriate primers.The data of the two methods were compared by appropriate statistical methods.Results:Both methods were able to measure different levels of parasite load in vaccinated/unvaccinated mice.In addition,wherever parasite load of a group was estimated high(or low)by one method,the estimated parasite load by another method was the same,although statistically significant differences were found between some groups.Conclusions:Both methods lead to approximately similar results in terms of differentiating parasite load of the experimental groups.However,due to the lower errors and faster process,the real-time PCR method is preferred.

A cell-based high-throughput approach to identify inhibitors of influenza A virus

Influenza is one of the most common infections threatening public health worldwide and is caused by the influenza virus.Rapid emergence of drug resistance has led to an urgent need to develop new anti-influenza inhibitors.In this study we established a 293T cell line that constitutively synthesizes a virus-based negative strand RNA,which expresses Gaussia luciferase upon influenza A virus infection.Using this cell line,an assay was developed and optimized to search for inhibitors of influenza virus replication.Biochemical studies and statistical analyses presented herein demonstrate the sensitivity and reproducibility of the assay in a high-throughput format(Z 0 factor value40.8).A pilot screening provides further evidence for validation of the assay.Taken together,this work provides a simple,convenient,and reliable HTS assay to identify compounds with anti-influenza activity.

Effects of neuregulin-1 on autonomic nervous system remodeling post-myocardial infarction in a rat model

Sympathetic nerve and vagus nerve remodeling play an important part in cardiac function post-myocardial infarction （MI）. Increasing evidence indicates that neuregulin-1 （NRG-1） improves cardiac function following heart failure. Since its impact on cardiac function and neural remodeling post-MI is poorly understood, we aimed to investigate the role of NRG-1 in autonomic nervous system remodeling post-MI. Forty-five Sprague-Dawley rats were equally randomized into three groups： sham （with the left anterior descending coronary artery exposed but without ligation）, MI （left anterior descending coronary artery ligation）, and MI plus NRG-1 （left anterior descending coronary artery ligation followed by intraperitoneal injection of NRG-1 （10 lag/kg, once daily for 7 days））. At 4 weeks after MI, echocardi- ography was used to detect the rat cardiac function by measuring the left ventricular end-systolic inner diameter, left ventricular diastolic diameter, left ventricular end-systolic volume, left ventricular end-diastolic volume, left ventricular ejection fraction, and left ventricular fractional shortening, mRNA and protein expression levels of tyrosine hydroxylase, growth associated protein-43 （neuronal specific pro- tein）, nerve growth factor, choline acetyltransferase （vagus nerve marker）, and vesicular acetylcholine transporter （cardiac vagal nerve fiber marker） in ischemic myocardia were detected by real-time PCR and western blot assay to assess autonomous nervous remodeling. After MI, the rat cardiac function deteriorated significantly, and it was significantly improved after NRG-1 injection. Compared with the MI group, mRNA and protein levels of tyrosine hydroxylase and growth associated protein-43, as well as choline acetyltransferase mRNA level significantly decreased in the MI plus NRG-1 group, while mRNA and protein levels of nerve growth factor and vesicular acetylcholine transporters, as well as choline acetyltransferase protein level slightly decreased. Our results indicate that NRG- 1 can improve cardiac function and regulate sympathetic and vagus nerve remodeling post-MI, thus reaching a new balance of the autonomic nervous system to protect the heart from injury.

Quality control of cell-based high-throughput drug screening

The pharmaceutical industry is presently suffering difficult times due to low productivity of new molecular entities.As a major source of drug leads,high-throughput screening(HTS)has been often criticized for its‘dead end’lead compounds.However,the fruitful achievements resulting from HTS technology indicate that it remains a feasible way for drug innovation.Because of increasing considerations of earlier stage ADMET(absorption,distribution,metabolism,excretion and toxicity)in drug development,cell-based HTS is highly recommended in modern drug discovery for its ability to detect more biologically relevant characteristics of compounds in living systems.This review provides a systematic and practical description of vital points for conducting high quality cell-based HTS,from assay development to optimization,compound management,data analyses,hit validation as well as lead identification.Potential problems and solutions are also covered.

Cross electro-nape-acupuncture ameliorates cerebral hemorrhageinduced brain damage by inhibiting necroptosis

BACKGROUND Receptor interacting protein kinase 1(RIPK1)-mediated cell death,including apoptosis and necroptosis,belongs to programmed cell death.It has been reported that RIPK1-mediated necroptosis exists in lesions of cerebral hemorrhage(CH).Electroacupuncture,a treatment derived from traditional Chinese medicine,could improve neurological impairment in patients with brain injury.AIM To investigate the protective role of cross electro-nape acupuncture(CENA)in CH,and clarify the potential mechanism.METHODS CH rat models were established,and CENA was applied to the experimental rats.Neurological functions and encephaledema were then measured.Necrotic cells in the brain of rats with CH were evaluated by propidium iodide staining.Necroptosis was assessed by immunofluorescence.Activation of the necroptosisrelated pathway was detected by western blot.Extraction of brain tissue,cerebrospinal fluid and serum samples was conducted to measure the expression and secretion of inflammatory cytokines by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay.RESULTS The necroptotic marker p-MLKL was detectable in the brains of rats with CH.Next,we found that CENA could ameliorate neurological functions in rat models of CH.Moreover,the upregulation of RIPK1-mediated necroptosis-related molecules in the brains of rats with CH were inhibited by CENA.Further investigation revealed that CENA partially blocked the interaction between RIPK1 and RIPK3.Finally,in vivo assays showed that CENA decreased the expression of the inflammatory cytokines tumor necrosis factor-α,interleukin-6 and interleukin-8 in CH rat models.CONCLUSION These findings revealed that CENA exerts a protective role in CH models by inhibiting RIPK1-mediated necroptosis.

Expression and Characterization of ArgR, An Arginine Regulatory Protein in Corynebacterium crenatum

Objective Corynebacterium crenatum MT, a mutant from C. crenatum AS 1.542 with a lethal argR gene, exhibits high arginine production. To confirm the effect of ArgR on arginine biosynthesis in C. crenatum, an intact argR gene from wild-type AS 1.542 was introduced into C. crenatum MT, resulting in C. crenatum MT. sp, and the changes of transcriptional levels of the arginine biosynthetic genes and arginine production were compared between the mutant strain and the recombinant strain. Methods Quantitative real-time polymerase chain reaction was employed to analyze the changes of the related genes at the transcriptional level, electrophoretic mobility shift assays were used to determine ArgR binding with the argCJBDF, argGH, and carAB promoter regions, and arginine production was determined with an automated amino acid analyzer. Results Arginine production assays showed a 69.9% reduction in arginine from 9.01±0.22 mg/mL in C. crenatum MT to 2.71±0.13 mg/mL （P〈0.05） in C. crenatum MT. sp. The argC, argB, argD, argF, argJ, argG, and carA genes were down-regulated significantly in C. crenatum MT. sp compared with those in its parental C. crenatum MT strain. The electrophoretic mobility shift assays showed that the promoter regions were directly bound to the ArgR protein. Conclusion The arginine biosynthetic genes in C crenatum are clearly controlled by the regulator ArgR, and intact ArgR in C. crenatum MT results in a significant descrease in production. negative arginine production.

Identification of various cell culture models for the study of Zika virus

AIM To identify cell culture models supportive for Zika virus(ZIKV) replication.METHODS Various human and non-human cell lines were infected with a defined amount of ZIKV Polynesia strain. Cells were analyzed 48 h post infection for the amount of intracellular and extracellular viral genomes and infectious viral particles by quantitative real-time PCR and virus titration assay. The extent of replication was monitored by immunofluorescence and western blot analysis by using Env and NS1 specific antibodies. Innate immunity was assayed by luciferase reporter assay and immunofluorescence analysis.RESULTS All investigated cell lines except CHO cells supported infection, replication and release of ZIKV. While in infected A549 and Vero cells a pronounced cytopathic effect was observed COS7, 293 T and Huh7.5 cells were most resistant. Although the analyzed cell lines released comparable amounts of viral genomes to the supernatant significant differences were found for the number of infectious viral particles. The neuronal cell lines N29.1 and SH-SY5 Y released 100 times less infectious viral particles than Vero-, A549-or 293 T-cells. However there is no strict correlation between the amount of produced viral particles and the induction of an interferon response in the analyzed cell lines.CONCLUSION The investigated cell lines with their different tissue origins and diverging ZIKV susceptibility display a toolbox for ZIKV research.

Mesenchymal stem cells and collagen patches for anterior cruciate ligament repair

AIM: To investigate collagen patches seeded with mesenchymal stem cells(MSCs) and/or tenocytes(TCs) with regards to their suitability for anterior cruciate ligament(ACL) repair. METHODS: Dynamic intraligamentary stabilization utilizes a dynamic screw system to keep ACL remnants in place and promote biological healing, supplemented by collagen patches. How these scaffolds interact with cells and what type of benefit they provide has not yet been investigated in detail. Primary ACL-derived TCs and human bone marrow derived MSCs were seeded onto two different types of 3D collagen scaffolds, Chondro-Gide?(CG) and Novocart?(NC). Cells were seeded onto the scaffolds and cultured for 7 d either as a pure populations or as "premix" containing a 1:1 ratio of TCs to MSCs. Additionally, as controls, cells were seeded in monolayers and in co-cultures on both sides of porous high-density membrane inserts(0.4 μm). We analyzed the patches by real time polymerase chain reaction, glycosaminoglycan(GAG), DNA and hydroxyproline(HYP) content. To determine cell spreading and adherence in the scaffolds microscopic imaging techniques, i.e., confocal laser scanning microscopy(c LSM) and scanning electron microscopy(SEM), were applied.RESULTS: CLSM and SEM imaging analysis confirmed cell adherence onto scaffolds. The metabolic cell activity revealed that patches promote adherence and proliferation of cells. The most dramatic increase in absolute metabolic cell activity was measured for CG samples seeded with tenocytes or a 1:1 cell premix. Analysis of DNA content and c LSM imaging also indicated MSCs were not proliferating as nicely as tenocytes on CG. The HYP to GAG ratio significantly changed for the premix group, resulting from a slightly lower GAG content, demonstrating that the cells are modifying the underlying matrix. Real-time quantitativepolymerase chain reaction data indicated that MSCs showed a trend of differentiation towards a more tenogenic-like phenotype after 7 d.CONCLUSION: CG and NC are both cyto-compatible with primary MSCs and TCs; TCs seemed to perform better on these collagen patches than MSCs.

Effect of electroacupuncture on the mRNA and protein expression of Rho-A and Rho-associated kinase Ⅱ in spinal cord injury rats

Electroacupuncture is beneficial for the recovery of spinal cord injury, but the underlying mechanism is unclear. The Rho/Rho-associated kinase（ROCK） signaling pathway regulates the actin cytoskeleton by controlling the adhesive and migratory behaviors of cells that could inhibit neurite regrowth after neural injury and consequently hinder the recovery from spinal cord injury. Therefore, we hypothesized electroacupuncture could affect the Rho/ROCK signaling pathway to promote the recovery of spinal cord injury. In our experiments, the spinal cord injury in adult Sprague-Dawley rats was caused by an impact device. Those rats were subjected to electroacupuncture at Yaoyangguan（GV3）, Dazhui（GV14）, Zusanli（ST36） and Ciliao（BL32） and/or monosialoganglioside treatment. Behavioral scores revealed that the hindlimb motor functions improved with those treatments. Real-time quantitative polymerase chain reaction, fluorescence in situ hybridization and western blot assay showed that electroacupuncture suppressed the m RNA and protein expression of Rho-A and Rho-associated kinase Ⅱ（ROCKⅡ） of injured spinal cord. Although monosialoganglioside promoted the recovery of hindlimb motor function, monosialoganglioside did not affect the expression of Rho-A and ROCKⅡ. However, electroacupuncture combined with monosialoganglioside did not further improve the motor function or suppress the expression of Rho-A and ROCKⅡ. Our data suggested that the electroacupuncture could specifically inhibit the activation of the Rho/ROCK signaling pathway thus partially contributing to the repair of injured spinal cord. Monosialoganglioside could promote the motor function but did not suppress expression of Rho A and ROCKⅡ. There was no synergistic effect of electroacupuncture combined with monosialoganglioside.

Nrf2-inducing and HMG-CoA reductase inhibitory activities of a polyphenol-rich fraction of Guazuma ulmifolia leaves

Objective: To fractionate and identify polyphenols from Guazuma ulmifolia Lam. leaves, and to explore their antioxidant, 5-hydroxy-3-methylglutaryl-coenzyme A(HMG-Co A) reductase inhibitory, and Nrf2 modulatory activities.Methods: The 1,1-diphenyl-2-picrylhydrazyl assay was used to evaluate the antioxidant activity of a polyphenolic fraction of the extract of Guazuma ulmifolia Lam. leaves. THP-1 gene reporter cell lines constructed with a transcriptional response element specific for Nrf2 and a minimal promoter for the firefly luciferase–green fluorescent protein transgene were used to determine the effect of the polyphenolic fraction on the Nrf2 signaling pathway. Furthermore, an assay of HMG-Co A reductase inhibitory activity was performed by using a commercial enzyme kit. Polyphenolic compounds were identified by liquid chromatographytandem mass spectrometry.Results: The polyphenolic fraction showed fairly strong antioxidant activity [IC50 =(14.90 ± 4.70) μg/m L] and inhibited HMG-Co A reductase activity by 69.10%, which was slightly lower than that by pravastatin(84.37%) and quercetin(84.25%). Additionally, the polyphenolic fraction activated the Nrf2 antioxidant signaling pathway at 500 μg/m L. Eleven subfractions resulting from the column chromatography separation of the polyphenolic fraction also showed relatively strong antioxidant activities(IC50: 17.46–217.14 μg/m L). The subfraction(F6) stimulated the Nrf2 signaling pathway and had HMG-Co A reductase inhibitory activity(65.43%). Moreover, the subfraction contained two main flavonoids: quercetin and quercimeritrin.Conclusions: The polyphenolic fraction of Guazuma ulmifolia could induce antioxidant genes via the Nrf2/antioxidant regulatory elements pathway, and is a promising candidate for an inhibitor of HMG-Co A reductase.

A Class of High-affinity Bicyclooctane G551D-CFTR Activators Identified by High Throughput Screening

The glycine-to-aspartic acid missense mutation at the codon 551(G551D) of the cystic fibrosis transmembrane conductance regulator(CFTR) is one of the five most frequent cystic fibrosis(CF) mutations associated with a severe CF phenotype. To explore the feasibility of pharmacological correction of disrupted activation of CFTR chloride channel caused by G551D mutation, we developed a halide-sensitive fluorescence miniassay for G551D-CFTR in Fisher rat thyroid(FRT) epithelial cells for the discovery of novel activators of G551D-CFTR. A class of bicyclooctane small molecule compounds that efficiently stimulate G551D-CFTR chloride channel activity was identified by high throughput screening via the FRT cell-based assay. This class of compounds selectively activates G551D-CFTR with a high affinity, whereas little effect of the compounds on wildtype CFTR can be seen. The discovery of a class of bicyclooctane G551D-CFTR activators will permit the analysis of structure-activity relationship of the compounds to identify ideal leads for in vivo therapeutic studies.

Optimized Conditions for the Delivery of Small Membrane Impermeable Compounds into Human Cells Using Hypotonic Shift

Cell-based assays represent a major end point of high throughput screening (HTS) but a key limitation of such assays is the potentially poor membrane permeability of test compounds. In this study, we optimized the conditions for the delivery of the membrane impermeable compound 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS) into human cells using hypotonic shift;a method that can promote the uptake of molecules from the extracellular fluid into cell cytoplasm via endocytosis. We showed that uptake of HPTS by cells was a function of hypotonic buffer osmolarity and that delivery was highly efficient with almost 100% of cells displaying uptake. Delivery of HPTS was equally effective at 25°C and 37°C, with delivery of compound proportional to incubation time and concentration of HPTS within the loading medium. The experimental conditions identified in this study could be applied to HTS drug discovery studies providing an effective method of delivering small membrane impermeable compounds into cells.

Creutzfeldt-Jakob disease presenting with bilateral hearing loss:A case report

BACKGROUND Sporadic Creutzfeldt-Jakob disease(s CJD)is a prion disease characterized as a fatal transmissible neurodegenerative disorder.Dizziness is often the first presenting symptom of s CJD,but hearing loss as an early manifestation is very rare.CASE SUMMARY A 76-year-old man presented with bilateral sudden hearing impairment and dizziness for 10 d.He was taking medications for hypertension and diabetes.He denied any difficulty with activities of daily living or hearing impairment before the onset of symptoms.Pure tone audiometry showed bilateral severe hearing impairment.Brain magnetic resonance imaging(MRI)and laboratory tests were within normal limits.Given his diagnosis of sudden sensory hearing loss,the patient received corticosteroid treatment but it was ineffective.Two weeks later,he complained of aggravated gait impairment,disorientation,and cognitive impairment.Repeat brain MRI showed diffuse cortical high signal intensities on diffusion-weighted imaging.In cerebrospinal fluid analysis,the real-time quaking-induced conversion assay was positive,and 14-3-3 protein was detected in the by western blotting.Considering all the data,we diagnosed probable s CJD,and the patient’s symptoms rapidly progressed into akinetic mutism.CONCLUSION For patients with abrupt bilateral hearing impairment,especially in the elderly,various differential diagnoses,including s CJD,should be considered.

Identified Bacteria and Virus in the Cerebrospinal Fluid of under Five Years Hospitalized Children for Clinical Meningitis at Panzi Hospital in the Eastern Part of DRC

Background: Meningitis remains a leading cause of death among children below 5 years of age in the Democratic Republic of the Congo (DR Congo). Distinguishing children with bacterial meningitis from those with viral meningitis in the emergency department is sometimes difficult. Here we identified bacteria and virus in the cerebral spinal fluid (CSF) of children with meningitis. Material and Methods: This is a prospective, analytical study carried out in the Pediatrics department of Panzi Hospital in the South-Kivu province of DR Congo. Between April 2021 and March 2022, 150 of 251 collected CSF from children aged from 1 to 59 months hospitalised due to clinical meningitis at Panzi referral university hospital, Bukavu, Eastern DR Congo were sent to the Lancet laboratory for bacteria identification by a multiplex real-time PCR assay for detection of the most different viruses and bacterial species causing meningitis. Result: The used multiplex real-time PCR assay allowed us to identify germs in 24.7% of cases (37/150). We isolated bacteria in 25/37 (67.5%) cases, and viruses in 9/37 (24.3%) while virus and bacteria co-infection was detected in 3/37 (8.1%). The most frequently identified bacteria were Streptococcus pneumoniae 14/37 (37.8%) followed by Haemophilus influenzae 6/37 (16.2%). The main virus was cytomegalovirus 5/37 (3.5%). Despite the age, the most found bacterial are common in children from rural areas and unvaccinated children. Bacterial and virus co-infection were identified in 66.7% of children aged between 25 - 60 months, mainly among male children, and in all children from rural areas (100%). The overall case fatality rate was 30% and was very high among cases with co-infection CMV-Pneumococcal (66.7%), followed by Streptococcus pneumoniae (50%). Conclusion: Meningitis remains frequent among children aged from one to 59 months among Bukavu Infants. We noticed that, Children with co-infection with bacteria and viruses might need higher attention when having meningitis symptoms, as this could lead to fatal outcomes. The introduction of molecular techniques, such as multiplex real-time PCR, has the potential to improve diagnosis and patient outcomes.

Rat cardiomyocyte H9c2(2-1)-based sulforhodamine B assay as a promising in vitro method to assess the biological component of effluent toxicity

The treatment of wastewaters is crucial to maintain the ecological status of receiving waters,and thereby guarantee the protection of aquatic life and human health.Wastewater quality evaluation is conventionally based on physicochemical parameters,but increasing attention has been paid to integrate physicochemical and biological data.Nevertheless,the regulatory use of fish in biological testing methods has been subject to various ethical and cost concerns,and in vitro cell-based assays have thus become an important topic of interest.Hence,the present study intends:(a) to evaluate the efficiency of two different sample pre-concentration techniques (lyophilisation and solid phase extraction) to assess the toxicity of municipal effluents on rat cardiomyoblast H9c2(2-1) cells,and (b) maximizing the use of the effluent sample collected,to estimate the environmental condition of the receiving environment.The gathered results demonstrate that the H9c2(2-1) sulforhodamine B-based assay is an appropriate in vitro method to assess biological effluent toxicity,and the best results were attained by lyophilising the sample as pre-treatment.Due to its response,the H9c2(2-1) cell line might be a possible alternative in vitro model for fish lethal testing to assess the toxicity of municipal effluents.The physicochemical status of the sample suggests a high potential for eutrophication,and iron exceeded the permissible level for wastewater discharge,possibly due to the addition of ferric chloride for wastewater treatment.In general,the levels of carbamazepine and sulfamethoxazole are higher than those reported for other countries,and both surpassed the aquatic protective values for long-term exposure.