An Analysis of Glycolytic Enzymes in the Cellular Response to Metal Toxicity

Metal toxicity is implicated in neurotoxicity, nephrotoxicity, aging and cancer. Protein oxidation resulting from oxidative stress is now known to be involved in metal toxicity. However, proteomic responses to metal induced oxidative stress have not been characterized. By using the yeast as a model, we characterized these changes occurring in response to sub-lethal doses of metals. Several proteins involved in protein synthesis, ribosome assembly decreased while antioxidant defenses, proteins involved in sulfur metabolism, and glutathione synthesis and ubiquitin increased following metal exposure. We also show that metals induced temporal and targeted protein oxidation independent of protein abundance. Among the targets were glycolytic enzymes and heat-shock proteins. As a consequence, glycolytic enzyme activities decreased whereas the levels and activities of the enzymes of the alternative pathway for glucose metabolism, pentose phosphate pathway (PPP) increased. True to prediction, we also found increased flow through the PPP as measured by elevated levels of NADPH and glutathione. NADPH and glutathione are crucial for maintaining the redox balance in the cell. Thus, rerouting of glucose metabolism into PPP is considered to be beneficial to the organism. Among the oxidation targets is a glycolytic protein, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) that is required for apoptosis in neuronal cells. We show that not only is GAPDH required for metal induced apoptosis in yeast but also the levels of GAPDH transcript and protein increase in the cytosol and the nucleus in an isoform specific fashion. Such changes strongly implicate the role of GAPDH in yeast apoptosis. This work provides evidence for the involvement of targeted protein oxidation in metal toxicity, shows the overlaps and differences in the mechanism of copper and cadmium toxicity, allows comprehension of how metabolic processes respond to metal stress and explores the potential of GAPDH as a sensor of oxidative stress and mediator for apoptosis

Online Personal Tutoring System

Nowadays, most of the primary schools in Malaysia are still using courseware such as CD during teaching as a learning material. Students also depend on web based learning programme to study the subject that required them to pay. However, not everybody able to pay for the online educational programme such as Score A programme. Besides that, teachers are not participating in the web based learning application as they not play role to update notes and assessment every time. With the growing of technologies, the situation should be changed. The Online Personal Tutoring System (OPTS) can provide the solution for the above situation. The objective of OPTS is to provide medium for teacher to share learning and teaching material towards providing platform for the for year 4 science students to the current KSSR (KURIKULUM STANDARD SEKOLAH RENDAH) which is based on band system. The OPTS is mainly for science subject at primary school which contain one topic. There are three targeted users in this system which is a single administrator, teacher and student. They are carried out different kind of roles as stated in user requirement. Waterfall model has been chosen as the method to develop this system which is planning, requirement analysis, implementation and testing. This methodology is chosen because it attempts to reduce inherent project risk by breaking a project into smaller segments, provides easy to change during the development process and it is suitable used on developing OPTS. The OPTS is expected to become an educational tutoring aid for pupils and teachers. Moreover, only teacher can participate in OPTS as the teaching material provider

R. v. Nur: A Positive Step but not the Solution to the Problem of Mandatory Minimums in Canada

Over the last several decades, Parliament has steadily increased the use of mandatory minimum sentences. Canada now ranks second in the world — behind only the United States — in the number of offences it has that carry mandatory minimums. In R. v. Nur, the Supreme Court of Canada declared unconstitutional the three-year mandatory minimum sentence for a first conviction for possession of a firearm. Prior to Nur, the Court had not struck down a mandatory minimum sentence since R. v. Smith, decided 30 years earlier. In the time between Smith and Nur, the Court was asked to consider the constitutionality of four other mandatory minimum sentences. But in each of these cases the Court upheld the constitutionality of these minimums. Viewed in this context, Nur is a key decision. It represents a critical step towards dismantling a mandatory minimum regime that has gained a foothold in Canada

The usefulness of pleural fluid alkaline phosphatase and its ratio to serum alkaline phosphatase levels in classifying pleural effusions as exudates and transudates and its correlation with light’s criteria

INTRODUCTION: Pleural effusion is a very common clinical presentation of diseases. A correct diagnosis of the underlying disease is essential for the management of pleural effusion. A limited number of diseases cause Transudative Pleural Effusion, whereas exudative effusions require more extensive diagnostic investigations. Therefore, the first step is to classify them as transudates or exudates, even if this differentiation does not contribute to the etiological diagnosis. Many criteria have been used to distinguish them, but none of them have been found to be satisfactory. Light’s criteria is the most commonly used method. The criteria is one or more of the following to diagnose exudates. 1. Pleural fluid protein / Serum protein > 0.5 2. Pleural fluid LDH/ Serum LDH > 0.6. 3. Pleural fluid LDH more than 2/3rd of the upper limit of serum. AIMS AND OBJECTIVES: To evaluate the advantages of Total Pleural fluid Alkaline Phosphatase (ALP) and its ratio to Serum Alkaline Phosphatase levels in classifying Pleural Effusions as Exudates or Transudates. MAREIALS & METHOD: This study is to be conducted among 60 patients with pleural Effusion, attending the Department of Medicine & Department of Thoracic Medicine in Govt. Rajaji Hospital, Madurai. METHODOLOGY: This study was conducted in Govt. Rajaji Hospital, Madurai which is affiliated to Madurai Medical College. This study subjects were selected from the patients admitted in Department of Medicine and Department of Medicine, Govt. Rajaji Hospital. The study was conducted in 60 patients; the patients had pleural effusion with clinical background of congestive cardiac failure, chronic liver disease, chronic kidney disease, tuberculosis, parapneumonic effusions, malignancy. RESULT: “By applying Light’s criteria in patients with exudative pleural effusion classified clinically, 81.8% of the cases were correctly diagnosed as exudative pleural effusion. By applying Pleural fluid Alkaline phosphatase in patients with exudative pleural effusion classified clinically, 87.8% of the cases were correctly diagnosed as exudative pleural effusion. Among the parameters used most specific test to classify an exudative pleural effusion from a transudative pleural effusion is pleural fluid total protein which is 95.45% and most sensitive test is pleural fluid / serum alkaline phosphatase ratio which is 93.90%. The positive predictive value, negative predictive value and diagnostic accuracy to classify an exudative pleural effusion from a transudative pleural effusion is higher for pleural fluid total protein which is 96.29%, 95.23%, 94% respectively. CONCLUSION: For many decades Light’s criteria had been used widely to differentiate exudative from transudative pleural effusion. But it also misclassified 25% of transudates as exudates, so there was a need to identify new parameters which would prove to be superior or supportive to the array of tests at present

Analysis of eukaryotic translation by integrating cryo-EM and ribosome profiling

Translational control plays a critical role in maintaining proteome homeostasis, and in influencing cellular differentiation, proliferation, growth and developmental pathways. Protein synthesis is closely linked to cellular metabolism and any aberrations in its regulation lead to diseased states. In this thesis presented here, translational regulation has been investigated in three different biological contexts by integrating two different techniques namely, cryo-electron microscopy and ribosomal profiling. Translational regulation has been investigated in maturing dendritic cells using ribosome profiling and RNAseq respectively. Dendritic cells (DC) are the professional antigen-presenting cells of the immune system. In the immature state (immature DC), they have the ability to monitor the environment and upon encountering antigens they mature to launch immune responses. Here, a defined cytokine mixture combined with TLR agonist (R848) has been used for in vitro DC maturation. Upon induction of maturation, pathways such as the ‘TNF signaling pathway’, the ‘cytokine-cytokine receptor interaction’ and the ‘IL-17 signaling pathway’ were up regulated both at the level of transcriptome and translatome respectively. Transcripts encoding for proteins involved in oxidative phosphorylation pathway were strongly repressed at the later stages of DC maturation (24 h). As observed in previous studies transcripts encoding for ribosomal proteins, antigen processing and presentation were also translationally up-regulated at 4 h while being translationally repressed at the 24 h time point. Transcripts of the glycolytic pathway are also translationally repressed at the 24 h time-point. Further, during the course of DC maturation, globally there was increased ribosome occupancy in the 5’ UTR. During the later stages of DC maturation, down regulation of ABCE1 led to accumulation of post-termination ribosomes in the 3’ UTR. Moreover, ribosome occupancy in the 3’ UTR showed strong correlation to its GC content. Ski proteins function as accessory factors and are essential for exosome function, which mediates the 3’ to 5’ mRNA decay pathway. Non-stop transcripts are primarily decayed via the 3’ to 5’ pathway. It has been shown here that the Ski complex, interacts with the ribosome independent of Ski7. Ribosomal profiling of 80S-Ski-complexes revealed a fraction of longer footprints, and contained more poly-A containing footprints. Further, RNAseq analysis of the purified 80S-Ski-complexes revealed strong asymmetric distribution of reads, where more reads mapped towards the 5’ end of the transcripts. Also, transcripts with shorter half-life (< 5 min) and with more non-optimal codon content showed enrichment for Ski-80S footprints. This hinted at the possibility that Ski complex might interact with ribosomes for turnover of canonical transcripts via the 3’-5’ decay pathway. The endoplasmic reticulum (ER) is responsible for properly modifying and folding most of the secretory and membrane proteins. Its functioning capacity is challenged during stressful circumstances such as in hypoxia, calcium imbalance and viral infection. Unfolded protein response (UPR) is the cellular mechanism that is activated to alleviate the ER stress. UPR acts via three main pathways in mammals, and of this IRE1α-XBP1u branch is the most evolutionarily conserved. XBP1u contains a C-terminal ribosomal pausing site and plays a critical role in mediating UPR. Using cryo-EM, XBP1u has been visualized in the ribosomal exit tunnel. Structural characterization revealed that XBP1u forms a turn in the vicinity of the peptidyl transferase center and causes a subtle distortion of the base C4398 to inhibit ribosomal activity. This explains the temporary nature of the ribosomal arrest mediated by XBP1u. During ribosomal pausing, HR2 of XBP1u is being recognized by SRP, but it fails to successfully engage with the Sec61 translocon. XBP1u has evolved with an intermediate ribosomal pausing strength, but this allows it to be efficiently targeted by SRP onto the Sec61 translocon, albeit without gating it