Plasma membrane Ca^(2+)-ATPases:Targets of oxidative stress in brain aging and neurodegeneration

The plasma membrane Ca2+-ATPase(PMCA)pumps play an important role in the maintenance of precise levels of intracellular Ca2+[Ca2+]i,essential to the functioning of neurons.In this article,we review evidence showing age-related changes of the PMCAs in synaptic plasma membranes(SPMs).PMCA activity and protein levels in SPMs diminish progressively with increasing age. The PMCAs are very sensitive to oxidative stress and undergo functional and structural changes when exposed to oxidants of physiological relevance.The major signatures of oxidative modification in the PMCAs are rapid inactivation,conformational changes,aggregation, internalization from the plasma membrane and proteolytic degradation.PMCA proteolysis appears to be mediated by both calpains and caspases.The predominance of one proteolytic pathway vs the other,the ensuing pattern of PMCA degradation and its consequence on pump activity depends largely on the type of insult,its intensity and duration.Experimental reduction of PMCA expression not only alters the dynamics of cellular Ca2+ handling but also has a myriad of downstream conse-quences on various aspects of cell function,indicating a broad role of these pumps.Age-and oxidation-related down-regulation of the PMCAs may play an important role in compromised neuronal function in the aging brain and its several-fold increased susceptibility to neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease,and stroke.Therapeutic approaches that protect the PMCAs and stabilize[Ca2+]i homeostasis may be capable of slowing and/or preventing neuronal degeneration.The PMCAs are therefore emerging as a new class of drug targets for therapeutic interventions in various chronic degenerative disorders.

NF-κB Controls Resistance of Human Salivary Gland (HSG) Cells to Apoptosis in an <i>in Vitro</i>Model of Sjogren’s Syndrome

Aim: To elucidate the anti-apoptotic properties of nuclear factor kappa light-chain-enhancer of activated B cells (NF-κB) and feedback regulation of NF-κB by nuclear factor of kappa light-chain-enhancer of activated B-cells inhibitor alpha (IκBα). Methods: We developed an in vitro model of Sjogren’s syndrome by transfecting human salivary gland (HSG) and acinar cells (NS-SV-AC) with a plasmid-encoding IκBαM (pCMV-IκBαM), a degradation-resistant IκBα (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha)-mutant, and examined TNF-induced apoptosis and anti-apoptotic properties of NF-κB. Apoptosis and induction of pro-apoptotic and anti-apoptotic genes were investigated by cDNA arrays, RT-PCR, electrophoretic mobility shift assays, and western blot. Results: In the presence of NF-κB inhibitors, TNF-induced apoptosis was markedly increased in both salivary gland and acinar cells. Increased caspase-3 activity was present in both HSG and NS-SV-AC cells. IκBαM-transfected salivary gland cells were more sensitive to TNF-induced apoptosis than IκBαM-transfected acinar cells. Transcription of pro-apoptotic genes was confirmed in both HSG and NS-SV-AC cells that were transfected with IκBαM. Results from caspase-3 activity assay confirmed previous experiments showing an apoptotic role for NF-κB. Conclusion: Data from gene expression arrays suggest that different mechanisms may operate during TNF-induced apoptosis in salivary gland ductal and acinar cells.

Antibiotics Susceptibility Profile of Staphylococcus aureus Clinical Isolates Collected in Hospitals in the City of Douala (Cameroon)

Introduction: Staphylococcus aureus is one of the most important agents involved in community and hospital-acquired infections. Due to the multi-drug resistance of strains to antibiotics, treatment to eradicate it is becoming more difficult and poses a global public health problem. Methodology: This was a cross-sectional study conducted from March to August 2020 in hospitals in the city of Douala, including all S. aureus isolates from diagnostic samples. Strain identification and antibiotic susceptibility testing were performed using the Vitek2 CompactTM (BioMerieux). Results: During the study period, 136 non-repetitive S. aureus strains were identified with a high frequency of methicillin-resistant S. aureus of 78.7%. The majority of the strains originated from the Douala General Hospital (66.9%) and was most frequently isolated from blood culture samples (55.1%). The study of biochemical characteristics showed that most of the strains identified had between 87% and 99% homology with the reference strain. The most active antibiotics were Quinupristin/Dalfopristin (94.2%), Linezolid (87.8%) and Vancomycin (84.2%). Methicillin resistance was associated with decreased susceptibility of S. aureus to other antibiotics such as Gentamycin (44.9%), Erythromycin (38.2%), Tetracycline (38.3%), Trimethoprim (21.4%), Ciprofloxacin (19.1%) and Levofloxacin (24.0%). Inducible MLSb and constitutive resistance phenotypes were identified with 26.7% and 22.8% respectively. Conclusion: The sensitivity of S. aureus strains differs from one antibiotic family to another, and remains good for molecules that are not available in our context. The high frequency of Methicillin-Resistant S. aureus shows the continuous progression of multi-resistant strains of S. aureus and their decreased sensitivity to usual antibiotics becomes more and more alarming.

Neuronal protection by a variant of GAPDH pseudogene P44 in AD

GAPDH is a conserved enzyme that binds diverse proteins, such as Siah during apoptotic nuclear translocation. There is one somatic GAPDH gene, but over 60 pseudogenes, the expression of which is nebulous. A single nucleotide polymorphism (SNP) in the GAPDHP44 pseudogene exhibits a beneficial allele in AD. The objective of this study was to examine the P44 gene and to propose a mechanism for the putative protein and its impact on AD. We examined the sequences in the putative coding region of the human GAPDHP44 gene and the upstream genetic elements usinga bioinformatics approach. We compared the amino acid sequences of the putative gene product with that of the parent GAPDH protein. There is a TATA box 24 nt upstream from, and a Kozak sequence at, putative transcription and translation start sites, respecttively. The upstream region also has sequences (7 - 16 nt) paralogous to those in parent gene introns;one shows homology to a known enhancer element. The resulting protein would contain 139 aa due to a stop codon, roughly the same size as the dinucleotide domain (151 aa) of the parent protein. The SNP is in a region (residues 80 - 120) that binds to the protein GOSPEL. We propose that the beneficial SNP may cause a glutamine to glutamate substitution. NMDA-stmulated neurons undergo GAPDH nitrosylation, Siah translocation, but can be rescued by GOSPEL binding to GAPDH. Our model suggests that the putative P44 protein may regulate GAPDH-GO-SPEL interaction and the beneficial SNPmay ameliorate AD.

Integrated chemical and transcriptomic analyses unveils synthetic characteristics of different medicinal root parts of Angelica sinensis

Objective:Why are different medicinal parts including heads,bodies and tails of Angelicae Sinensis Radix(ASR)distinct in pharmaceutical activities?Here we explored their discrepancy in chemical constituents and transcriptome.Methods:ASR were separated into three medicinal parts:heads(rootstocks with petiole traces of ASR),bodies(taproots of ASR)and tails(lateral roots of ASR),and chemical and transcriptomic analyses were conducted simultaneously.Results:High performance liquid chromatography(HPLC)fingerprint results showed that five widely used active ingredients(ferulic acid,senkyunolide H,senkyunolide A,n-butylphathlide,and ligustilide)were distributed unevenly in the three ASR medicinal parts.Partial least squares-discriminant analysis(PLS-DA)demonstrated that the heads can be differentiated from the two other root parts due to different amounts of the main components.However,the content of ferulic acid(a main quality marker)was significantly higher in tails than in the heads and bodies.The transcriptome analysis found that 25,062,10,148 and 29,504 unigenes were specifically expressed in the heads,bodies and tails,respectively.WGCNA analysis identified 17 co-expression modules,which were constructed from the 19,198 genes in the nine samples of ASR.Additionally,we identified 28 unigenes involved in two phenylpropanoid biosynthesis(PB)pathways about ferulic acid metabolism pathways,of which 17 unigenes(60.7%)in the PB pathway were highly expressed in the tails.The expression levels of PAL,C3 H,and CQT transcripts were significantly higher in the tails than in other root parts.RT-q PCR analysis confirmed that PAL,C3 H,and CQT genes were predominantly expressed in the tail parts,especially PAL,whose expression was more than doubled as compared with that in other root parts.Conclusion:Chemical and transcriptomic analyses revealed the distribution contents and pivotal transcripts of the ferulic acid biosynthesis-related pathways.The spatial gene expression pattern partially explained the discrepancy of integral medicinal activities of three medicinal root parts.

Bone graft substitutes for spine fusion: A brief review

Bone graft substitutes are widely used in the field of orthopedics and are extensively used to promote vertebral fusion. Fusion is the most common technique in spine surgery and is used to treat morbidities and relieve discomfort. Allograft and autograft bone substitutes are currently the most commonly used bone grafts to promote fusion. These approaches pose limitations and present complications to the patient. Numerous alternative bone graft substitutes are on the market or have been developed and proposed for application. These options have attempted to promote spine fusion by enhancing osteogenic properties. In this review, we reviewed biology of spine fusion and the current advances in biomedical materials and biological strategies for application in surgical spine fusion. Our findings illustrate that, while many bone graft substitutes perform well as bone graft extenders, only osteoinductive proteins(recombinant bone morphogenetic proteins-2 and osteogenic protein-1) provide evidence for use as both bone enhancers and bone substitutes for specific types of spinal fusion. Tissue engineered hydrogels, synthetic polymer composites and viral based gene therapy also holds the potential to be used for spine fusion in future, though warrants further investigation to be used in clinical practice.

Malaria over-diagnosis in Cameroon:diagnostic accuracy of Fluorescence and Staining Technologies(FAST)Malaria Stain and LED microscopy versus Giemsa and bright field microscopy validated by polymerase chain reaction

Background:Malaria is a major world health issue and its continued burden is due,in part,to difficulties in the diagnosis of the illness.The World Health Organization recommends confirmatory testing using microscopy-based techniques or rapid diagnostic tests(RDT)for all cases of suspected malaria.In regions where Plasmodium species are indigenous,there are multiple etiologies of fever leading to misdiagnoses,especially in populations where HIV is prevalent and children.To determine the frequency of malaria infection in febrile patients over an 8-month period at the Regional Hospital in Bamenda,Cameroon,we evaluated the clinical efficacy of the Flourescence and Staining Technology(FAST)Malaria stain and ParaLens AdvanceTM microscopy system(FM)and compared it with conventional bright field microscopy and Giemsa stain(GS).Methods:Peripheral blood samples from 522 patients with a clinical diagnosis of“suspected malaria”were evaluated using GS and FM methods.A nested PCR assay was the gold standard to compare the two methods.PCR positivity,sensitivity,specificity,positive predictive value(PPV),and negative predictive value(NPV)were determined.Results:Four hundred ninety nine samples were included in the final analysis.Of these,30 were positive via PCR(6.01%)with a mean PPV of 19.62%and 27.99%for GS and FM,respectively.The mean NPV was 95.01%and 95.28%for GS and FM,respectively.Sensitivity was 26.67%in both groups and specificity was 92.78%and 96.21%for GS and FM,respectively.An increased level of diagnostic discrepancy was observed between technicians based upon skill level using GS,which was not seen with FM.Conclusions:The frequency of malarial infections confirmed via PCR among patients presenting with fever and other symptoms of malaria was dramatically lower than that anticipated based upon physicians’clinical suspicions.A correlation between technician skill and accuracy of malaria diagnosis using GS was observed that was less pronounced using FM.Additionally,FM increased the specificity and improved the PPV,suggesting this relatively low cost approach could be useful in resource-limited environments.Anecdotally,physicians were reluctant to not treat all patients symptomatically before results were known and in spite of a negative microscopic diagnosis,highlighting the need for further physician education to avoid this practice of overtreatment.A larger study in an area with a known high prevalence is being planned to compare the two microscopy methods against available RDTs.

Housing Environment Affects Baseline Anxiety in Zebrafish

Neuroscience researchers that wish to address compelling questions in psychopharmacology would benefit from the use of zebrafish, which have a behavioral repertoire that is rich and complex and that reflects many fundamental processes in humans, such as those that evoke anxiety. Zebrafish behavior is easily quantified in diverse test environments, but it is important to consider the conditions used to house the fish, prior to the behavioral testing, when designing experiments. Studies show that fish housed at high densities exhibit elevated cortisol levels as compared with fish housed at lower densities. The literature is less clear about the behavioral consequences of housing and handling. We examined the effects of housing (group versus paired) on several behavioral parameters. We observed that group-housed fish exhibited an anxious phenotype as evidenced by tank diving and meandering. Other more general indicators of movement and spatial navigation were unaffected by housing. This study focused on two specific housing environments, and the results support the need for continued research in this area to further elucidate the contributions of housing and handling on the subtleties of zebrafish behavioral markers.