Nuclear import mechanism of Php4 under iron deprivation in fission yeast Schizosaccharomyces pombe

Php4 is a subunit of the CCAAT-binding protein complex that has a negative regulatory function during iron deprivation in the fission yeast Schizosaccharomyces pombe. Under low iron conditions, Php4 fosters the repression of genes encoding iron using proteins. In contrast, under iron-replete conditions, Php4 is inactivated at both transcriptional and post-transcriptional levels. Our group has already described that Php4 is a nucleo-cytoplasmic shuttling protein, which accumulates into the nucleus during iron deficiency. On the contrary, Php4 is exported from the nucleus to the cytoplasm in response to iron abundance. Php4 possesses a leucine-rich NES (93LLEQLEML100) that is necessary for its nuclear export by the exportin Crm1. Our current study aims at understanding the mechanism by which Php4 is imported in the nucleus during iron starvation. Through microscopic analyses using different mutant strains, we showed that the nuclear localization of Php4 is independent of the other subunits of the CCAAT-binding core complex namely Php2, Php3 and Php5. Deletion mapping analysis of Php4 identifies two putative nuclear localization sequences (NLSs) in Php4 (171KRIR174 and 234KSVKRVR240). Using chimeric proteins that consist of GFP fused to Php4, we engineered substitutions of the basic amino acid residues 171AAIA174 and 234ASVAAAA240 and analyzed the functionality of both NLSs. We observed that both monopartite NLSs play critical role for Php4 nuclear localization. We also observed that mutant strains of cut15+, imp1+ or sal3+ exhibited defects in nuclear targeting of Php4, revealing that nuclear accumulation of Php4 is dependent on two karyopherin α (Imp1 and Cut15) and one karyopherin β (Sal3) receptors. Consistently, the Php4-mediated repression activity is abolished in the absence of two functional NLSs. Moreover, loss of Imp1, Cut15 or Sal3 resulted in increased expression of isa1+, which is a target gene of Php4. Co-immunoprecipitation assay (Co-IP) reveals physical interaction of Php4 with Imp1, Cut15 and Sal3 in vitro. Collectively, our results demonstrate that Php4 has two distinct NLS regions responsible for its nuclear localization. Furthermore, karyopherin α and β receptors play a role in the nuclear import of Php4. Because Php4 is essential for growth under low iron conditions, the presence of two NLSs would ensure the protein to reach its nuclear destination when cells undergo a transition from iron-sufficient to iron-limiting conditions

Evaluation of rK-39 strip test using urine for diagnosis of visceral leishmaniasis in an endemic area in Bangladesh

Diagnosis of visceral leishmaniasis (VL) by demonstration of parasites in tissue smears obtained from bone marrow, spleen or lymph nodes is risky, painful, and difficult. The rK-39 strip test is widely used for the diagnosis of VL using blood/serum samples in endemic countries. The aim of the study was to evaluate the rK-39 strip test using urine sample as a non-invasive means for the diagnosis of VL. The rk-39 strip test was performed using urine from 100 suspected VL cases along with 25 disease control (malarial febrile cases) and 50 healthy control (from endemic and non-endemic areas). All the VL suspected cases were positive with the rK-39 strip test using serum. The sensitivity and specificity of the rK-39 strip test using urine samples was 95% and 93.3%, respectively, compared to serum based rK-39 test. The findings suggest that the urine based rK-39 test could be a practical and efficient tool for the diagnosis of VL patients in rural areas, particularly where resources are limited

Short Communication: Evaluation of a New Rapid Diagnostic Test for Quality Assurance by Kala Azar Elimination Programme in Bangladesh

In Bangladesh, serological tests have been widely used for the primary screening of visceral leishmaniasis (VL). Several serologic tests are available for the diagnosis of VL. Selection of the best test is important to permit diagnostic differentiation between symptomatic and asymptomatic patients and to reduce cross-reactivity. We evaluated the effectiveness of a new serological test “Onsite Leishmania Ab Rapid Test” as a part of “quality assurance” activities for the kala azar elimination programme of the Government of Bangladesh. Plasma samples of 100 parasitologically confirmed cases of VL along with 101 healthy controls were tested, and “Onsite Leishmania Ab Rapid Test” strip tests were positive in 94 out of 100 confirmed VL cases, whereas four out of 51 healthy subjects from the VL endemic areas also tested positive. All the 50 healthy volunteers tested negative. Thus, the sensitivity and specificity of “Onsite Leishmania Ab Rapid Test” strip test were found to be 94% (95% CI: 87–98) and 96% (95% CI: 90–99), respectively. This study showed that the performance of the “Onsite Leishmania Ab Rapid Test” strip tests was up to the recommended level

Considerations for application of benchmark dose modeling in radiation research: workshop highlights

publishedVersio

Prevalence of anopheline species and their Plasmodium infection status in epidemic-prone border areas of Bangladesh

<p>Abstract</p> <p>Background</p> <p>Information related to malaria vectors is very limited in Bangladesh. In the changing environment and various <it>Anopheles </it>species may be incriminated and play role in the transmission cycle. This study was designed with an intention to identify anopheline species and possible malaria vectors in the border belt areas, where the malaria is endemic in Bangladesh.</p> <p>Methods</p> <p><it>Anopheles </it>mosquitoes were collected from three border belt areas (Lengura, Deorgachh and Matiranga) during the peak malaria transmission season (May to August). Three different methods were used: human landing catches, resting collecting by mouth aspirator and CDC light traps. Enzyme-linked immunosorbent assay (ELISA) was done to detect <it>Plasmodium falciparum</it>, <it>Plasmodium vivax</it>-210 and <it>Plasmodium vivax</it>-247 circumsporozoite proteins (CSP) from the collected female species.</p> <p>Results</p> <p>A total of 634 female <it>Anopheles </it>mosquitoes belonging to 17 species were collected. <it>Anopheles vagus </it>(was the dominant species (18.6%) followed by <it>Anopheles nigerrimus </it>(14.5%) and <it>Anopheles philippinensis </it>(11.0%). Infection rate was found 2.6% within 622 mosquitoes tested with CSP-ELISA. Eight (1.3%) mosquitoes belonging to five species were positive for <it>P. falciparum</it>, seven (1.1%) mosquitoes belonging to five species were positive for <it>P. vivax </it>-210 and a single mosquito (0.2%) identified as <it>Anopheles maculatus </it>was positive for <it>P. vivax</it>-247. No mixed infection was found. Highest infection rate was found in <it>Anopheles karwari </it>(22.2%) followed by <it>An. maculatus </it>(14.3%) and <it>Anopheles barbirostris </it>(9.5%). Other positive species were <it>An. nigerrimus </it>(4.4%), <it>An. vagus </it>(4.3%), <it>Anopheles subpictus </it>(1.5%) and <it>An. philippinensis </it>(1.4%). <it>Anopheles vagus </it>and <it>An. philippinensis </it>were previously incriminated as malaria vector in Bangladesh. In contrast, <it>An. karwari</it>, <it>An. maculatus</it>, <it>An. barbirostris</it>, <it>An. nigerrimus </it>and <it>An. subpictus </it>had never previously been incriminated in Bangladesh.</p> <p>Conclusion</p> <p>Findings of this study suggested that in absence of major malaria vectors there is a possibility that other <it>Anopheles </it>species may have been playing role in malaria transmission in Bangladesh. Therefore, further studies are required with the positive mosquito species found in this study to investigate their possible role in malaria transmission in Bangladesh.</p

Mécanismes de suppression des tumeurs médiés par SOCS1 dans le carcinome hépatocellulaire

Abstract : SOCS1 and its closest homolog, SOCS3, are considered tumor suppressors in the liver due to frequent repression of their genes in hepatocellular carcinoma (HCC) and increased susceptibility of SOCS1- or SOCS3- deficient mice to HCC. SOCS1 is also known to regulate the paradoxical oncogenic functions of the cell cycle inhibitor p21. However, the underlying mechanisms are not yet clear. The goal of this study is to understand how SOCS1-mediated regulation of p21 (CDKN1A) controls HCC development. To determine whether SOCS1 and SOCS3 differentially modulate oncogenic signaling pathways in hepatocytes, we correlated SOCS1 and SOCS3 expression with genes associated with key oncogenic signaling pathways and assessed their relationship to patient survival using transcriptomic and clinical data from the TCGA database. We have identified several oncogenic drivers that showed mutual exclusivity with SOCS1 or SOCS3. Notably, CDK2 and AURKA demonstrated corresponding modulations in the regenerating livers and DEN-induced tumors of hepatocyte-specific Socs1 or Socs3-deficient mice as well as showing promise in HCC prognosis Furthermore, a combination of SOCS1 or SOCS3 expression along with certain oncogenic signaling pathway genes revealed a poor prognosis. Mice with a conditional knockout for either SOCS1 or SOCS3 in hepatocytes displayed increased susceptibility to HCC, confirming that SOCS1 and SOCS3 function as tumor suppressors. Surprisingly, double knockout mice, lacking both SOCS1 and SOCS3, in hepatocytes showed decreased HCC progression. Further investigation revealed that SOCS1 deficiency results in the upregulation of SOCS3 following genotoxic stress and that SOCS3 promoted p53 activation leading to the induction of Cdkn1a, which codes for p21. Deletion of Cdkn1a in SOCS1-deficient mice prevented HCC development. Our data also showed that SOCS3 promotes oncogenesis in SOCS1-deficient hepatocytes by activating p53-mediated induction of Cdkn1a. p21 is known to exert its paradoxical oncogenic potential through the activation of NRF2 and augmentation of NRF2-mediated antioxidant responses. In support of this notion, our data showed increased NRF2 activation in SOCS1-deficient mice that was abolished by the combined loss of either SOCS3 or CDKN1A. In contrast, overexpression of SOCS1 reduced the ability of HCC cells to tolerate oxidative stress by down-modulating p21-NRF2. Overall, our findings show that SOCS1 deficiency in hepatocytes activates the SOCS3-p53-CDKN1A axis leading to an NRF2-mediated antioxidant response that promotes hepatocarcinogenesis. This knowledge will help to develop new therapeutic strategies to treat HCC, which is one of the most fatal cancers.SOCS1 et son homologue le plus fortement apparenté, SOCS3, sont considérés comme des gènes suppresseurs de tumeurs dans le foie en raison de leur répression génique fréquemment observée dans le carcinome hépatocellulaire (CHC) et de la susceptibilité accrue des souris déplétées en SOCS1- ou en SOCS3- au développement du CHC. Il est connu que SOCS1 participe aux fonctions oncogènes paradoxales de l’inhibiteur du cycle cellulaire p21. Toutefois, les mécanismes sous-jacents demeurent mal compris. L'objectif de cette étude est de mieux comprendre les mécanismes de régulation de p21 (CDKN1A) médiés par SOCS1 participant au développement du CHC. Afin de déterminer si SOCS1 et SOCS3 modulent différentiellement les voies de signalisation oncogéniques dans les hépatocytes, nous avons corrélé l’expression de SOCS1 et SOCS3 aux gènes associés aux principales voies de signalisation oncogéniques, en plus d’évaluer leur relation avec la survie d’une cohorte de patients atteints de CHC en utilisant les données transcriptomiques et cliniques issues de la banque de données du TCGA. Nous avons identifié plusieurs modulateurs oncogéniques qui sont mutuellement exclusifs à l’expression de SOCS1 ou SOCS3. Plus particulièrement, l’expression de Cdk2 et Aurka était fortement induite dans les foies en régénération ainsi que dans les tumeurs induites au DEN chez les souris déficientes en Socs1 ou Socs3. Une expression plus élevée de CDK2 et AURKA a également été corrélée à un mauvais pronostic dans la cohorte de patients atteints de CHC. De plus, l’expression combinée de SOCS1 ou SOCS3 avec certains gènes des voies de signalisation oncogéniques révèle un mauvais pronostic. Les souris dont les hépatocytes sont dépourvus de Socs1 ou Socs3 ont présenté une susceptibilité accrue au développement du CHC, confirmant ainsi que SOCS1 et SOCS3 agissent comme suppresseur de tumeurs. Étonnamment, les souris dont les hépatocytes n’expriment pas SOCS1 et SOCS3 ont démontré une diminution de la progression du CHC. Des recherches plus approfondies ont confirmé que l’absence de SOCS1 entraîne une régulation positive de SOCS3 en réponse à un stress génotoxique et que SOCS3 favorise l’activation de p53 conduisant à l’induction de l’expression de Cdkn1a (le gène codant pour p21). Surprenamment, la suppression de Cdkn1a chez les souris déficientes en SOCS1 a empêché le développement du CHC, démontrant ainsi que SOCS3 favorise l’oncogenèse dans les hépatocytes dépourvus de SOCS1 par l’induction de l’expression de Cdkn1a, médiée par p53. Il est connu que p21 exerce son pouvoir oncogénique par l’activation de NRF2 et, de ce fait, l’augmentation des réponses antioxydantes médiées par NRF2. En accord avec cette notion, nos résultats démontrent que l’augmentation de l’activation de NRF2 observée chez les souris déplétées en SOCS1, était abolie par la perte additionnelle de SOCS3 ou de p21. À l’inverse, la surexpression de SOCS1 a réduit la capacité des cellules CHC à tolérer un stress oxydatif en modulant négativement p21/NRF2. De façon générale, nos résultats démontrent que l’activation de l’axe SOCS3-p53-p21 dans les hépatocytes déplétés en SOCS1 conduit à la réponse antioxydante médiée par NRF2, favorisant ainsi l’hépatocarcinogenèse. Ces découvertes contribueront au développement de nouvelles stratégies et approches thérapeutiques contre le CHC, qui est l’un des cancers les plus mortels

Nuclear import mechanism of Php4 under iron deprivation in fission yeast Schizosaccharomyces pombe

Php4 is a subunit of the CCAAT-binding protein complex that has a negative regulatory function during iron deprivation in the fission yeast Schizosaccharomyces pombe. Under low iron conditions, Php4 fosters the repression of genes encoding iron using proteins. In contrast, under iron-replete conditions, Php4 is inactivated at both transcriptional and post-transcriptional levels. Our group has already described that Php4 is a nucleo-cytoplasmic shuttling protein, which accumulates into the nucleus during iron deficiency. On the contrary, Php4 is exported from the nucleus to the cytoplasm in response to iron abundance. Php4 possesses a leucine-rich NES (93LLEQLEML100) that is necessary for its nuclear export by the exportin Crm1. Our current study aims at understanding the mechanism by which Php4 is imported in the nucleus during iron starvation. Through microscopic analyses using different mutant strains, we showed that the nuclear localization of Php4 is independent of the other subunits of the CCAAT-binding core complex namely Php2, Php3 and Php5. Deletion mapping analysis of Php4 identifies two putative nuclear localization sequences (NLSs) in Php4 (171KRIR174 and 234KSVKRVR240). Using chimeric proteins that consist of GFP fused to Php4, we engineered substitutions of the basic amino acid residues 171AAIA174 and 234ASVAAAA240 and analyzed the functionality of both NLSs. We observed that both monopartite NLSs play critical role for Php4 nuclear localization. We also observed that mutant strains of cut15+, imp1+ or sal3+ exhibited defects in nuclear targeting of Php4, revealing that nuclear accumulation of Php4 is dependent on two karyopherin α (Imp1 and Cut15) and one karyopherin β (Sal3) receptors. Consistently, the Php4-mediated repression activity is abolished in the absence of two functional NLSs. Moreover, loss of Imp1, Cut15 or Sal3 resulted in increased expression of isa1+, which is a target gene of Php4. Co-immunoprecipitation assay (Co-IP) reveals physical interaction of Php4 with Imp1, Cut15 and Sal3 in vitro. Collectively, our results demonstrate that Php4 has two distinct NLS regions responsible for its nuclear localization. Furthermore, karyopherin α and β receptors play a role in the nuclear import of Php4. Because Php4 is essential for growth under low iron conditions, the presence of two NLSs would ensure the protein to reach its nuclear destination when cells undergo a transition from iron-sufficient to iron-limiting conditions

Advances in the Current Understanding of How Low-Dose Radiation Affects the Cell Cycle

Cells exposed to ionizing radiation undergo a series of complex responses, including DNA damage, reproductive cell death, and altered proliferation states, which are all linked to cell cycle dynamics. For many years, a great deal of research has been conducted on cell cycle checkpoints and their regulators in mammalian cells in response to high-dose exposures to ionizing radiation. However, it is unclear how low-dose ionizing radiation (LDIR) regulates the cell cycle progression. A growing body of evidence demonstrates that LDIR may have profound effects on cellular functions. In this review, we summarize the current understanding of how LDIR (of up to 200 mGy) regulates the cell cycle and cell-cycle-associated proteins in various cellular settings. In light of current findings, we also illustrate the conceptual function and possible dichotomous role of p21Waf1, a transcriptional target of p53, in response to LDIR

Evidence-based diagnostic algorithm for visceral leishmaniasis in Bangladesh

Evidence-based diagnostic algorithm is highly recommended for the visceral leishmaniasis (VL). This crosssectional study was performed in Bangladesh to evaluate VL diagnostic tools including serology, buffy coat smear microscopy for LD body and various DNA-based techniques using buffy coat in 100 confirmed VL cases and 100 controls. The performance of tools against spleen smear (gold standard) was evaluated using kappa coefficient. Diagnostic precision and other inherent indicators were considered for index scoring (IS) of performance of tools using factor analysis. A diagnostic algorithm was formulated based on the IS and availability of the tools at different health care facilities of Bangladesh. A high level of agreement (kappa ≥ 0.80) was observed for all the diagnostic tools. The highest kappa coefficients were found for rK39 RDT and rK39 ELISA (0.95), followed by ssuRNA-PCR (0.94), Buffy coat smear (0.93), rK28 ELISA (0.92), rK28 RDT (0.89), LAMP (0.89), Mini-exon PCR (0.86), ITS1 (0.85), and ITS2 PCR (0.80). rK39 RDT was found to be the best diagnostic test (IS: 1.7) followed by rK28 RDT (IS: 1.5), buffy coat smear microscopy (IS: 0.5), rK39 & rK28 ELISA (IS: 0.3), ssuRNA-PCR (IS: -0.7) and LAMP, Mini-exon, ITS1, & ITS2 PCR (IS: -0.9). rK39 RDT has been proposed as the best option for primary health care facilities, while buffy coat smear microscopy was found to be a good adjunct for confirmation of serology-positive cases and proposed for secondary and tertiary facilities. ssuRNA-PCR or LAMP can be an alternate confirmation tool only applicable to the tertiary facilities