Cytomegalovirus seropositivity drives the CD8 T cell repertoire toward greater clonality in healthy elderly individuals

The deterioration in immune function with aging is thought to make a major contribution to the increased morbidity and mortality from infectious disease in old age. One aspect of immune senescence is the reduction in CD8 T cell repertoire as due to the accumulation of oligoclonal, memory T cells and a reduction in the naive T cell pool. CD8 T cell clonal expansions accumulate with age, but their antigenic specificity remains unknown. In this study, we show that in elderly individuals seropositivity for human CMV leads to the development of oligoclonal populations of CMV-specific CTL that can constitute up to one-quarter of the total CD8 T cell population. Furthermore, CMV-specific CTL have a highly polarized membrane phenotype that is typical of effector memory cells (CD28(-), CD57(+), CCR7(-)). TCR analyses show that CMV-specific CTL have highly restricted clonality with greater restriction in the larger expansions. Clonal analysis of the total CD8 T cell repertoire was compared between CMV-seropositive and CMV-seronegative donors. Thirty-three percent more clonal expansions were observed in CMV-seropositive donors in comparison with seronegative individuals. These data implicate CMV as a major factor in driving oligoclonal expansions in old age. Such a dramatic accumulation of virus-specific effector CTL might impair the ability to respond to heterologous infection and may underlie the negative influence of CMV seropositivity on survival in the very elderly.     

DNA fragmentation, an indicator of apoptosis, in cultured black tiger shrimp Penaeus monodon infected with white spot syndrome virus (WSSV)

Fifty black tiger shrimp Penaeus monodon from commercial cultivation ponds in Malaysia were examined by Tdt-mediated dUTP nick-end labeling (TUNEL) fluorescence assay and agarose gel electrophoresis of DNA extracts for evidence of DNA fragmentation as an indicator of apoptosis. From these specimens, 30 were grossly normal and 20 showed gross signs of white spot syndrome virus (WSSV) infection. Of the 30 grossly normal shrimp, 5 specimens were found to be positive for WSSV infection by normal histology and by nested polymerase chain reaction (PCR) analysis. All of the specimens showing gross signs of WSSV infection were positive for WSSV by normal histology, while 5 were positive by nested PCR only (indicating light infections) and 15 were positive by 1-step PCR (indicating heavy infections). Typical histological signs of WSSV infection included nuclear hypertrophy, chromatin condensation and margination. None of the 25 grossly normal shrimp negative for WSSV by 1-step PCR showed any signs of DNA fragmentation by TUNEL assay or agarose gel electrophoresis of DNA extracts. The 10 specimens that gave PCR-positive results for WSSV by nested PCR only (i.e., 5 grossly normal shrimp and 5 grossly positive for WSSV) gave mean counts of 16 +/- 8% TUNEL-positive cells, while the 25 specimens PCR positive by 1-step PCR gave mean counts of 40 +/- 7% TUNEL-positive cells. Thus, the number of TUNEL positive cells present in tissues increased with increasing severity of infection, as determined by gross signs of white spots on the cuticle, the number of intranuclear inclusions in histological sections, and results from single and nested PCR assays. DNA extracts of PCR-positive specimens tested by agarose gel electrophoresis showed indications of DNA fragmentation either as smears or as 200 bp ladders. Given that DNA fragmentation is generally considered to be a hallmark of apoptosis, the results suggested that apoptosis might be implicated in shrimp death caused by WSSV.     

Protein engineering of the colony-stimulating factor-1 receptor kinase domain for structural studies

A parallel approach to designing crystallization constructs for the c-FMS kinase domain was implemented, resulting in proteins suitable for structural studies. Sequence alignment and limited proteolysis were used to identify and eliminate unstructured and surface-exposed domains. A small library of chimeras was prepared in which the kinase insert domain of FMS was replaced with the kinase insert domain of previously crystallized receptor-tyrosine kinases. Characterization of the newly generated FMS constructs by enzymology and thermoshift assays demonstrated similar activities and compound binding to the FMS full-length cytoplasmic domain. Two chimeras were evaluated for crystallization in the presence and absence of a variety of ligands resulting in crystal structures, and leading to a successful structure-based drug design project for this important inflammation target.     

Crystal structure of a soluble form of human monoglyceride lipase in complex with an inhibitor at 1.35 Å resolution

A high-resolution structure of a ligand-bound, soluble form of human monoglyceride lipase (MGL) is presented. The structure highlights a novel conformation of the regulatory lid-domain present in the lipase family as well as the binding mode of a pharmaceutically relevant reversible inhibitor. Analysis of the structure lacking the inhibitor indicates that the closed conformation can accommodate the native substrate 2-arachidonoyl glycerol. A model is proposed in which MGL undergoes conformational and electrostatic changes during the catalytic cycle ultimately resulting in its dissociation from the membrane upon completion of the cycle. In addition, the study outlines a successful approach to transform membrane associated proteins, which tend to aggregate upon purification, into a monomeric and soluble form.     

Candidate nsSNPs that can affect the functions and interactions of cell cycle proteins

Nonsynonymous single nucleotide polymorphisms (nsSNPs) alter the encoded amino acid sequence, and are thus likely to affect the function of the proteins, and represent potential disease-modifiers. There is an enormous number of nsSNPs in the human population, and the major challenge lies in distinguishing the functionally significant and potentially disease-related ones from the rest. In this study, we analyzed the genetic variations that can alter the functions and the interactions of a group of cell cycle proteins (n = 60) and the proteins interacting with them (n = 26) using computational tools. As a result, we extracted 249 nsSNPs from 77 cell cycle proteins and their interaction partners from public SNP databases. Only 31 (12.4%) of the nsSNPs were validated. The majority (64.5%) of the validated SNPs were rare (minor allele frequencies < 5%). Evolutionary conservation analysis using the SIFT tool suggested that 16.1% of the validated nsSNPs may disrupt the protein function. In addition, 58% of the validated nsSNPs were located in functional protein domains/motifs, which together with the evolutionary conservation analysis enabled us to infer possible biological consequences of the nsSNPs in our set. Our study strongly suggests the presence of naturally occurring genetic variations in the cell cycle proteins that may affect their interactions and functions with possible roles in complex human diseases, such as cancer.     

Determinants of Compliance Behaviours among Patients Undergoing Hemodialysis in Malaysia

Background

Patients with end stage renal disease often fail to follow prescribed dietary and fluid regimen, leading to undesirable outcomes. This study aimed to examine and identify factors influencing dietary, fluid, medication and dialysis compliance behaviours in patients undergoing hemodialysis.

Methods

This was a cross-sectional study which employed purposive sampling design. A total of 188 respondents were recruited from 14 dialysis centres in Malaysia between 2008–2011. Self-reported compliance behaviours and biochemical measurements were used as evaluation tools.

Results

Compliance rates of dietary, fluid, medication and dialysis were 27.7%, 24.5%, 66.5% and 91.0%, respectively. Younger, male, working patients and those with longer duration on hemodialysis were found more likely to be non-compliant. Lacks of adequate knowledge, inadequate self-efficacy skills, forgetfulness and financial constraints were the major perceived barriers towards better compliance to fluid, dietary, medication and dialysis, respectively.

Conclusions

Healthcare professionals should recognise the factors hindering compliance from the patients'' perspective while assisting them with appropriate skills in making necessary changes possible.     

Is Content Really King? An Objective Analysis of the Public's Response to Medical Videos on YouTube

Medical educators and patients are turning to YouTube to teach and learn about medical conditions. These videos are from authors whose credibility cannot be verified & are not peer reviewed. As a result, studies that have analyzed the educational content of YouTube have reported dismal results. These studies have been unable to exclude videos created by questionable sources and for non-educational purposes. We hypothesize that medical education YouTube videos, authored by credible sources, are of high educational value and appropriately suited to educate the public. Credible videos about cardiovascular diseases were identified using the Mayo Clinic''s Center for Social Media Health network. Content in each video was assessed by the presence/absence of 7 factors. Each video was also evaluated for understandability using the Suitability Assessment of Materials (SAM). User engagement measurements were obtained for each video. A total of 607 videos (35 hours) were analyzed. Half of all videos contained 3 educational factors: treatment, screening, or prevention. There was no difference between the number of educational factors present & any user engagement measurement (p NS). SAM scores were higher in videos whose content discussed more educational factors (p<0.0001). However, none of the user engagement measurements correlated with higher SAM scores. Videos with greater educational content are more suitable for patient education but unable to engage users more than lower quality videos. It is unclear if the notion “content is king” applies to medical videos authored by credible organizations for the purposes of patient education on YouTube.     

Hypothalamic Sirt1 protects terminal Schwann cells and neuromuscular junctions from age‐related morphological changes

Neuromuscular decline occurs with aging. The neuromuscular junction (NMJ), the interface between motor nerve and muscle, also undergoes age‐related changes. Aging effects on the NMJ components—motor nerve terminal, acetylcholine receptors (AChRs), and nonmyelinating terminal Schwann cells (tSCs)—have not been comprehensively evaluated. Sirtuins delay mammalian aging and increase longevity. Increased hypothalamic Sirt1 expression results in more youthful physiology, but the relationship between NMJ morphology and hypothalamic Sirt1 was previously unknown. In wild‐type mice, all NMJ components showed age‐associated morphological changes with ~80% of NMJs displaying abnormalities by 17 months of age. Aged mice with brain‐specific Sirt1 overexpression (BRASTO) had more youthful NMJ morphologic features compared to controls with increased tSC numbers, increased NMJ innervation, and increased numbers of normal AChRs. Sympathetic NMJ innervation was increased in BRASTO mice. In contrast, hypothalamic‐specific Sirt1 knockdown led to tSC abnormalities, decreased tSC numbers, and more denervated endplates compared to controls. Our data suggest that hypothalamic Sirt1 functions to protect NMJs in skeletal muscle from age‐related changes via sympathetic innervation.