Role of a Putative Cyclic Di-GMP Forming Locus MSMEG_2196 in Mycobacterium Smegmatis

Cyclic di-guanosine-monophosphate (c-di-GMP) has been recognized as a second messenger in bacteria controlling multiple cellular processes such as biofilm formation, motility, and virulence. Proteins containing GGDEF and EAL domains are engaged in the synthesis and degradation, respectively, of cyclic di-GMP. Some bacteria contain multiple proteins with GGDEF and EAL domains. The genome of Mycobacterium tuberculosis encodes only one protein (Rv1354c) which contains a GGDEF domain. This protein also contains a tandem EAL. The function of this protein in mycobacteria has not yet been determined. In this study, the orthologue of Rv1354c was investigated in Mycobacterium smegmatis (MSMEG_2196). The expression of MSMEG_2196 in M. smegmatis was altered by constructing sense and antisense expressing strains. The effect of the altered expression of MSMEG_2196 on M. smegmatis was tested under carbon, oxygen, phosphorous, and nitrogen limited growth conditions. There was no significant effect on growth in either the antisense or sense expressing strains grown under nutrient-rich, or carbon-, or oxygen-, or phosphorous limitation conditions. However, a growth effect was observed in the antisense expressing strain when grown under nitrogen-limited conditions. In particular, at mid stationary-phase (1,800 min) the MSMEG_2196 antisense strain had an OD600 value of 0.60, compared to that of the control M. smegmatis/pMind strain (OD600 value of 1.09). These results were further confirmed by the low colony forming units measures observed in MSMEG_2196 antisense strain. Proteomic analysis was carried out on the MSMEG_2196 antisesne expressing strain grown in the nitrogen-limited condition. Proteins that were differentially expressed were identified by mass spectrometry. A number of the proteins that were down-regulated in the antisense expressing strain are important in the survival of the bacteria under nitrogen-limited conditions. This study indicates a role for MSMEG_2196 in growth or survival of mycobacteria under nitrogen-limitations

MutY-Homolog (MYH) inhibition reduces pancreatic cancer cell growth and increases chemosensitivity

Patients with pancreatic ductal adenocarcinoma (PC) have a poor prognosis due to metastases and chemoresistance. PC is characterized by extensive fibrosis, which creates a hypoxic microenvironment, and leads to increased chemoresistance and intracellular oxidative stress. Thus, proteins that protect against oxidative stress are potential therapeutic targets for PC. A key protein that maintains genomic integrity against oxidative damage is MutY-Homolog (MYH). No prior studies have investigated the function of MYH in PC cells. Using siRNA, we showed that knockdown of MYH in PC cells 1) reduced PC cell proliferation and increased apoptosis; 2) further decreased PC cell growth in the presence of oxidative stress and chemotherapy agents (gemcitabine, paclitaxel and vincristine); 3) reduced PC cell metastatic potential; and 4) decreased PC tumor growth in a subcutaneous mouse model in vivo. The results from this study suggest MYH may be a novel therapeutic target for PC that could potentially improve patient outcome by reducing PC cell survival, increasing the efficacy of existing drugs and reducing metastatic spread

βIII-tubulin: a novel mediator of chemoresistance and metastases in pancreatic cancer

Pancreatic cancer is a leading cause of cancer-related deaths in Western societies. This poor prognosis is due to chemotherapeutic drug resistance and metastatic spread. Evidence suggests that microtubule proteins namely, β-tubulins are dysregulated in tumor cells and are involved in regulating chemosensitivity. However, the role of β-tubulins in pancreatic cancer are unknown. We measured the expression of different β-tubulin isotypes in pancreatic adenocarcinoma tissue and pancreatic cancer cells. Next, we used RNAi to silence βIII-tubulin expression in pancreatic cancer cells, and measured cell growth in the absence and presence of chemotherapeutic drugs. Finally, we assessed the role of βIII-tubulin in regulating tumor growth and metastases using an orthotopic pancreatic cancer mouse model. We found that βIII-tubulin is highly expressed in pancreatic adenocarcinoma tissue and pancreatic cancer cells. Further, we demonstrated that silencing βIII-tubulin expression reduced pancreatic cancer cell growth and tumorigenic potential in the absence and presence of chemotherapeutic drugs. Finally, we demonstrated that suppression of βIII-tubulin reduced tumor growth and metastases in vivo. Our novel data demonstrate that βIII-tubulin is a key player in promoting pancreatic cancer growth and survival, and silencing its expression may be a potential therapeutic strategy to increase the long-term survival of pancreatic cancer patients

Role of a Putative Cyclic Di-GMP Forming Locus MSMEG_2196 in Mycobacterium Smegmatis

Cyclic di-guanosine-monophosphate (c-di-GMP) has been recognized as a second messenger in bacteria controlling multiple cellular processes such as biofilm formation, motility, and virulence. Proteins containing GGDEF and EAL domains are engaged in the synthesis and degradation, respectively, of cyclic di-GMP. Some bacteria contain multiple proteins with GGDEF and EAL domains. The genome of Mycobacterium tuberculosis encodes only one protein (Rv1354c) which contains a GGDEF domain. This protein also contains a tandem EAL. The function of this protein in mycobacteria has not yet been determined. In this study, the orthologue of Rv1354c was investigated in Mycobacterium smegmatis (MSMEG_2196). The expression of MSMEG_2196 in M. smegmatis was altered by constructing sense and antisense expressing strains. The effect of the altered expression of MSMEG_2196 on M. smegmatis was tested under carbon, oxygen, phosphorous, and nitrogen limited growth conditions. There was no significant effect on growth in either the antisense or sense expressing strains grown under nutrient-rich, or carbon-, or oxygen-, or phosphorous limitation conditions. However, a growth effect was observed in the antisense expressing strain when grown under nitrogen-limited conditions. In particular, at mid stationary-phase (1,800 min) the MSMEG_2196 antisense strain had an OD600 value of 0.60, compared to that of the control M. smegmatis/pMind strain (OD600 value of 1.09). These results were further confirmed by the low colony forming units measures observed in MSMEG_2196 antisense strain. Proteomic analysis was carried out on the MSMEG_2196 antisesne expressing strain grown in the nitrogen-limited condition. Proteins that were differentially expressed were identified by mass spectrometry. A number of the proteins that were down-regulated in the antisense expressing strain are important in the survival of the bacteria under nitrogen-limited conditions. This study indicates a role for MSMEG_2196 in growth or survival of mycobacteria under nitrogen-limitations

βIII-tubulin: a novel mediator of chemoresistance and metastases in pancreatic cancer

Pancreatic cancer is a leading cause of cancer-related deaths in Western societies. This poor prognosis is due to chemotherapeutic drug resistance and metastatic spread. Evidence suggests that microtubule proteins namely, β-tubulins are dysregulated in tumor cells and are involved in regulating chemosensitivity. However, the role of β-tubulins in pancreatic cancer are unknown. We measured the expression of different β-tubulin isotypes in pancreatic adenocarcinoma tissue and pancreatic cancer cells. Next, we used RNAi to silence βIII-tubulin expression in pancreatic cancer cells, and measured cell growth in the absence and presence of chemotherapeutic drugs. Finally, we assessed the role of βIII-tubulin in regulating tumor growth and metastases using an orthotopic pancreatic cancer mouse model. We found that βIII-tubulin is highly expressed in pancreatic adenocarcinoma tissue and pancreatic cancer cells. Further, we demonstrated that silencing βIII-tubulin expression reduced pancreatic cancer cell growth and tumorigenic potential in the absence and presence of chemotherapeutic drugs. Finally, we demonstrated that suppression of βIII-tubulin reduced tumor growth and metastases in vivo. Our novel data demonstrate that βIII-tubulin is a key player in promoting pancreatic cancer growth and survival, and silencing its expression may be a potential therapeutic strategy to increase the long-term survival of pancreatic cancer patients

I-AbACUS: a Reliable Software Tool for the Semi-Automatic Analysis of Invasion and Migration Transwell Assays

The quantification of invasion and migration is an important aspect of cancer research, used both in the study of the molecular processes involved in this collection of diseases and the evaluation of the efficacy of new potential treatments. The transwell assay, while being one of the most widely used techniques for the evaluation of these characteristics, shows a high dependence on the operator\u2019s ability to correctly identify the cells and a low protocol standardization. Here we present I-AbACUS, a software tool specifically designed to aid the analysis of transwell assays that automatically and specifically recognizes cells in images of stained membranes and provides the user with a suggested cell count. A complete description of this instrument, together with its validation against the standard analysis technique for this assay is presented. Furthermore, we show that I-AbACUS is versatile and able to elaborate images containing cells with different morphologies and that the obtained results are less dependent on the operator and their experience. We anticipate that this instrument, freely available (Gnu Public Licence GPL v2) at www.marilisacortesi.com as a standalone application, could significantly improve the quantification of invasion and migration of cancer cells

MutY-Homolog (MYH) inhibition reduces pancreatic cancer cell growth and increases chemosensitivity

Patients with pancreatic ductal adenocarcinoma (PC) have a poor prognosis due to metastases and chemoresistance. PC is characterized by extensive fibrosis, which creates a hypoxic microenvironment, and leads to increased chemoresistance and intracellular oxidative stress. Thus, proteins that protect against oxidative stress are potential therapeutic targets for PC. A key protein that maintains genomic integrity against oxidative damage is MutY-Homolog (MYH). No prior studies have investigated the function of MYH in PC cells. Using siRNA, we showed that knockdown of MYH in PC cells 1) reduced PC cell proliferation and increased apoptosis; 2) further decreased PC cell growth in the presence of oxidative stress and chemotherapy agents (gemcitabine, paclitaxel and vincristine); 3) reduced PC cell metastatic potential; and 4) decreased PC tumor growth in a subcutaneous mouse model in vivo. The results from this study suggest MYH may be a novel therapeutic target for PC that could potentially improve patient outcome by reducing PC cell survival, increasing the efficacy of existing drugs and reducing metastatic spread

A Rationally Optimized Nanoparticle System for the Delivery of RNA Interference Therapeutics into Pancreatic Tumors in Vivo

Pancreatic cancer is a devastating disease with a dismal prognosis. Short-interfering RNA (siRNA)-based therapeutics hold promise for the treatment of cancer. However, development of efficient and safe delivery vehicles for siRNA remains a challenge. Here, we describe the synthesis and physicochemical characterization of star polymers (star 1, star 2, star 3) using reversible addition–fragmentation chain transfer polymerization (RAFT) for the delivery of siRNA to pancreatic cancer cells. These star polymers were designed to contain different lengths of cationic poly­(dimethylaminoethyl methacrylate) (PDMAEMA) side-arms and varied amounts of poly­[oligo­(ethylene glycol) methyl ether methacrylate] (POEGMA). We showed that star-POEGMA polymers could readily self-assemble with siRNA to form nanoparticles. The star-POEGMA polymers were nontoxic to normal cells and delivered siRNA with high efficiency to pancreatic cancer cells to silence a gene (<i>TUBB3</i>/βIII-tubulin) which is currently undruggable using chemical agents, and is involved in regulating tumor growth and metastases. Notably, systemic administration of star-POEGMA-siRNA resulted in high accumulation of siRNA to orthotopic pancreatic tumors in mice and silenced βIII-tubulin expression by 80% at the gene and protein levels in pancreatic tumors. Together, these novel findings provide strong rationale for the use of star-POEGMA polymers as delivery vehicles for siRNA to pancreatic tumors