AJUR Volume 19 Issue 3 (December 2022)

Click on this link to download the full high-definition interactive pdf for AJUR Volume 19 Issue 3 (December 2022) or https://doi.org/10.33697/ajur.2022.063

Links to individual manuscripts, abstracts, and keywords are provided below.

p.3. Comparison of Genotypic and Phenotypic Predictions for Heavy Metal Resistance in Salmonella enterica and Escherichia coli

Jeevan Rivera-Díaz, Haley Phillippi, Nyduta Mbogo, Erin M. Nawrocki, & Edward G. Dudley


ABSTRACT: Salmonella enterica and Escherichia coli are two pathogenic bacteria of worldwide importance that can infect the gastrointestinal tract. Contamination in the food supply chain is an area of concern. Animal feed may be supplemented with essential trace elements, which function as nutritional additives to promote growth & health and optimize production. Bacteria have acquired many metal resistance genes to adapt to the exposure of metals. In this study, our objectives were to evaluate in S. enterica and E. coli, the correlation between the resistance genotype and phenotype to certain heavy metals, and the ability of conjugative plasmids to transfer antimicrobial resistance genes (AMRGs) and heavy metal resistance genes (HMRGs). A total of 10 strains, five S. enterica and five E. coli, were used for this study. Minimal inhibitory concentrations (MICs) were determined for heavy metals: copper, silver, arsenic, and tellurite. The tested isolates showed resistance to copper (5/10; 50%), arsenic (7/10; 70%), and silver (9/10; 90%). Cohen’s Kappa statistics were used to analyze genotype to phenotype agreements. Among the 10 strains sampled, the accordance between geno- and phenotypic heavy metal resistance was fair for copper (kappa = 0.4), none to slight for arsenic (kappa = 0.19) and tellurite (kappa = 0), and no agreement for silver (kappa = -0.19). The transfer of HMRGs was determined in a conjugation experiment performed for all five Salmonella strains as donors using mixed broth cultures. Transconjugants were obtained only from the genotypically tellurite-resistant strain PSU-3260, which yielded a transfer frequency of 10⁻³ transconjugants per donor. In such strain, the tellurite-resistant genes reside on an IncHI2-type plasmid that shares high DNA sequence identity with known HMRG-disseminating Salmonella plasmids. Our results indicated no considerable correlation between the geno- and phenotypic resistance towards heavy metals in the sampled S. enterica and E. coli. The necessity of research in this area is supported by the lack of standardized protocols and MIC clinical breakpoints for heavy metals.

KEYWORDS: Heavy metal; resistance; Salmonella; E. coli; agriculture; genotype; phenotype; MIC

p.17. Preventing the Activation of a Stress Gene Response in Escherichia coli Using Acetate, Butyrate, and Propionate

Kaylee M. Weigel, Kathleen M Ruff-Schmidt, Birgit M. Prüß, & Danielle L J Condry


ABSTRACT: Regulation of microbial symbiosis in the human intestinal tract is imperative to maintain overall human health and prevent dysbiosis-related diseases, such as inflammatory bowel disease and obesity. Short-chain fatty acids (SCFA) in the intestine are produced by bacterial fermentation and aid in inflammation reduction, dietary fiber digestion, and metabolizing nutrients for the colon. SCFA, notably acetate, butyrate, and propionate, are starting to be used in clinical interventions for GI diseases. While acetate has been shown to mitigate a stress response in the proteome of Escherichia coli cells, little is known about the effects of butyrate and propionate on the same cells. This study aims to evaluate the effects that butyrate and propionate have on the activation of stress promoters in E. coli when induced with a known stressor. Three different strains of E. coli containing the pUCD615 plasmid were used, each with a different promoter fused to the structural genes of the lac operon on the plasmid. Each promoter detected a unique stress response: grpE’::lux fusion (heat shock), recA’::lux fusion (SOS response), and katG’::lux fusion (oxidative damage). Activation of these stress promoters by treatment groups resulted in the emission of bioluminescence which was quantified and compared across treatment groups. All three SCFAs at 25 mM added to cultures prior to stressing the bacteria caused significantly lower bioluminescence levels when compared to the stressed culture without prior addition of SCFA. This indicates that these SCFAs may reduce the stress response in E. coli.

KEYWORDS: Short-chain fatty acids; acetate; butyrate; propionate; Escherichia coli; stress response; Vibrio fischeri luxCDABE; grpE; katG; recA

p.27 Quantification of Microfibers from Marine Sediments from Three Locations in Southern California: An Exposed Beach (Ventura County), a Watershed (Los Angeles County), and an Enclosed Harbor (Orange County)

Adrianna Ebrahim & Mia LeClerc


ABSTRACT: Microfibers are small (<5 mm) fibers made of synthetic materials that are ubiquitous in the environment. The purpose of this observational study was to quantify the number of microfibers in marine sediments and determine which locations have the highest risk for this type of pollution. Sediment samples were taken from three locations in Southern California (Sycamore Watershed, Ventura State Beach Jetty, and Newport Beach Harbor) to determine which had the highest number of microfibers. It was hypothesized that microfibers would be found at each sample site and that the most microfibers would be found at Sycamore Watershed due to its proximity to a wastewater discharge point. The microfibers were separated from the sediment through a process of stratification and filtration and analyzed by a one-way ANOVA and Tukey’s test. Per sample, there was an average of 111.5 (土99.3, n=14) microfibers found per sample at Sycamore Watershed, 59 (土17.4, n=18) at Newport Beach Harbor, and 53 (土14.4, n=18) at Ventura State Beach Jetty. A total of 3,590 microfibers were found from all three sample sites. Analysis revealed that Sycamore Watershed had significantly more microfibers than any other site (p<.05). It is likely that Sycamore Watershed had the most microfibers because of its proximity to a sewage-sludge disposal site that contains the polluted water from our washing machines. In conclusion, microfibers are polluting the sediments in harbors, open coastlines, and watersheds in California, negatively affecting the ecosystems in these areas.

KEYWORDS: Microfiber; Microplastic; Macroplastic; Marine Pollution; Synthetic Materials; Wastewater Treatment Plants; Sediments; Watershed; Harbor; Jetty

p. 37. Stabilization of Cisplatin via Coordination of Ethylenediamine

Samantha L. Rea, Alexia Smith, Brooke Hornberger, Grace Fillmore, Jeremy Burkett, & Timothy Dwyer


ABSTRACT: While the chemotherapeutic cisplatin is used to treat a variety of cancers, metal toxicity and cisplatin resistance via genetic and epigenetic changes limits its use and calls for alternative therapies. To combat the observed toxicities and create a more stable compound, which avoids isomerization into a trans configuration, three cisplatin analogues including cispalladium, dichloro-(ethylenediamine)-platinum(II), and dichloro-(ethylenediamine)-palladium(II) were synthesized as potential cisplatin alternatives. Each compound was evaluated for cytotoxicity on SK-OV-3 cells against cisplatin. Synthesis of dichloro-(ethylenediamine)-platinum(II) yielded 20.5% of the theoretical yield, while dichloro-(ethylenediamine)-palladium(II) yielded 49.1%. Results from the cytotoxicity trial revealed that cispalladium was not effective against SK-OV-3 cells, and dichloro-(ethylenediamine)-palladium had minimal effects. The dichloro-(ethylenediamine)-platinum(II) was the most efficacious with an IC50 value of 0.77 µg/ml compared to the IC50 of 0.61 µg/ml for cisplatin. With a similar IC50 to cisplatin, these results suggest that dichloro-(ethylenediamine)-platinum(II) has the potential to serve as a cisplatin alternative for cancer patients who develop resistance following their clinical course of cisplatin. Future studies on the cytotoxicity of dichloro-(ethylenediamine)-platinum(II) to induce cell death on cisplatin-resistance cell lines are necessary to determine the ability of the compound to be utilized as a cisplatin alternative.

KEYWORDS: Cisplatin; Ovarian Cancer; SK-OV-3; Drug Resistance; Stability; Palladium; Ethylenediamine; Cispalladium; Dichloro-(ethylenediamine)-platinum(II); Dichloro-(ethylenediamine)-palladium(II)

p.47. Novel Interactors of the SH2 Domain of the Signaling Adaptors CRK and CRKL Identified in Neuro2A Cells

Caroline M. Dumas, Anna M. Schmoker, Shannon R. Bennett, Amara S. Chittenden, Chelsea B. Darwin, Helena K. Gaffney, Hannah L. Lewis, Eliana Moskovitz, Jonah T. Rehak, Anna A. Renzi, Claire E. Rothfelder, Adam J. Slamin, Megan E. Tammaro, Leigh M. Sweet, & Bryan A. Ballif*


ABSTRACT: CT10 regulator of kinase (CRK) and CRK-like (CRKL) form a family of signaling adaptor proteins that serve important roles in the regulation of fundamental cellular processes, including cell motility and proliferation, in a variety of cell types. The Src Homology 2 (SH2) domain of CRK and CRKL interacts with proteins containing phosphorylated tyrosine-X-X-proline (pYXXP) motifs, facilitating complex formation during signaling events. A handful of CRK/CRKL-SH2-specific interactors have been identified to date, although in silico analyses suggest that many additional interactors remain to be found. To identify CRK/CRKL-SH2 interactors with potential involvement in neuronal development, we conducted a mass spectrometry-based proteomics screen using a neuronal cell line (Neuro2A, or N2A). This resulted in the identification of 132 (6 known and 126 novel) YXXP-containing CRK/CRKL-SH2 interactors, of which 77 were stimulated to bind to the CRK/CRKL-SH2 domain following tyrosine phosphatase inhibition. Approximately half of the proteins identified were common interactors of both the CRK- and CRKL-SH2 domains. However, both CRK family member SH2 domains exhibited unique binding partners across experimental replicates. These findings reveal an abundance of novel neuronal CRK/CRKL-SH2 domain binding partners and suggest that CRK family SH2 domains possess undescribed docking preferences beyond the canonical pYXXP motif.

KEYWORDS: CRK; CRKL; SH2; LC-MS/MS; Proteomics; Neurodevelopment; Signal Transduction

SUPPLEMENT 1 (Figures)