Publications by authors named "Emily Putnam"

6 Publications

  • Page 1 of 1

Hypomethylating Agent Azacitidine Is Effective in Treating Brain Metastasis Triple-Negative Breast Cancer Through Regulation of DNA Methylation of Keratin 18 Gene.

Transl Oncol 2020 Jun 11;13(6):100775. Epub 2020 May 11.

Department of Biomedical Sciences, West Virginia School of Osteopathic Medicine, 400 Lee Street North, Lewisburg, WV. Electronic address:

Breast cancer patients presenting with symptomatic brain metastases have poor prognosis, and current chemotherapeutic agents are largely ineffective. In this study, we evaluated the hypomethylating agent azacitidine (AZA) for its potential as a novel therapeutic in preclinical models of brain metastasis of breast cancer. We used the parental triple-negative breast cancer MDA-MB-231 (231) cells and their brain colonizing counterpart (231Br) to ascertain phenotypic differences in response to AZA. We observed that 231Br cells have higher metastatic potential compared to 231 cells. With regard to therapeutic value, the AZA IC value in 231Br cells is significantly lower than that in parental cells (P < .01). AZA treatment increased apoptosis and inhibited the Wnt signaling transduction pathway, angiogenesis, and cell metastatic capacity to a significantly higher extent in the 231Br line. AZA treatment in mice with experimental brain metastases significantly reduced tumor burden (P = .0112) and increased survival (P = .0026) compared to vehicle. Lastly, we observed a decreased expression of keratin 18 (an epithelial maker) in 231Br cells due to hypermethylation, elucidating a potential mechanism of action of AZA in treating brain metastases from breast cancer.
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http://dx.doi.org/10.1016/j.tranon.2020.100775DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7225776PMC
June 2020

B vitamin acquisition by gut commensal bacteria.

PLoS Pathog 2020 01 23;16(1):e1008208. Epub 2020 Jan 23.

Department of Microbial Pathogenesis and Microbial Sciences Institute, Yale University School of Medicine, New Haven, Connecticut, United States of America.

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http://dx.doi.org/10.1371/journal.ppat.1008208DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6977713PMC
January 2020

The Conserved Spore Coat Protein SpoVM Is Largely Dispensable in Spore Formation.

mSphere 2017 Sep-Oct;2(5). Epub 2017 Sep 20.

Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts, USA.

The spore-forming bacterial pathogen is a leading cause of health care-associated infections in the United States. In order for this obligate anaerobe to transmit infection, it must form metabolically dormant spores prior to exiting the host. A key step during this process is the assembly of a protective, multilayered proteinaceous coat around the spore. Coat assembly depends on coat morphogenetic proteins recruiting distinct subsets of coat proteins to the developing spore. While 10 coat morphogenetic proteins have been identified in , only two of these morphogenetic proteins have homologs in the : SpoIVA and SpoVM. SpoIVA is critical for proper coat assembly and functional spore formation, but the requirement for SpoVM during this process was unknown. Here, we show that SpoVM is largely dispensable for spore formation, in contrast with . Loss of SpoVM resulted in modest decreases (~3-fold) in heat- and chloroform-resistant spore formation, while morphological defects such as coat detachment from the forespore and abnormal cortex thickness were observed in ~30% of mutant cells. Biochemical analyses revealed that SpoIVA and SpoVM directly interact, similarly to their counterparts. However, in contrast with , SpoVM was not essential for SpoIVA to encase the forespore. Since coat morphogenesis requires SpoIVA-interacting protein L (SipL), which is conserved exclusively in the , but not the more broadly conserved SpoVM, our results reveal another key difference between and spore assembly pathways. The spore-forming obligate anaerobe is the leading cause of antibiotic-associated diarrheal disease in the United States. When spores are ingested by susceptible individuals, they germinate within the gut and transform into vegetative, toxin-secreting cells. During infection, must also induce spore formation to survive exit from the host. Since spore formation is essential for transmission, understanding the basic mechanisms underlying sporulation in could inform the development of therapeutic strategies targeting spores. In this study, we determine the requirement of the homolog of SpoVM, a protein that is essential for spore formation in due to its regulation of coat and cortex formation. We observed that SpoVM plays a minor role in spore formation, in contrast with , indicating that this protein would not be a good target for inhibiting spore formation.
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http://dx.doi.org/10.1128/mSphere.00315-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5607322PMC
September 2017

Global analysis of the sporulation pathway of Clostridium difficile.

PLoS Genet 2013 8;9(8):e1003660. Epub 2013 Aug 8.

Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, Vermont, USA.

The Gram-positive, spore-forming pathogen Clostridium difficile is the leading definable cause of healthcare-associated diarrhea worldwide. C. difficile infections are difficult to treat because of their frequent recurrence, which can cause life-threatening complications such as pseudomembranous colitis. The spores of C. difficile are responsible for these high rates of recurrence, since they are the major transmissive form of the organism and resistant to antibiotics and many disinfectants. Despite the importance of spores to the pathogenesis of C. difficile, little is known about their composition or formation. Based on studies in Bacillus subtilis and other Clostridium spp., the sigma factors σ(F), σ(E), σ(G), and σ(K) are predicted to control the transcription of genes required for sporulation, although their specific functions vary depending on the organism. In order to determine the roles of σ(F), σ(E), σ(G), and σ(K) in regulating C. difficile sporulation, we generated loss-of-function mutations in genes encoding these sporulation sigma factors and performed RNA-Sequencing to identify specific sigma factor-dependent genes. This analysis identified 224 genes whose expression was collectively activated by sporulation sigma factors: 183 were σ(F)-dependent, 169 were σ(E)-dependent, 34 were σ(G)-dependent, and 31 were σ(K)-dependent. In contrast with B. subtilis, C. difficile σ(E) was dispensable for σ(G) activation, σ(G) was dispensable for σ(K) activation, and σ(F) was required for post-translationally activating σ(G). Collectively, these results provide the first genome-wide transcriptional analysis of genes induced by specific sporulation sigma factors in the Clostridia and highlight that diverse mechanisms regulate sporulation sigma factor activity in the Firmicutes.
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http://dx.doi.org/10.1371/journal.pgen.1003660DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3738446PMC
March 2014

Structural and functional analysis of the CspB protease required for Clostridium spore germination.

PLoS Pathog 2013 Feb 7;9(2):e1003165. Epub 2013 Feb 7.

Graduate Program in Cell, Molecular and Biomedical Sciences, University of Vermont, Burlington, Vermont, United States of America.

Spores are the major transmissive form of the nosocomial pathogen Clostridium difficile, a leading cause of healthcare-associated diarrhea worldwide. Successful transmission of C. difficile requires that its hardy, resistant spores germinate into vegetative cells in the gastrointestinal tract. A critical step during this process is the degradation of the spore cortex, a thick layer of peptidoglycan surrounding the spore core. In Clostridium sp., cortex degradation depends on the proteolytic activation of the cortex hydrolase, SleC. Previous studies have implicated Csps as being necessary for SleC cleavage during germination; however, their mechanism of action has remained poorly characterized. In this study, we demonstrate that CspB is a subtilisin-like serine protease whose activity is essential for efficient SleC cleavage and C. difficile spore germination. By solving the first crystal structure of a Csp family member, CspB, to 1.6 Å, we identify key structural domains within CspB. In contrast with all previously solved structures of prokaryotic subtilases, the CspB prodomain remains tightly bound to the wildtype subtilase domain and sterically occludes a catalytically competent active site. The structure, combined with biochemical and genetic analyses, reveals that Csp proteases contain a unique jellyroll domain insertion critical for stabilizing the protease in vitro and in C. difficile. Collectively, our study provides the first molecular insight into CspB activity and function. These studies may inform the development of inhibitors that can prevent clostridial spore germination and thus disease transmission.
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http://dx.doi.org/10.1371/journal.ppat.1003165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3567191PMC
February 2013

SpoIVA and SipL are Clostridium difficile spore morphogenetic proteins.

J Bacteriol 2013 Mar 4;195(6):1214-25. Epub 2013 Jan 4.

Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, Vermont, USA.

Clostridium difficile is a major nosocomial pathogen whose infections are difficult to treat because of their frequent recurrence. The spores of C. difficile are responsible for these clinical features, as they resist common disinfectants and antibiotic treatment. Although spores are the major transmissive form of C. difficile, little is known about their composition or morphogenesis. Spore morphogenesis has been well characterized for Bacillus sp., but Bacillus sp. spore coat proteins are poorly conserved in Clostridium sp. Of the known spore morphogenetic proteins in Bacillus subtilis, SpoIVA is one of the mostly highly conserved in the Bacilli and the Clostridia. Using genetic analyses, we demonstrate that SpoIVA is required for proper spore morphogenesis in C. difficile. In particular, a spoIVA mutant exhibits defects in spore coat localization but not cortex formation. Our study also identifies SipL, a previously uncharacterized protein found in proteomic studies of C. difficile spores, as another critical spore morphogenetic protein, since a sipL mutant phenocopies a spoIVA mutant. Biochemical analyses and mutational analyses indicate that SpoIVA and SipL directly interact. This interaction depends on the Walker A ATP binding motif of SpoIVA and the LysM domain of SipL. Collectively, these results provide the first insights into spore morphogenesis in C. difficile.
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http://dx.doi.org/10.1128/JB.02181-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3592010PMC
March 2013