In vitro engineered models of neurodegenerative diseases

Neurodegeneration is a catastrophic process that develops progressive damage leading to functional andstructural loss of the cells of the nervous system and is among the biggest unavoidable problems of our age.Animalmodels do not reflect the pathophysiology observed in humans due to distinct differences between the neuralpathways,gene expression patterns,neuronal plasticity,and other disease-related mechanisms in animals andhumans.Classical in vitro cell culture models are also not sufficient for pre-clinical drug testing in reflecting thecomplex pathophysiology of neurodegenerative diseases.Today,modern,engineered techniques are applied to developmulticellular,intricate in vitro models and to create the closest microenvironment simulating biological,biochemical,and mechanical characteristics of the in vivo degenerating tissue.In THIS review,the capabilities and shortcomings ofscaffold-based and scaffold-free techniques,organoids,and microfluidic models that best reflect neurodegeneration invitro in the biomimetic framework are discussed.

Catalpa bignonioides extract improves exercise performance through regulation of growth and metabolism in skeletal muscles

Objective:To evaluate the effects of Catalpa bignonioides fruit extract on the promotion of muscle growth and muscular capacity in vitro and in vivo.Methods:Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.Cell proliferation was assessed using a 5-bromo-2’-deoxyuridine(BrdU)assay kit.Western blot analysis was performed to determine the protein expressions of related factors.The effects of Catalpa bignonioides extract were investigated in mice using the treadmill exhaustion test and whole-limb grip strength assay.Chemical composition analysis was performed using high-performance liquid chromatography(HPLC).Results:Catalpa bignonioides extract increased the proliferation of C2C12 mouse myoblasts by activating the Akt/mTOR signaling pathway.It also induced metabolic changes,increasing the number of mitochondria and glucose metabolism by phosphorylating adenosine monophosphate-activated protein kinase.In an in vivo study,the extract-treated mice showed improved motor abilities,such as muscular endurance and grip strength.Additionally,HPLC analysis showed that vanillic acid may be the main component of the Catalpa bignonioides extract that enhanced muscle strength.Conclusions:Catalpa bignonioides improves exercise performance through regulation of growth and metabolism in skeletal muscles,suggesting its potential as an effective natural agent for improving muscular strength.

Resilience-Oriented Approach to the Control of Ventricular Assist Devices

Context: Advanced heart failure (AHF) poses a global challenge, where heart transplantation is a treatment option but limited by donor scarcity. Proposal: This study aims to enhance the performance of ventricular assist devices (VADs) in the face of adverse events (AEs) using a resilience-based approach. The objective is to develop a method for integrating resilience attributes into VAD control systems, employing dynamic risk analysis and control strategies. Results: The outcomes include a resilient control architecture enabling anticipatory, regenerative, and degenerative actions in response to AEs. A method of applied resilience (MAR) based on dynamic risk management and resilience attribute analysis was proposed. Conclusion: Dynamic integration between medical and technical teams allows continuous adaptation of control systems to meet patient needs over time, improving reliability, safety, and effectiveness of VADs, with potential positive impact on the health of heart failure patients.

Longitudinal Evaluation of Hemiplegic Ankle Rehabilitation Efficacy by Wearable Inertial Sensor Systems with an Assortment of Machine Learning Algorithms

With the amalgamation of wearable systems equipped with inertial sensors, such as a gyroscope, and machine learning a therapy regimen can be objectively quantified, and then the initial phase and final phase of a one year therapy regimen can be distinguished through machine learning. In the context of rehabilitation of a hemiplegic ankle, a longitudinal therapy regimen incorporating stretching and then a series of repetitions for raising and lowering the foot of the hemiplegic ankle can be applied over the course of a year. Using a smartphone equipped with an application to function as a wearable and wireless gyroscope platform mounted to the dorsum of the foot by an armband, the initial phase and final phase of a one year longitudinally applied therapy regimen can be objectively quantified and recorded for subsequent machine learning. Considerable classification accuracy is attained to distinguish between the initial phase and final phase by a support vector machine for a one year longitudinally applied hemiplegic ankle therapy regimen based on the gyroscope signal data obtained by a smartphone functioning as a wearable and wireless inertial sensor system. .

Flow cytometric detection of hepatitis C virus antigens in infected peripheral blood leukocytes: Binding and entry

AIM： We designed two synthetic-core-specific peptides core 1 （C1） and core 2 （C2）, and an E1-specific peptide （El）. We produced specific polyclonal antibodies against these peptides and used the antibodies for detection of HCV antigens on surface and within infected peripheral blood leukocytes. METHODS： Peripheral blood from a healthy individual who tested negative for HCV RNA was incubated with HCV type 4 infected serum for i h and 24 h at 37 ℃. Cells were stained by direct and indirect immunofluorescence and measured by flow cytometry. RESULTS： After 1 h of incubation, antibodies against C1, C2, and El detected HCV antigens on the surface of 27%, 26% and 73% of monocytes respectively, while 10%, 5% and 9% of lymphocytes were positive with anti-C1, anti-C2 and anti-E1 respectively. Only 1-3% of granulocytes showed positive staining with anti-C1, anti-C2 and anti E1 antibodies. After 24 h of incubation, we found no surface staining with anti-C1, anti-C2 or anti-E1. Direct immunostaining using anti-C2 could not detect intracellular HCV antigens, after 1 h of incubation with the virus, while after 24 h of incubation, 28% of infected cells showed positive staining. Only plus strand RNA was detectable intracellularly as early as 1 h after incubation, and remained detectable throughout 48 h post-infection. Interestingly, minus RNA strand could not be detected after 1 h, but became strongly detectable intracellularly after 24 h post-infection. CONCLUSION： Monocytes and lymphocytes are the preferred target cells for HCV infection in peripheral blood leukocytes. Our specific anti-core and anti-E1 antibodies are valuable reagents for demonstration of HCV cell cycle. Also, HCV is capable of infecting and replicating in peripheral blood mononuclear cells as confirmed by detection of minus strand HCV RNA as well as intracellular staining of core HCV antigen.

HepG2 cells support viral replication and gene expression of hepatitis C virus genotype 4 in vitro

AIM： TO establish a cell culture system with longterm replication of hepatitis C virus （HCV） genome and expression of viral antigens in vitro. METHODS： HepG2 cell line was tested for its susceptibility to HCV by incubation with a serum from a patient with chronic hepatitis C. Cells and supernatant were harvested at various time points during the culture. Culture supernatant was tested for its ability to infect na＇ive cells. The presence of minus （antisense） RNA strand, and the detection of core and E1 antigens in cells were examined by RT-PCR and immunological techniques （flow cytometry and Western blot） respectively. RESULTS： The intracellular HCV RNA was first detected on d 3 after infection and then could be consistently detected in both cells and supernatant over a period of at least three months. The fresh cells could be infected with supernatant from cultured infected cells. Flow cytometric analysis showed surface and intracellular HCV antigen expression using in house made polyclonal antibodies （anti-core, and anti-E1）. Western blot analysis showed the expression of a cluster of immunogenic peptides at molecular weights extended between 31 and 45 kDa in an one month old culture of infected cells whereas this cluster was undetectable in uninfected HepG2 cells. CONCLUSION： HepG2 cell line is not only susceptible to HCV infection but also supports its replication in vitro. Expression of HCV structural proteins can be detected in infected HepG2 cells. These cells are also capable of shedding viral particles into culture media which in turn become infectious to uninfected cells.

Positional effect of mutations in 5'UTR of hepatitis C virus 4a on patients' response to therapy

AIM： To investigate the effects of mutations in domain Ⅲ of the hepatitis C virus （HCV） internal ribosome entry sequences （IRES） on the response of chronic HCV genotype 4a patients to interferon therapy.METHODS： HCV RNA was extracted from 19 chronic HCV 4a patients receiving interferon/ribavirin therapy who showed dramatic differences in their response to combination therapy after initial viral clearance. IRES domain Ⅲ was cloned and 15 clones for each patient were sequenced. The obtained sequences were aligned with genotype 4a prototype using the ClustaIW program and mutations scored. Prediction of stem-loop secondary structure and thermodynamic stability of the major quasispecies in each patient was performed using the MFOLD 3.2 program with Turner energies and selected constraints on base pairing.RESULTS： Analysis of RNA secondary structure revealed that insertions in domain Ⅲ altered WatsonCrick base pairing of stems and reduced molecular stability of RNA, which may ultimately reduce binding affinity to ribosomal proteins. Insertion mutations in domain - were statistically more prevalent in sustained viral response patients （SVR, n = 14） as compared to breakthrough （BT, n = 5） patients.CONCLUSION： The influence of mutations within domain Ⅲ on the response of HCV patients to combination therapy depends primarily on the position, but not the frequency, of these mutations within IRES domain Ⅲ.

Antibody to El peptide of hepatitis C virus genotype 4 inhibits virus binding and entry to HepG2 cells in vitro

AIM： To analyze the neutralizing activity of antibodies against E1 region of hepatitis C virus （HCV）. Specific polydonal antibody was raised via immunization of New Zealand rabbits with a synthetic peptide that had been derived from the E1 region of HCV and was shown to be highly conserved among HCV published genotypes. METHODS： Hyper-immune HCV E1 antibodies were incubated over night at 4 ℃ with serum samples positive for HCV RNA, with viral loads ranging from 615 to 3.2 million IU/mL. Treated sera were incubated with HepG2 cells for 90 min. Blocking of viral binding and entry into cells by anti E1 antibody were tested by means of RTPCR and flow cytometry. RESULTS： Direct immunostaining using FITC conjugated E1 antibody followed by Flow cytometric analysis showed reduced mean fluorescence intensity in samples pre-incubated with E1 antibody compared with untreated samples. Furthermore, 13 out of 18 positive sera （72%） showed complete inhibition of infectivity as detected by RT-PCR. CONCLUSION： In house produced E1 antibody, blocks binding and entry of HCV virion infection to target cells suggesting the involvement of this epitope in virus binding and entry. Isolation of these antibodies that block virus attachment to human cells are useful as therapeutic reagents.

Evaluation of liver tissue by polymerase chain reaction for hepatitis B virus in patients with negative viremia

AIM: To assess the clinical significance of Hepatitis B virus (HBV) DNA localization in the liver tissue of patients with positive HBsAg and negative viremia.METHODS: HBV virological parameters of 33 HBsAg positive chronic hepatitis patients, including seromarkers and HBV DNA amplification in both sera and liver biopsies, were evaluated.RESULTS: Ten patients had negative viremia and positive HBV DNA in their liver biopsies. Most of them had HBeAg-negative/HBeAb-positive chronic hepatitis.Their liver biochemical and histopathological profiles were different from the viremic patients. Their disease pattern was designated as 'hepatitis B in situ'.CONCLUSION: Hepatitis B in situ is a consequential entity which can be missed in clinical practice. It is a new clinical pattern of chronic HBV infection that considers HBV in liver biopsy and adds a new indication for antiviral therapy.

Effect of <i>Chromolaena odorata</i>Extracton Hematotoxicity and Spleen Histopathology Induced by <i>Salmonella typhi</i>in Wistar Rats

Salmonella typhi is a facultative intracellular pathogen that causes typhoid fever in humans. In the present study, the effect of Salmonella typhi infection on hematological indices and spleen histology in Wistar rats was investigated and was followed by an evaluation of the ameliorative potential of the methanol leaf extract of Chromolaena odorata (MLECO) compared with ciprofloxacin treatment. The animals were divided into six groups: group 1 was normal control, group 2 was infected with Salmonella typhi without treatment (negative control), groups 3, 4 and 5 were Salmonella typhi infected and treated with 100 mg/kg, 200 mg/kg and 400 mg/kg of the extract respectively and group 6 was also Salmonella typhi infected and treated with 500 mg/70kg of ciprofloxacin. The animals were inoculated with a single infectious dose of Salmonella typhi bacteria and thereafter, treated with the extract and ciprofloxacin for a period of seventeen days, after the animals were confirmed infected. The rats were humanely sacrificed and blood samples taken for haematological investigations, and the spleen harvested and processed for histological examinations. Chromolaena odorata administration reversed the adverse hematotoxicity and histopathological changes in the spleen induced by?Salmonella typhi?infection.

The Frequency of Survivorship in Heterozygous Diploids of Cdc13-1exo1Δ Mutants of S. cerevisiae Is One Survivor Cell in 72 Cells/Generation at 36°C

Telomeres cap ends of eukaryotic chromosomes prevent them from degradation and ensure genomic stability. Cdc13 is an essential telomere recruitment and maintenance protein. A temperature-sensitive point mutation in cdc13 gene leads to telomere impairment, giving rise to cdc13-1 mutants that suffer lethality at enhanced temperatures. Deleting Exo1 gene from these mutants, however, leads to the emergence of temperature-tolerant mutants called survivors. Yeasts are known to exist as either diploids or haploids. These yeast genotypes generate survivors. The frequency of survivorship in the haploid genotype is one cell in 104 cells/generation at 36°C, however, the frequency at which they emerge in their diploid counterparts at the same temperature is not known. In this study, we investigated the frequency of Survivorship in heterozygous diploids of cdc13-1exo1Δ mutants of S. cerevisiae at 36°C. Diploids were constructed by mating haploid strains of opposite mating type cdc13-1 exo1:LEU strains with strains of cdc13-1 exo1:HIS. The crosses were 1296 × 3181, 2561 × 3182, 1296 × 3182 and 2561 × 3181. Genetic markers and phenotypic appearance were considered while mating the mutant cells. Using a stick, a smear of one haploid strain was made on each YEPD plate labelled C2, C8, C9, D1, D14, and D15. A smear of another opposite mating type was made on the previous strain. They were mixed and allowed to mate overnight, before culturing on media lacking Luecine and Histidine (-L and -H). Survivors were generated by culturing these diploids at 36°C. Using SPSS 20.0 software for windows SPSS, 2011, the frequency was determined as one Survivor cell in 72 cells/generation, as their frequency of survivorship averaged 5.9 × 10-5 ± 0.04.

Visualization of the Machine Learning Process Using J48 Decision Tree for Biometrics through ECG Signal

The inherently unique qualities of the heart infer the candidacy for the domain of biometrics, which applies physiological attributes to establish the recognition of a person’s identity. The heart’s characteristics can be ascertained by recording the electrical signal activity of the heart through the acquisition of an electrocardiogram (ECG). With the application of machine learning the subject specific ECG signal can be differentiated. However, the process of distinguishing subjects through machine learning may be considered esoteric, especially for contributing subject matter experts external to the domain of machine learning. A resolution to this dilemma is the application of the J48 decision tree available through the Waikato Environment for Knowledge Analysis (WEKA). The J48 decision tree elucidates the machine learning process through a visualized decision tree that attains classification accuracy through the application of thresholds applied to the numeric attributes of the feature set. Additionally, the numeric attributes of the feature set for the application of the J48 decision tree are derived from the temporal organization of the ECG signal maxima and minima for the respective P, Q, R, S, and T waves. The J48 decision tree achieves considerable classification accuracy for the distinction of subjects based on their ECG signal, for which the machine learning model is briskly composed.

Target specificity of selective bioactive compounds in blocking α-dystroglycan receptor to suppress Lassa virus infection: an in silico approach

Lassa hemorrhagic fever,caused by Lassa mammarenavirus(LASV)infection,accumulates up to 5000 deaths every year.Currently,there is no vaccine available to combat this disease.In this study,a library of 200 bioactive compounds was virtually screened to study their drug-likeness with the capacity to block theα-dystroglycan(α-DG)receptor and prevent LASV influx.Following rigorous absorption,distribution,metabolism,and excretion(ADME)and quantitative structure-activity relationship(QSAR)profiling,molecular docking was conducted with the top ligands against theα-DG receptor.The compounds chrysin,reticuline,and 3-caffeoylshikimic acid emerged as the top three ligands in terms of binding affinity.Post-docking analysis revealed that interactions with Arg76,Asn224,Ser259,and Lys302 amino acid residues of the receptor protein were important for the optimum binding affinity of ligands.Molecular dynamics simulation was performed comprehensively to study the stability of the protein-ligand complexes.In-depth assessment of root-mean-square deviation(RMSD),root mean square fluctuation(RMSF),polar surface area(PSA),B-Factor,radius of gyration(Rg),solvent accessible surface area(SASA),and molecular surface area(MolSA)values of the protein-ligand complexes affirmed that the candidates with the best binding affinity formed the most stable protein-ligand complexes.To authenticate the potentialities of the ligands as target-specific drugs,an in vivo study is underway in real time as the continuation of the research.

Investigating the Bioburden of “Neglected” Hospital Low Contact Surfaces

Microbes inhabit every surface, reproduce, and if undisturbed, could form biofilm. Hospital contact surfaces have been reported to play a major role in the spread of healthcare-acquired infections (HAIs). Most studies on these surfaces as a route for the spread of nosocomial infections have focused on the high-contact surfaces. There is a paucity of information on the bioburden of “neglected” low-contact surfaces such as bedside bible, ward television, and ward clock, etc. This study was carried out to investigate the bioburden of “neglected” low-contact hospital surfaces and compare it with that of the high-contact surfaces. Using a sterile swab stick moistened in normal saline, we collected 400 samples from contact surfaces of 20 randomly selected hospitals in Owerri, southeast in Nigeria, and by standard microbiological methods and with reference to standard identification manuals, microbial species were isolated and characterized. The results show that the mean of the bioburden in cfu/square swabbed surface of these “neglected” low-contact surfaces is significantly higher (p = 0.005) than that of the high-contact surfaces which may be a result of target hygienic cleaning, with attention on the high-contact surfaces and the low-contact surfaces are often “neglected”. This result gives an insight into the continued prevalence of hospital-acquired infections as these “neglected” low-contact surfaces continue to serve as a reservoir for pathogenic microbes and a source of continued microbial contamination of hospital surfaces. It therefore calls for a revamp of existing hospital cleaning protocols and redesigning of cleaning regimes.

Oral frailty and neurodegeneration in Alzheimer’s disease

Frailty is a critical intermediate status of the aging process with a multidimensional and multisystem nature and at higher risk for adverse health-related outcomes,including falls,disability,hospitalizations,institutionalization,mortality,dementia,and Alzheimer’s disease.Among different frailty phenotypes,oral frailty has been recently suggested as a novel construct defined as a decrease in oral function with a coexisting decline in cognitive and physical functions.We briefly reviewed existing evidence on operational definitions of oral frailty,assessment and screening tools,and possible relationships among oral frailty,oral microbiota,and Alzheimer’s disease neurodegeneration.Several underlying mechanism may explain the oral health-frailty links including undernutrition,sarcopenia linked to both poor nutrition and frailty,psychosocial factors,and the chronic inflammation typical of oral disease.Oral microbiota may influence Alzheimer’s disease risk through circulatory or neural access to the brain and the interplay with periodontal disease,often causing tooth loss also linked to an increased Alzheimer’s disease risk.On this bases,COR388,a bacterial protease inhibitor targeting Porphyromonas gingivalis implicated in periodontal disease,is now being tested in a double-blind,placebocontrolled Phase II/III study in mild-to-moderate Alzheimer’s disease.Therefore,oral status may be an important contributor to general health,including Alzheimer’s disease and latelife cognitive disorders,suggesting the central role of preventive strategies targeting the novel oral frailty phenotype and including maintenance and improvement of oral function and nutritional status to reduce the burden of both oral dysfunction and frailty.

Molecular study on Y chromosome microdeletions in Egyptian males with idiopathic infertility

Aim: To determine the frequency of genetic deletions within the azoospermia factors in Egyptian infertile males. Methods: The Yq microdeletions in 33 infertile males with undetectable chromosomal anomalies were examined by mutiplex polymerase chain reaction (PCR). Deletions were confirmed using single PCR amplifications. Results: Four out of the total 33 (12 %) men had Yq11 microdeletions, thus supporting the average reported figures in other populations. Three of those 4 cases had single short tandem sequence deletions with discrete histological findings of their testes. Single sY272 deletion within AZFc was associated with Sertoli cell only syndrome, whereas a patient with isolated sY84 deletion within AZFa had immature testicular structure. The remaining case had a large deletion in AZFa-c and short stature. Conclusion: The present study supports the hypothesis that the Yqn encompasses genetic determinants of stature besides genes controlling spermatogenesis.

A computer-based image analysis for tear ferning featuring

The present work focuses on the development of a novel computer-based approach for tear ferning(TF)featuring.The original TF images of the recently developedfive-point grading scale have been used to assign a grade for any TF image automatically.A vector characteristic(VC)representing each grade was built using the reference images.A weighted combination between features selected from textures analysis using gray level co-occurrence matrix(GLCM),power spectrum(PS)analysis and linear specificity of the image were used to build the VC of each grade.A total of 14 features from texture analysis were used.PS at di®erent frequency points and number of line segments in each image were also used.Five features from GLCM have shown significant di®erences between the recently developed grading scale images which are:angular second moment at 0
and 45
,contrast,and correlation at 0
and 45
;thesefive features were all included in the characteristic vector.Three specific power frequencies were used in the VC because of the discrimination power.Number of line segments was also chosen because of dissimilarities between images.A VC for each grade of TF reference images was constructed and was found to be significantly different from each other's.This is a basic and fundamental step toward an automatic grading for computer-based diagnosis for dry eye.

Medical Diagnosis Expert System for Malaria and Related Diseases for Developing Countries

There is a strong need for cost-effective technologies to manage disease processes and thus reduce morbidity and mortality in the developing countries. Yet bringing intelligent healthcare informatics to bear on the dual problems of reducing healthcare costs and improving quality and outcomes is a challenge even in countries with a reasonably developed technology infrastructure. This paper focused at how appropriate an ap-plication of Medical Diagnosis Expert System (MDES) is to manage diseases in developing countries. MDES is usually designed to enable clinicians to identify diseases and describe methods of treatment to be carried out taking into account the user capability. The MDES described here is implemented using the C Language Integrated Production System (CLIPS). The CLIPS is an expert system, which has a shell composed of four modules: the user interface, the explanation system, the inference engine and the knowledge base editor. In the system, a number of patient cases will be selected as prototypes and stored in a separate database. The knowledge is acquired from literature review, human experts and the internet of the specific domain and is used as a base for analysis, diagnosis and recommendations.

Microsurgical tunica albuginea transplantation in an animal model

Several andrological diseases require surgical repair or reconstruction of tunica albuginea, which envelops the corpora cavernosa penis. Despite intense research efforts involving a variety of biological materials, such as skin, muscle aponeurosis, human dura mater, tunica vaginalis, and pericardium, engineered tunica albuginea suitable for graft use is yet to be obtained. The study investigates microsurgical tunica albuginea allotransplantation in an animal model with the purpose of creation of an organ-specific tissue bank to store penile tissue, from cadaveric donors and male-to-female trans-sexual surgery, for allogeneic transplantation. Materials were tunica albuginea tissue explanted from 15 donor rats, cryopreserved at -80~C, gamma-irradiated, and implanted in 15 recipient rats, of which three rats were used as controls. Penile grafts were explanted at different time intervals; after macroscopic evaluation of the organ, the grafts were processed to morphological, histochemical, and immunohistochemical examinations by light microscopy. Detection of pro-inflammatory cytokines was also performed. Examination of the tunica albuginea allografts collected 1, 3, or 6 months after surgery and of control tunica albuginea fragments showed that tunica albuginea implants achieved biointegration with adjacent tissue at all-time points. The integration of cryopreserved rat tunica albuginea allografts, documented by our study, encourages the exploration of tunica albuginea allotransplantation in humans. In conclusion, the effectiveness and reliability of the tunica albuginea conditioning protocol described here suggest the feasibility of setting up a tunica albuginea bank as a further tissue bank.