Eukaryotic Polyribosome Profile Analysis

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase or the elongation phase by a variety of cellular stresses such as amino acid starvation or growth factor withdrawal. Alternatively, translation of individual mRNAs can be regulated by mRNA localization or the presence of cognate microRNAs. Studies of protein synthesis frequently utilize polyribosome analysis to shed light on the mechanisms of translation regulation or defects in protein synthesis. In this assay, mRNA/ribosome complexes are isolated from eukaryotic cells. A sucrose density gradient separates mRNAs bound to multiple ribosomes known as polyribosomes from mRNAs bound to a single ribosome or monosome. Fractionation of the gradients allows isolation and quantification of the different ribosomal populations and their associated mRNAs or proteins. Differences in the ratio of polyribosomes to monosomes under defined conditions can be indicative of defects in either translation initiation or elongation/termination. Examination of the mRNAs present in the polyribosome fractions can reveal whether the cohort of individual mRNAs being translated changes with experimental conditions. In addition, ribosome assembly can be monitored by analysis of the small and large ribosomal subunit peaks which are also separated by the gradient. In this video, we present a method for the preparation of crude ribosomal extracts from yeast cells, separation of the extract by sucrose gradient and interpretation of the results. This procedure is readily adaptable to mammalian cells

Assessing Equity in Protected Area Governance: Approaches to Promote Just and Effective Conservation

With the inclusion of equity concerns in Aichi Target 11 of the Convention on Biological Diversity, equitable management has become an important objective for the world's protected areas. The way equity is defined and operationalised influences whether this strategic shift can help identify pathways commensurate with conservation effectiveness. We examined equity around a protected area in Laos, combining quantitative and qualitative methods to explore the three dimensions of procedure, recognition and distribution. Local understandings of equity depended on discrete, evolving issues, with attention to informal decision making and dynamic values required to uncover suitable solutions. We show that equity definitions focused on material distribution and assessments reliant on standardised indicators may result in inadequate responses that sustain local perceptions of inequitable management and miss opportunities for effective conservation. Equity should be considered a management goal to continually adapt towards, informed by stakeholder dialogue

Discriminating single-base difference miRNA expressions using microarray Probe Design Guru (ProDeG)

MicroRNAs (miRNA) are endogenous tissue-specific short RNAs that regulate gene expression. Discriminating each let-7 family member expression is especially important due to let-7's abundance and connection with development and cancer. However, short lengths (22 nt) and similarities between multiple sequences have prevented identification of individual members. Here, we present ProDeG, a computational algorithm which designs imperfectly matched sequences (previously yielding only noise levels in microarray experiments) for genome-wide microarray “signal” probes to discriminate single nucleotide differences and to improve probe qualities. Our probes for the entire let-7 family are both homogeneous and specific, verified using microarray signals from fluorescent dye-tagged oligonucleotides corresponding to the let-7 family, demonstrating the power of our algorithm. In addition, false let-7c signals from conventional perfectly-matched probes were identified in lymphoblastoid cell-line samples through comparison with our probe-set signals, raising concerns about false let-7 family signals in conventional microarray platform

Trauma induces apoptosis in human thoracolumbar intervertebral discs

BACKGROUND: Vertebral fractures resulting from high energy trauma often comprise the risk of posttraumatic degenerative changes in the affected intervertebral discs (IVD). Particularly in conservatively treated patients, or in cases after implant removal of an exclusively posterior stabilization, consecutive disc degeneration and the associated functional losing of the spinal segment clearly represent detrimental treatment results. In this regard, apoptosis of IVD cells has been suggested to be involved in the critical changes of the extracellular matrix. METHODS: To investigate whether fractures of the vertebrae induce apoptosis in the affected IVD, disc tissue from patients (n = 17) undergoing open reduction and internal fixation of thoracolumbar spine fractures were analysed in regards to caspase activity, apoptosis-receptor expression levels and gene expression of apoptosis-regulating proteins such as Bax and Bcl-2. Healthy IVD tissue (n = 3) obtained from patients undergoing surgical resection of adjacent vertebrae were used as control samples. RESULTS: In contrast to healthy control IVD tissues, samples from traumatic thoracolumbar IVD showed positive TUNEL staining and a significant increase of caspase-3/7 activity. Interestingly, analyses of the initiator caspase-8 and -9 revealed significantly increased activation levels compared to control values, suggesting the coexistent activation of both the extrinsic (receptor-mediated) and intrinsic (mitochondria-mediated) apoptosis pathway. Accordingly, expression levels of the Fas receptor (FasR) mRNA were significantly increased. Although the TNF receptor I (TNFR I) was only slightly upregulated, corresponding TNFα from trauma IVD presented significantly increased mRNA expression values. Furthermore, traumatic IVD cells demonstrated significantly reduced expression of the mitochondria-bound anti-apoptotic Bcl-2, thereby maintaining baseline transcriptional levels of the pro-apoptotic Bax protein when compared to control IVD cells. CONCLUSION: Our data suggest that thoracolumbar fractures induce early caspase-dependent apoptosis in IVD cells of the affected intervertebral disc, in part, by downregulation of the anti-apoptotic protein Bcl-2 (intrinsic apoptosis pathway), as well as signalling via the death receptor complex (TNFR I and FasR)

Thoracic myelopathy caused by ossification of ligamentum flavum of which fluorosis as an etiology factor

PURPOSE: To evaluate the clinical feature, operative method and prognosis of thoracic ossification of ligamentum flavum caused by skeletal fluorosis. METHODS: All the patients with thoracic OLF, who underwent surgical management in the authors' hospital from 1993–2003, were retrospectively studied. The diagnosis of skeletal fluorosis was made by the epidemic history, clinical symptoms, radiographic findings, and urinalysis. En bloc laminectomy decompression of the involved thoracic levels was performed in all cases. Cervical open door decompression or lumbar laminectomy decompression was performed if relevant stenosis existed. The neurological statuses were evaluated with the Japanese Orthopaedic Association (JOA) scoring system preoperatively and at the end point of follow up. Also, the recovery rate was calculated. RESULTS: 23 cases have been enrolled in this study. Imaging study findings showed all the cases have ossification of ligamentum flavum together with ossification of many other ligaments and interosseous membranes, i.e. interosseous membranes of the forearm in 18 of 23 (78.3%), of the leg in 14 of 23 (60.1%) and of the ribs in 11 of 23 (47.8%). Urinalysis showed markedly increased urinary fluoride in 14 of 23 patients (60.9%). All the patients were followed up from 12 months to 9 years and 3 months, with an average of 4 years and 5 months. The JOA score increased significantly at the end of follow up (P = 0.0001). The recovery rate was 51.83 ± 32.36%. Multiple regression analysis revealed that the preoperative JOA score was an important predictor of surgical outcome (p = 0.0022, r = 0.60628). ANOVA analysis showed that patients with acute onset or too long duration had worse surgical result (P = 0.0003). CONCLUSION: Fluorosis can cause ossification of thoracic ligamentum flavum, as well as other ligaments. En bloc laminectomy decompression was an effective method. Preoperative JOA score was the most important predictor of surgical outcome. Patients with acute onset or too long duration had worse surgical outcome

Factors Associated with Refusal of Rapid HIV Testing in an Emergency Department

HIV screening studies in the emergency department (ED) have demonstrated rates of HIV test refusal ranging from 40–67%. This study aimed to determine the factors associated with refusal to undergo routine rapid HIV testing in an academic ED in Boston. HIV counselors offered routine testing to 1,959 patients; almost one-third of patients (29%) refused. Data from a self-administered survey were used to determine independent correlates of HIV testing refusal. In multivariate analysis, women and patients with annual household incomes of $50,000 or more were more likely to refuse testing, as were those who reported not engaging in HIV risk behaviors, those previously HIV tested and those who did not perceive a need for testing. Enrollment during morning hours was also associated with an increased risk of refusal. Increased educational efforts to convey the rationale and benefits of universal screening may improve testing uptake among these groups

Inactivation of the dnaK gene in Clostridium difficile 630 Δerm yields a temperature-sensitive phenotype and increases biofilm-forming ability

Abstract Clostridium difficile infection is a growing problem in healthcare settings worldwide and results in a considerable socioeconomic impact. New hypervirulent strains and acquisition of antibiotic resistance exacerbates pathogenesis; however, the survival strategy of C. difficile in the challenging gut environment still remains incompletely understood. We previously reported that clinically relevant heat-stress (37–41 °C) resulted in a classical heat-stress response with up-regulation of cellular chaperones. We used ClosTron to construct an insertional mutation in the dnaK gene of C. difficile 630 Δerm. The dnaK mutant exhibited temperature sensitivity, grew more slowly than C. difficile 630 Δerm and was less thermotolerant. Furthermore, the mutant was non-motile, had 4-fold lower expression of the fliC gene and lacked flagella on the cell surface. Mutant cells were some 50% longer than parental strain cells, and at optimal growth temperatures, they exhibited a 4-fold increase in the expression of class I chaperone genes including GroEL and GroES. Increased chaperone expression, in addition to the non-flagellated phenotype of the mutant, may account for the increased biofilm formation observed. Overall, the phenotype resulting from dnaK disruption is more akin to that observed in Escherichia coli dnaK mutants, rather than those in the Gram-positive model organism Bacillus subtilis

Yokukansan Inhibits Neuronal Death during ER Stress by Regulating the Unfolded Protein Response

Recently, several studies have reported Yokukansan (Tsumura TJ-54), a traditional Japanese medicine, as a potential new drug for the treatment of Alzheimer's disease (AD). Endoplasmic reticulum (ER) stress is known to play an important role in the pathogenesis of AD, particularly in neuronal death. Therefore, we examined the effect of Yokukansan on ER stress-induced neurotoxicity and on familial AD-linked presenilin-1 mutation-associated cell death.We employed the WST-1 assay and monitored morphological changes to evaluate cell viability following Yokukansan treatment or treatment with its components. Western blotting and PCR were used to observe the expression levels of GRP78/BiP, caspase-4 and C/EBP homologous protein.Yokukansan inhibited neuronal death during ER stress, with Cnidii Rhizoma (Senkyu), a component of Yokukansan, being particularly effective. We also showed that Yokukansan and Senkyu affect the unfolded protein response following ER stress and that these drugs inhibit the activation of caspase-4, resulting in the inhibition of ER stress-induced neuronal death. Furthermore, we found that the protective effect of Yokukansan and Senkyu against ER stress could be attributed to the ferulic acid content of these two drugs.Our results indicate that Yokukansan, Senkyu and ferulic acid are protective against ER stress-induced neuronal cell death and may provide a possible new treatment for AD