In vitro evaluation of asparagine endopeptidase as a candidate biomarker of treatment failure in childhood acute lymphoblastic leukaemia

PhDBackground Although cure rates in childhood acute lymphoblastic leukaemia (ALL) approach 90%, relapses, particularly central nervous system (CNS) relapses, pose a significant therapeutic challenge. Prevention of relapses requires better predictive biomarkers. Towards this end, global gene-expression microarray screens of primary ALL samples were performed. Overexpression of the lysosomal cysteine protease, asparagine endopeptidase (AEP) was identified in adverse-risk progenitor-B ALL genotypes. AEP overexpression in adult human epithelial cancers has been linked to metastasis and adverse prognosis. Hypothesis AEP overexpression promotes leukaemic cell infiltration of the CNS and other extramedullary sites. AEP degrades the bacterial protein, E. coli L-asparaginase (ASNase) and potentially mediates lymphoblast resistance to this key antileukaemic drug. Summary of findings Quantitative AEP transcript estimation validated microarray findings in the discovery cohort. Sample availability precluded conclusive demonstration of protein overexpression. Intracellular expression in SD1 cells, a model AEPoverepressing ALL cell line, was strikingly heterogeneous and included aberrant peripheral localisation in endolysosomal macrovesicles. Similar findings were observed anecdotally in primary lymphoblasts. In SD1 cells, precursor AEP protein was shed in microvesicles. AEP’s role in cell motility was examined in human embryonic kidney (HEK293) cells. Contrary to published reports, ectopic AEP overexpression in HEK293 cells was not associated with enhanced motility. ASNase was consistently degraded when incubated with ALL cell lysates. In overexpressing disease, ASNase degradation is potentially accelerated by AEP, resulting in inadequate drug activity during early treatment. AEP-cleaved ASNase Synopsis fragments retain known sensitising epitopes, suggesting that AEP cleavage could also potentiate formation of neutralising ASNase antibodies, compounding ASNase treatment failure. Sole substitution at an AEP cleavage site generated an AEP-resistant ASNase variant. Computational protein modelling enabled identification of an appropriate substituting amino acid that best retains drug activity. Conclusions AEP is a candidate marker of poor treatment response in childhood ALL and is presently the subject of a prospective nationwide clinical biomarker study. Its postulated role in cell motility appears to be cell-specific and dependent on the concomitant upstream expression of additional candidate pro-motility molecules. The protease also appears to be a marker of aberrant lymphoblast vesicle phenotype, an observation that is currently the focus of further studies.Cancer research U

Isolation of subgenus B adenovirus during a fatal outbreak of enterovirus 71-associated hand, foot, and mouth disease in Sibu, Sarawak.

BACKGROUND: In mid-1997, several children died in Sarawak, Malaysia, during an epidemic of enterovirus-71 (EV71) hand, foot, and mouth disease. The children who died had a febrile illness that rapidly progressed to cardiopulmonary failure and the cause was not satisfactorily resolved. We describe the isolation and identification of a subgenus B adenovirus from the children who died. METHODS: We studied two groups of children presenting to Sibu Hospital from April 14 to Sept 30, 1997. For children who died, the inclusion criterion was death after febrile illness, and for those who did not die it was acute flaccid paralysis (AFP). Serum and cerebrospinal fluid samples were tested for IgM antibodies to Japanese encephalitis and dengue viruses. Viruses isolated were identified by immunofluorescence, reverse-transcriptase PCR, or PCR and DNA sequencing. FINDINGS: Enterovirus was isolated in three (19%) of 16 children who died and in none of the eight surviving children with AFP. However, an agent that was initially difficult to identify was found in ten (63%) children who died and five (63%) surviving children who had AFP. The agents isolated from ten (66.7%) of these 15 children were eventually identified as adenoviruses and were isolated mainly from clinically important sterile sites or tissues. All the enterovirus-positive children who died had this second agent. INTERPRETATION: Our data raises doubts that EV71 was the only aetiological agent in these deaths

A dyad of lymphoblastic lysosomal cysteine proteases degrades the antileukemic drug L-asparaginase

l-Asparaginase is a key therapeutic agent for treatment of childhood acute lymphoblastic leukemia (ALL). There is wide individual variation in pharmacokinetics, and little is known about its metabolism. The mechanisms of therapeutic failure with l-asparaginase remain speculative. Here, we now report that 2 lysosomal cysteine proteases present in lymphoblasts are able to degrade l-asparaginase. Cathepsin B (CTSB), which is produced constitutively by normal and leukemic cells, degraded asparaginase produced by Escherichia coli (ASNase) and Erwinia chrysanthemi. Asparaginyl endopeptidase (AEP), which is overexpressed predominantly in high-risk subsets of ALL, specifically degraded ASNase. AEP thereby destroys ASNase activity and may also potentiate antigen processing, leading to allergic reactions. Using AEP-mediated cleavage sequences, we modeled the effects of the protease on ASNase and created a number of recombinant ASNase products. The N24 residue on the flexible active loop was identified as the primary AEP cleavage site. Sole modification at this site rendered ASNase resistant to AEP cleavage and suggested a key role for the flexible active loop in determining ASNase activity. We therefore propose what we believe to be a novel mechanism of drug resistance to ASNase. Our results may help to identify alternative therapeutic strategies with the potential of further improving outcome in childhood ALL

Appraisal of climate change and cyclone trends in Indian coastal states: a systematic approach towards climate action

Abstract: Indian coastal regions have often been affected by frequent climate-induced natural disasters such as cyclones, floods, droughts and other related hazards in recent decades. Existing literature was not sufficient to fully understand these event trends from diverse perspectives in a systematised manner at current scenarios. Therefore, a systematic approach has been employed to assess the climate change and cyclone trends of nine Indian coastal states by using various geographical information system (GIS) tools for 2006–2020. The results showed that 61 cyclones occurred in nine coastal states from 2006 to 2020; the highest numbers were recorded in Odisha (20), West Bengal (14) and Andhra Pradesh (11). Accordingly, these three coastal states emerged as the most vulnerable for high-intensity cyclones. The results also identified that the highest average temperature (29.3 °C) was recorded at Tamil Nadu and Gujarat, and the lowest temperature (26.7 °C) was recorded in West Bengal and Odisha. Most of the coastal states showed fluctuations in temperatures during the study period. At the same time, Kerala and Karnataka states recorded the highest average rainfall (2341 mm and 2261 mm) and highest relative humidity (78.11% and 76.57%). Conversely, the Gujarat and West Bengal states recorded the lowest relative humidity at 59.65% and 70.78%. Based on these results, the current study generated GIS vulnerability maps for climate change and cyclone activity, allowing one to rank each state’s vulnerability. Cumulatively, these results and maps assist in understanding the driving mechanisms of climate change, cyclones and will contribute towards more effective and efficient sustainable disaster management in the future

Full Reference Image Quality Metrics and their Performance

This paper mainly aims to study the performance of objective assessment methods of image quality. It take into consideration the correlations between each objective assessment and the subjective assessment in order to determine objective test performance. Three objective assessment methods used in this study are the Structural Similarity (SSIM) index, the Peak Signal-to-Noise Ratio (PSNR) and the Mean Squared Error (MSE) calculating algorithm. The resulting data indicate what type of objective assessment was most suitable for which type of impairment imposed upon an image. This is clarified using the Pearson Correlation Coefficient as described in the paper. As an overall, SSIM index had the best correlation characteristics to the subjective assessment, followed by the MSE calculating algorithm. From this study, a better understanding of the requirements for developing an efficient image quality assessment method was gained