Reversal of cisplatin resistance in oral squamous cell carcinoma by piperlongumine loaded smart nanoparticles through inhibition of Hippo-YAP signaling pathway.

Cisplatin alone or in combination with 5FU and docetaxel is the preferred chemotherapy regimen for advanced-stage OSCC patients. However, its use has been linked to recurrence and metastasis due to the development of drug resistance. Therefore, sensitization of cancer cells to conventional chemotherapeutics can be an effective strategy to overcome drug resistance. Piperlongumine (PL), an alkaloid, have shown anticancer properties and sensitizes numerous neoplasms, but its effect on OSCC has not been explored. However, low aqueous solubility and poor pharmacokinetics limit its clinical application. Therefore, to improve its therapeutic efficacy, we developed piperlongumine-loaded PLGA-based smart nanoparticles (smart PL-NPs) that can rapidly release PL in an acidic environment of cancer cells and provide optimum drug concentrations to overcome chemoresistance. Our results revealed that smart PL-NPs has high cellular uptake in acidic environment, facilitating the intracellular delivery of PL and sensitizing cancer cells to cisplatin, resulting in synergistic anticancer activity in vitro by increasing DNA damage, apoptosis, and inhibiting drug efflux. Further, we have mechanistically explored the Hippo-YAP signaling pathway, which is the critical mediator of chemoresistance, and investigated the chemosensitizing effect of PL in OSCC. We observed that PL alone and in combination with cisplatin significantly inhibits the activation of YAP and its downstream target genes and proteins. In addition, the combination of cisplatin with smart PL-NPs significantly inhibited tumor growth in two preclinical models (patient-derived cell based nude mice and zebrafish xenograft). Taken together, our findings suggest that smart PL-NPs with cisplatin will be a novel formulation to reverse cisplatin resistance in patients with advanced OSCC.

Piperlongumine based nanomedicine impairs glycolytic metabolism in triple negative breast cancer stem cells through modulation of GAPDH & FBP1.

BACKGROUND: Triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancer and exhibits high rate of chemoresistance, metastasis, and relapse. This can be attributed to the failure of conventional therapeutics to target a sub-population of slow cycling or quiescent cells called as cancer stem cells (CSCs). Therefore, elimination of CSCs is essential for effective TNBC treatment. PURPOSE: Research suggests that breast CSCs exhibit elevated glycolytic metabolism which directly contributes in maintenance of stemness, self-renewability and chemoresistance as well as in tumor progression. Therefore, this study aimed to target rewired metabolism which can serve as Achilles heel for CSCs population and have far reaching effect in TNBC treatment. METHODS: We used two preclinical models, zebrafish and nude mice to evaluate the fate of nanoparticles as well as the therapeutic efficacy of both piperlongumine (PL) and its nanomedicine (PL-NPs). RESULTS: In this context, we explored a phytochemical piperlongumine (PL) which has potent anti-cancer properties but poor pharmacokinetics impedes its clinical translation. So, we developed PLGA based nanomedicine for PL (PL-NPs), and demonstrated that it overcomes the pharmacokinetic limitations of PL, along with imparting advantages of selective tumor targeting through Enhanced Permeability and Retention (EPR) effect in zebrafish xenograft model. Further, we demonstrated that PL-NPs efficiently inhibit glycolysis in CSCs through inhibition of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by modulating glutathione S-transferase pi 1 (GSTP1) and upregulation of fructose-1,6-bisphosphatase 1 (FBP1), a rate-limiting enzyme in gluconeogenesis. We also illustrated that inhibition of glycolysis results in overall tumor regression in two preclinical models. CONCLUSION: This study discusses novel mechanism of action by which PL acts on CSCSs. Taken together our study provides insight into development of PL based nanomedicine which could be exploited in clinics to achieve complete eradication of TNBC by targeting CSCs.

Ajay Kumar Parida (1963-2022), an eminent plant biotechnologist with a passion for mangrove biology.

We remember Dr Ajay Parida, a leading plant biotechnologist, whose premature passing has deprived the Indian plant science community of a committed scientist and an able administrator. Born on 12 December 1963 in Bhagabanpur, Cuttack District (now Jajpur district), Odisha, he passed away in Guwahati on 19 July 2022. A collegial scientist, his down-to-earth and approachable nature, as well as his resourcefulness were instrumental in advancing the cause of Indian science and harnessing frontier biotechnological tools as vehicles of social consciousness. His expertise in quantitative DNA variation and molecular marker analysis, paved the way for subsequent research on mangrove molecular diversity at the M. S. Swaminathan Research Foundation (MSSRF), Chennai. His contributions to mangrove biology, genetics and genomics as well as extremophile plant species in the Indian context over two decades are a benchmark in his field. He also provided commendable leadership in his capacity as Director, Institute of Life Sciences (ILS), Bhubaneshwar during the COVID-19 pandemic.

SELEX based aptamers with diagnostic and entry inhibitor therapeutic potential for SARS-CoV-2.

Frequent mutation and variable immunological protection against vaccination is a common feature for COVID-19 pandemic. Early detection and confinement remain key to controlling further spread of infection. In response, we have developed an aptamer-based system that possesses both diagnostic and therapeutic potential towards the virus. A random aptamer library (~ 1017 molecules) was screened using systematic evolution of ligands by exponential enrichment (SELEX) and aptamer R was identified as a potent binder for the SARS-CoV-2 spike receptor binding domain (RBD) using in vitro binding assay. Using a pseudotyped viral entry assay we have shown that aptamer R specifically inhibited the entry of a SARS-CoV-2 pseudotyped virus in HEK293T-ACE2 cells but did not inhibit the entry of a Vesicular Stomatitis Virus (VSV) glycoprotein (G) pseudotyped virus, hence establishing its specificity towards SARS-CoV-2 spike protein. The antiviral potential of aptamers R and J (same central sequence as R but lacking flanked primer regions) was tested and showed 95.4% and 82.5% inhibition, respectively, against the SARS-CoV-2 virus. Finally, intermolecular interactions between the aptamers and the RBD domain were analyzed using in silico docking and molecular dynamics simulations that provided additional insight into the binding and inhibitory action of aptamers R and J.

Nimbolide-based nanomedicine inhibits breast cancer stem-like cells by epigenetic reprogramming of DNMTs-SFRP1-Wnt/ß-catenin signaling axis.

Triple-negative breast cancer (TNBC) harbors a high percentage of breast cancer stem-like cells (BCSCs) that significantly contribute to poor prognosis, metastasis, and relapse of the disease. Thus, targeting BCSCs could be a promising approach to combat TNBC. In this context, we investigated nimbolide (Nim), a limonoid triterpenoid that has potent anticancer properties, but poor pharmacokinetics and low bioavailability limit its therapeutic application. So, to enhance the therapeutic potential of Nim, Nim-encapsulated poly(lactic-co-glycolic acid) (PLGA) nanoparticles (Nim NPs) were formulated and the anticancer stem cell (CSC) effects evaluated in vitro and in vivo. In vitro studies suggested that Nim NPs significantly inhibited several inherent characteristics of BCSCs, such as stemness, self-renewability, chemoresistance, epithelial-to-mesenchymal transition (EMT), and migration in comparison to native Nim. Next, the mechanism behind the anti-CSC effect of Nim was explored. Mechanistically, we found that Nim epigenetically restores tumor suppressor gene secreted frizzled-related protein 1 (SFRP1) expression by downregulating DNA methyltransferases (DNMTs), leading to Wnt/ß-catenin signaling inhibition. Further, in vivo results demonstrated that Nim NPs showed enhanced anti-tumor and anti-metastatic effects compared to native Nim in two preclinical models without any systemic toxicity. Overall, these findings provide proof of concept that Nim-based phytonanomedicine can inhibit BCSCs by epigenetic reprogramming of the DNMTs-SFRP1-Wnt/ß-catenin signaling axis.

Targeting transcriptional kinase of CDK7 halts proliferation of multiple myeloma cells by modulating the function of canonical NF-kB pathway and cell cycle regulatory proteins.

Multiple myeloma (MM) is an incurable plasma cell neoplasm. Despite several effective frontline therapeutic regimens, including Bortezomib (BTZ), relapse is almost inevitable; therefore, better therapeutic modalities to improve the outcomes are needed. Cyclin-dependent kinases (CDKs) are an essential constituent of the cellular transcriptional machinery and tumors including MM are critically dependent on transcription to maintain their oncogenic state. In the present study, we explored the efficacy of THZ1, a covalent CDK7 inhibitor in MM treatment using Bortezomib resistant (H929BTZR) cells and zebrafish xenografts. THZ1 showed anti-myeloma activity in the models of MM but had no effect on healthy CD34+ cells. THZ1 suppresses phosphorylation of carboxy-terminal domain of RNA polymerase II and downregulates the transcription of BCL2 family of proteins both in H929BTZS and H929BTZR cells leading to G1/S arrest and apoptosis. THZ1 mediates inhibition of bone marrow stromal cells-induced proliferation and activation of NF-kB signaling. The data derived from zebrafish xenografts of MM demonstrate that THZ1 combined with BTZ synergistically reduces tumor growth in zebrafish embryos. Collectively, our results reveal that THZ1 alone as well as in combination with BTZ has effective anti-myeloma activity.

CRISPR-based kinome-screening revealed MINK1 as a druggable player to rewire 5FU-resistance in OSCC through AKT/MDM2/p53 axis.

Cisplatin, 5FU and docetaxel (TPF) are the most common chemotherapy regimen used for advanced OSCC. However, many cancer patients experience relapse, continued tumor growth, and spread due to drug resistance, which leads to treatment failure and metastatic disease. Here, using a CRISPR/Cas9 based kinome knockout screening, Misshapen-like kinase 1 (MINK1) is identified as an important mediator of 5FU resistance in OSCC. Analysis of clinical samples demonstrated significantly higher MINK1 expression in the tumor tissues of chemotherapy non-responders as compared to chemotherapy responders. The nude mice and zebrafish xenograft experiments indicate that knocking out MINK1 restores 5FU mediated cell death in chemoresistant OSCC. An antibody based phosphorylation array screen revealed MINK1 as a negative regulator of p53. Mechanistically, MINK1 modulates AKT phosphorylation at Ser473, which enables p-MDM2 (Ser 166) mediated degradation of p53. We also identified lestaurtinib as a potent inhibitor of MINK1 kinase activity. The patient derived TPF resistant cell based xenograft data suggest that lestaurtinib restores 5FU sensitivity and facilitates a significant reduction of tumor burden. Overall, our study suggests that MINK1 is a major driver of 5FU resistance in OSCC. The novel combination of MINK1 inhibitor lestaurtinib and 5FU needs further clinical investigation in advanced OSCC.

Zebrafish Larvae as a Model to Evaluate Potential Radiosensitizers or Protectors.

Zebrafish are extensively used in several kinds of research because they are one of the easily maintained vertebrate models and exhibit several features of a unique and convenient model system. As highly proliferative cells are more susceptible to radiation-induced DNA damage, zebrafish embryos are a front-line in vivo model in radiation research. In addition, this model projects the effect of radiation and different drugs within a short time, along with major biological events and associated responses. Several cancer studies have used zebrafish, and this protocol is based on the use of radiation modifiers in the context of radiotherapy and cancer. This method can be readily used to validate the effects of different drugs on irradiated and control (non-irradiated) embryos, thus identifying drugs as radio sensitizing or protective drugs. Although this methodology is used in most drug screening experiments, the details of the experiment and the toxicity assessment with the background of X-ray radiation exposure are limited or only briefly addressed, making it difficult to perform. This protocol addresses this issue and discusses the procedure and toxicity evaluation with a detailed illustration. The procedure describes a simple approach for using zebrafish embryos for radiation studies and radiation-based drug screening with much reliability and reproducibility.

CMTM6 attenuates cisplatin-induced cell death in OSCC by regulating AKT/c-Myc-driven ribosome biogenesis.

CMTM6, a type 3 transmembrane protein, is known to stabilize the expression of programmed cell death ligand 1 (PD-L1) and hence facilitates the immune evasion of tumor cells. Recently, we demonstrated that CMTM6 is a major driver of cisplatin resistance in oral squamous cell carcinomas (OSCC). However, the detailed mechanism of how CMTM6 rewires cisplatin resistance in OSCC is yet to be explored. RNA sequencing analysis of cisplatin-resistant OSCC lines stably expressing Nt shRNA and CMTM6 shRNA revealed that CMTM6 might be a potential regulator of the ribosome biogenesis network. Knocking down CMTM6 significantly inhibited transcription of 47S precursor rRNA and hindered the nucleolar structure, indicating reduced ribosome biogenesis. When CMTM6 was ectopically over-expressed in CMTM6KD cells, almost all ribosomal machinery components were rescued. Mechanistically, CMTM6 induced the expression of C-Myc, which promotes RNA polymerase I mediated rDNA transcription. In addition to this, CMTM6 was also found to regulate the AKT-mTORC1-dependent ribosome biogenesis and protein synthesis in cisplatin-resistant lines. The nude mice and zebrafish xenograft experiments indicate that blocking ribosome synthesis either by genetic inhibitor (CMTM6KD) or pharmacological inhibitor (CX-5461) significantly restores cisplatin-mediated cell death in chemoresistant OSCC. Overall, our study suggests that CMTM6 is a major regulator of the ribosome biogenesis network and targeting the ribosome biogenesis network is a viable target to overcome chemoresistance in OSCC. The novel combination of CX-5461 and cisplatin deserves further clinical investigation in advanced OSCC.

Isolation and Characterization of Five Severe Acute Respiratory Syndrome Coronavirus 2 Strains of Different Clades and Lineages Circulating in Eastern India.

The emergence of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) as a serious pandemic has altered the global socioeconomic dynamics. The wide prevalence, high death counts, and rapid emergence of new variants urge for the establishment of research infrastructure to facilitate the rapid development of efficient therapeutic modalities and preventive measures. In agreement with this, SARS-CoV-2 strains were isolated from patient swab samples collected during the first COVID-19 wave in Odisha, India. The viral isolates were adapted to in vitro cultures and further characterized to identify strain-specific variations in viral growth characteristics. The neutralization susceptibility of viral isolates to vaccine-induced antibodies was determined using sera from individuals vaccinated in the Government-run vaccine drive in India. The major goal was to isolate and adapt SARS-CoV-2 viruses in cell culture with minimum modifications to facilitate research activities involved in the understanding of the molecular virology, host-virus interactions, drug discovery, and animal challenge models that eventually contribute toward the development of reliable therapeutics.

Underlying Co-Morbidity Reveals Unique Immune Signatures in Type II Diabetes Patients Infected With SARS-CoV2.

Background: SARS-CoV2 infection in patients with comorbidities, particularly T2DM, has been a major challenge globally and has been shown to be associated with high morbidity and mortality. Here, we did whole blood immunophenotyping along with plasma cytokine, chemokine, antibody isotyping, and viral load from oropharyngeal swab to understand the immune pathology in the T2DM patients infected with SARS-CoV2. Methods: Blood samples from 25 Covid-19 positive patients having T2DM, 10 Covid-19 positive patients not having T2DM, and 10 Covid-19 negative, non-diabetic healthy controls were assessed for various immune cells by analyzing for their signature surface proteins in mass cytometry. Circulating cytokines, chemokines, and antibody isotypes were determined from plasma while viral copy number was determined from oropharyngeal swabs. All our representative data corroborated with laboratory findings. Results: Our observations encompass T2DM patients having elevated levels of both type I and type II cytokines and higher levels of circulating IgA, IgM, IgG1, and IgG2 as compared to NDM and healthy volunteers. They also displayed higher percentages of granulocytes, mDCs, plasmablasts, Th2-like cells, CD4+ EM cells, and CD8+ TE cells as compared to healthy volunteers. T2DM patients also displayed lower percentages of pDCs, lymphocytes, CD8+ TE cells, CD4+, and CD8+ EM. Conclusion: Our study demonstrated that patients with T2DM displayed higher inflammatory markers and a dysregulated anti-viral and anti-inflammatory response when compared to NDM and healthy controls and the dysregulated immune response may be attributed to meta inflammation.

Long-read 16S-seq reveals nasopharynx microbial dysbiosis and enrichment of Mycobacterium and Mycoplasma in COVID-19 patients: a potential source of co-infection.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a major global health concern. This virus infects the upper respiratory tract and causes pneumonia-like symptoms. So far, few studies have shown alterations in nasopharyngeal (NP) microbial diversity, enrichment of opportunistic pathogens and their role in co-infections during respiratory infections. Therefore, we hypothesized that microbial diversity changes, with increase in the population of opportunistic pathogens, during SARS-CoV2 infection in the nasopharynx, which may be involved in co-infection in COVID-19 patients. The 16S rRNA variable regions, V1-V9, of NP samples of control and COVID-19 (symptomatic and asymptomatic) patients were sequenced using the Oxford Nanopore™ technology. Comprehensive bioinformatics analysis for determining alpha/beta diversities, non-metric multidimensional scaling, correlation studies, canonical correspondence analysis, linear discriminate analysis, and dysbiosis index were used to analyze the control and COVID-19-specific NP microbiomes. We observed significant dysbiosis in the COVID-19 NP microbiome with an increase in the abundance of opportunistic pathogens at genus and species levels in asymptomatic/symptomatic patients. The significant abundance of Mycobacteria spp. and Mycoplasma spp. in symptomatic patients suggests their association and role in co-infections in COVID-19 patients. Furthermore, we found strong correlation of enrichment of Mycobacteria and Mycoplasma with the occurrences of chest pain and fever in symptomatic COVID-19 patients. This is the first study from India to show the abundance of Mycobacteria and Mycoplasma opportunistic pathogens in non-hospitalized COVID-19 patients and their relationship with symptoms, indicating the possibility of co-infections.

Fluvastatin sensitizes pancreatic cancer cells toward radiation therapy and suppresses radiation- and/or TGF-ß-induced tumor-associated fibrosis.

Pancreatic cancer (PC) is highly resistant to chemo and radiotherapy. Radiation-induced fibrosis (RIF) is a major cause of clinical concern for various malignancies, including PC. In this study, we aimed to evaluate the radiosensitizing and anti-RIF potential of fluvastatin in PC. Short-term viability and clonogenic survival assays were used to evaluate the radiosensitizing potential of fluvastatin in multiple human and murine PC cell lines. The expression of different proteins was analyzed to understand the mechanisms of fluvastatin-mediated radiosensitization of PC cells and its anti-RIF effects in both mouse and human pancreatic stellate cells (PSCs). Finally, these effects of fluvastatin and/or radiation were assessed in an immune-competent syngeneic murine model of PC. Fluvastatin radiosensitized multiple PC cell lines, as well as radioresistant cell lines in vitro, by inhibiting radiation-induced DNA damage repair response. Nonmalignant cells, such as PSCs and NIH3T3 cells, were less sensitive to fluvastatin-mediated radiosensitization than PC cells. Interestingly, fluvastatin suppressed radiation and/or TGF-ß-induced activation of PSCs, as well as the fibrogenic properties of these cells in vitro. Fluvastatin considerably augmented the antitumor effect of external radiation therapy and also suppressed intra-tumor RIF in vivo. These findings suggested that along with radiation, fluvastatin co-treatment may be a potential therapeutic approach against PC.

Single-Cell Immunogenomic Approach Identified SARS-CoV-2 Protective Immune Signatures in Asymptomatic Direct Contacts of COVID-19 Cases.

The response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is largely impacted by the level of virus exposure and status of the host immunity. The nature of protection shown by direct asymptomatic contacts of coronavirus disease 2019 (COVID-19)-positive patients is quite intriguing. In this study, we have characterized the antibody titer, SARS-CoV-2 surrogate virus neutralization, cytokine levels, single-cell T-cell receptor (TCR), and B-cell receptor (BCR) profiling in asymptomatic direct contacts, infected cases, and controls. We observed significant increase in antibodies with neutralizing amplitude in asymptomatic contacts along with cytokines such as Eotaxin, granulocyte-colony stimulating factor (G-CSF), interleukin 7 (IL-7), migration inhibitory factor (MIF), and macrophage inflammatory protein-1α (MIP-1α). Upon single-cell RNA (scRNA) sequencing, we explored the dynamics of the adaptive immune response in few representative asymptomatic close contacts and COVID-19-infected patients. We reported direct asymptomatic contacts to have decreased CD4+ naive T cells with concomitant increase in CD4+ memory and CD8+ Temra cells along with expanded clonotypes compared to infected patients. Noticeable proportions of class switched memory B cells were also observed in them. Overall, these findings gave an insight into the nature of protection in asymptomatic contacts.

Expression of two uncharacterized protein coding genes in zebrafish lateral line system.

The lateral line system is a mechanosensory organ of fish and amphibians that detects changes in water flow and is formed by the coordinated action of many signalling pathways. These signalling pathways can easily be targeted in zebrafish using pharmacological inhibitors to decipher their role in lateral line system development at cellular and molecular level. We have identified two uncharacterized proteins, whose mRNA are expressed in the lateral line system of zebrafish. One of these proteins, uncharacterized protein LOC564095 precursor, is conserved across vertebrates and its mRNA is expressed in posterior lateral line primordium (pLLP). The other uncharacterized protein, LOC100536887, is present only in the teleost fishes and its mRNA is expressed in neuromasts. We show that inhibition of retinoic acid (RA) signalling reduces the expression of both of these uncharacterized genes. It is reported that inhibition of RA signalling during gastrulation starting at 7 hours post fertilization (hpf) abrogates pLLP formation, and inhibition of RA signalling at 10 hpf delays the initiation of pLLP migration. Here, we show that inhibition of RA signalling before and during segmentation (9-16 hpf) results in delayed initiation and reduced speed of pLLP migration, as well as inhibition of posterior neuromasts formation.

Methods to generate and evaluate zebrafish models of human kidney diseases.

Kidney-related disorders affect millions of people worldwide. A survey of chronic kidney disease (CKD) patients showed that the burden of kidney diseases is increasing every year. The global burden of disease (GBD) study 2017 ranked CKD as the 12th leading cause of deaths worldwide. Hence, identification of the causes of kidney diseases, development of accurate diagnostic methods and novel therapeutics is highly relevant. Model organisms that faithfully recapitulate human diseases play important roles in understanding the disease process and provide valuable ground to find their cure. Zebrafish is an excellent model to study the development, pathophysiology and molecular aspects of human kidney diseases. In this review, we summarize various genetic and experimental manipulations that can be carried out in zebrafish to better understand the pathophysiology of human kidney diseases. We suggest that these methods will be helpful in the development of potential therapies to treat kidney diseases.

IL-6 Signaling Protects Zebrafish Larvae during Staphylococcus epidermidis Infection in a Bath Immersion Model.

The host immune responses to Staphylococcus epidermidis, a frequent cause of nosocomial infections, are not well understood. We have established a bath immersion model of this infection in zebrafish (Danio rerio) larvae. Macrophages play a primary role in the host immune response and are involved in clearance of infection in the larvae. S. epidermidis infection results in upregulation of tlr-2 There is marked inflammation characterized by heightened NF-κB signaling and elevation of several proinflammatory cytokines. There is rapid upregulation of il-1b and tnf-a transcripts, whereas an increase in il-6 levels is relatively more delayed. The IL-6 signaling pathway is further amplified by elevation of IL-6 signal transducer (il-6st) levels, which negatively correlates with miRNA dre-miR-142a-5p. Enhanced IL-6 signaling is protective to the host in this model as inhibition of the signaling pathway resulted in increased mortality upon S. epidermidis infection. Our study describes the host immune responses to S. epidermidis infection, establishes the importance of IL-6 signaling, and identifies a potential role of miR-142-5p-il-6st interaction in this infection model.

BMP2K phosphorylates AP-2 and regulates clathrin-mediated endocytosis.

Phosphorylation of the central adaptor protein complex, AP-2 is pivotal for clathrin-mediated endocytosis (CME). Here, we uncover the role of an uncharacterized kinase (BMP-2 inducible kinase-BMP2K) in AP-2 phosphorylation. We demonstrate that BMP2K can phosphorylate AP-2 in vitro and in vivo. Functional impairment of BMP2K impedes AP-2 phosphorylation leading to defects in clathrin-coated pit (CCP) morphology and cargo internalization. BMP2K engages AP-2 via its extended C-terminus and this interaction is important for its CCP localization and function. Notably, endogenous BMP2K levels decline upon functional impairment of AP-2 indicating AP-2 dependent BMP2K stabilization in cells. Further, functional inactivation of BMP2K in zebrafish embryos yields gastrulation phenotypes which mirror AP-2 loss-of-function suggesting physiological relevance of BMP2K in vertebrates. Together, our findings propose involvement of a novel kinase in AP-2 phosphorylation and in the operation of CME.

Genomic organization and hypoxia inducible factor responsive regulation of teleost hsp90ß gene during hypoxia stress.

BACKGROUND: The physiological significance of a large family of heat-shock proteins (HSPs), comprised of the cytosolic HSP90A and the endoplasmic reticulum component of HSPB, is evident in prokaryotes and eukaryotes. The HSP90A is believed to play critical roles in diverse physiological functions of cell viability and chromosomal stability including stress management. Heightened abundance of hsp90ß transcript was documented in Channa striatus, a freshwater fish, which is capable of surviving within an extremely hypoxic environment. METHODS AND RESULTS: To better understand the mechanism of hsp90ß gene expression, we investigated its genomic organization. Eleven exons were identified, including a long upstream intron with a remarkable similarity with human, but not with chicken counterpart. Dual-luciferase assays identified promoter activity in a 1366 bp 5'-flanking segment beyond the transcription initiation site. Examination detected a minimal promoter of 754 bp containing a TATA-box, CAAT-enhancer in addition to providing clues regarding other enhancer and repressor elements. The driving capability of this minimal promoter was further validated by its binding ability with TATA-box binding protein and the generation of GFP expressing transgenic zebrafish (F2). Further, deletion of an inverted HIF (hypoxia inducible factor) motif RCGTG (upstream of the TATA-box) dramatically reduced luciferase expression in a hypoxic environment (CoCl2 treated cultivable cells) and was identified as a cis-acting HIF responsive element, necessary for the hypoxia-induced expression. CONCLUSIONS: The results obtained herein provide an insight regarding how hsp90ß gene expression is controlled by HIF responsive element in teleost both during hypoxia stress management and normal physiological functions, and suggested that the hsp90ß gene promoter could be used as a potential candidate for generating ornamental and food-fish transgenics.