Docosahexaenoic acid and butyrate synergistically modulate intracellular calcium compartmentalization to induce colonocyte apoptosis

Docosahexaenoic acid (DHA, 22:6n-3) from fish oil, and butyrate, a short-chain fatty acid fiber-fermentation product, protect against colon tumorigenesis in part by coordinately inducing apoptosis. We have demonstrated that the combination of these two bioactive compounds demonstrates an enhanced ability to induce colonocyte apoptosis by potentiating mitochondrial lipid oxidation. In order to explore the potential involvement of intracellular Ca2+ in the pro-apoptotic effect of DHA and butyrate, young adult mouse colonocytes (YAMC) and human colonocytes (HCT-116: p53+/+ and p53- /-) were treated with DHA or linoleic acid (LA) for 72 h ± butyrate for the final 6, 12 or 24 h. Cytosolic and mitochondrial Ca2+ levels were measured using Fluo-4 and Rhod-2. In addition, IP3 pool, store-operated channel (SOC)-mediated changes and apoptosis were measured. DHA did not alter basal Ca2+ or apoptosis following 6 h butyrate cotreatment. In contrast, at 12 and 24 h, DHA and butyrate treated cultures exhibited a decrease in cytosolic Ca2+ and enhanced apoptosis compared to LA and butyrate. DHA and butyrate also increased the mitochondrial-to-cytosolic Ca2+ ratio at 6, 12 and 24 h. The accumulation of mitochondrial Ca2+ preceded the onset of apoptosis which increased only following 12 h of butyrate co-treatment. RU-360, a mitochondrial uniporter inhibitor, abrogated mitochondrial Ca2+ accumulation and also partially blocked apoptosis in DHA and butyrate co-treated cells. p53+/+ and p53-/- cells demonstrated similar data with respect to all parameters. Additionally, mitochondrial Ca2+ measurements were also made in rat primarycolonocyte- culture. Rats were fed semipurified diets containing either fish oil (a source of DHA) or corn oil (a source of LA), and colonic crypts were incubated in butyrate exvivo and mitochondrial Ca2+ was quantified. Crypts from rats fed fish oil incubated in butyrate exhibited an increase in the mitochondrial-to-cytosolic Ca2+ ratio compared to fish oil only. In summary, our results indicate for the first time that the combination of DHA and butyrate, compared to butyrate alone, further enhances apoptosis by additionally recruiting a p53-independent Ca2+-mediated intrinsic mitochondrial pathway. These data explain in part why fermentable fiber when combined with fish oil exhibits an enhanced ability to induce apoptosis and protect against colon tumorigenesis

Role of Pattern Recognition Receptors in the Modulation of Antimicrobial Peptide Expression in the Corneal Epithelial Innate Response to F. solani

Purpose: Fusarium solani (F. solani) keratitis is a potentially sight-threatening fungal infection of the cornea. Antimicrobial peptides (AMPs), such as human β-defensins (hBDs) and cathelicidins, essential components of the immune system, likely have a protective role against F. solani keratitis. We examined the role of pattern recognition receptors (PRRs), Dectin-1, and TLR2 in F. solani–induced modulation of AMP expression in vitro. Methods: Human corneal epithelial cells (HCECs) were exposed to heat-inactivated F. solani or pathogen-associated molecular patterns (PAMPs) of F. solani (Zymosan or Zymosan Depleted) for 6, 12, or 24 hours following which AMP mRNA and protein levels were determined. Involvement of TLR2 and Dectin-1 was confirmed by using siRNA knock-down (TLR2 and Dectin-1) or chemical inhibitor BAY 61-3606 (Dectin-1). The functional significance of AMP upregulation was tested using culture supernatant from F. solani or PAMP-treated HCECs against F. solani in the presence of hBD2 or LL37 neutralizing antibody. Results: We confirm that HCECs express Dectin-1 and TLR2. HCECs demonstrated upregulation of AMPs hBD2 and cathelicidin LL37 following exposure to heat-inactivated F. solani or PAMPs. TLR2 and Dectin-1 knockdown and BAY 61-3606 treatment decreased AMP mRNA upregulation confirming PRR involvement. The culture supernatant from F. solani or PAMP-treated HCECs showed substantial killing of F. solani and hBD2 or LL37 neutralizing antibody significantly decreased this effect implicating involvement of these AMPs. Conclusions: These findings demonstrate that Dectin-1 and TLR2 have an important role in regulating F. solani-induced AMP expression in corneal epithelial cells

Simultaneous submission of seven CTSA proposals: UM1, K12, R25, T32-predoctoral, T32-postdoctoral, and RC2: strategies, evaluation, and lessons learned

Translation is the process of turning observations in the research laboratory, clinic, and community into interventions that improve people\u27s health. The Clinical and Translational Science Awards (CTSA) program is a National Center for Advancing Translational Sciences (NCATS) initiative to advance translational science and research. Currently, 64 CTSA hubs exist across the nation. Since 2006, the Houston-based Center for Clinical Translational Sciences (CCTS) has assembled a well-integrated, high-impact hub in Texas that includes six partner institutions within the state, encompassing ∼23,000 sq. miles and over 16 million residents. To achieve the NCATS goal of more treatments for all people more quickly, the CCTS promotes diversity and inclusion by integrating underrepresented populations into clinical studies, workforce training, and career development. In May 2023, we submitted the UM1 application and six companion proposals: K12, R25, T32-Predoctoral, T32-Postdoctoral, and RC2 (two applications). In October 2023, we received priority scores for the UM1 (22), K12 (25), T32-Predoctoral (20), and T32-Postdoctoral (23), which historically fall within the NCATS funding range. This report describes the grant preparation and submission approach, coupled with data from an internal survey designed to assimilate feedback from principal investigators, writers, reviewers, and administrative specialists. Herein, we share the challenges faced, the approaches developed, and the lessons learned

Contact lens care solution killing efficacy against Acanthamoeba castellanii by in vitro testing and live-imaging

In the past decade there has been an increased incidence of Acanthamoeba keratitis, particularly in contact lens wearers. The aim of this study was to utilize in vitro killing assays and to establish a novel, time-lapse, live-cell imaging methodology to demonstrate the efficacy of contact lens care solutions in eradicating Acanthamoeba castellanii (A. castellanii) trophozoites and cysts. Standard qualitative and quantitative in vitro assays were performed along with novel time-lapse imaging coupled with fluorescent dye staining that signals cell death. Quantitative data obtained demonstrated that 3% non-ophthalmic hydrogen peroxide demonstrated the highest percent killing at 87.4% corresponding to a 4.4 log kill. The other contact lens care solutions which showed a 72.9 to 29.2% killing which was consistent with 4.3–2.8 log reduction in trophozoite viability. Both analytical approaches revealed that polyquaternium/PHMB-based was the least efficacious in terms of trophicidal activity. The cysticidal activity of the solutions was much less than activity against trophozoites and frequently was not detected. Live-imaging provided a novel visual endpoint for characterizing the trophocidal activity of the care solutions. All solutions caused rapid rounding or pseudocyst formation of the trophozoites, reduced motility and the appearance of different morphotypes. Polyquaternium/alexidine-based and peroxide-based lens care system induced the most visible damage indicated by significant accumulation of debris from ruptured cells. Polyquaternium/PHMB-based was the least effective showing rounding of the cells but minimal death. These observations are in keeping with care solution biocides having prominent activity at the plasma membrane of Acanthamoeba

Antimicrobial Peptides and Acanthamoeba: Peptide expression and anti-amoebicidal activity via time-lapse imaging

Purpose: A prototype of an atmospheric pressure cold plasma (APCP) source has been developed for the treatment of corneal infections and for ocular surface sterilization. Two minutes of APCP treatment was previously found effective against various microorganisms but caused, in corneal fibroblast cultures, transitory ROS formation, expression of the ogg1 marker of oxidative damage and increase of apoptosis. In the present study we investigated the transcriptome changes of whole human cornea (HC) ex vivo exposed to the disinfectant dose of APCP. Methods: The effects of exposure to APCP for 2 min on HC, in the absence or presence of the antioxidant N-acetyl L-cysteine (NAC) and related to unexposed controls were assessed at 6 h post-treatment by histological and immunohistochemical analysis, Western blotting and Illlumina RNA sequencing (RNA-seq). Additional HC were subsequently exposed to APCP in the same conditions to measure the expression

Esc1-21 a Novel Antimicrobial Peptide for Microbial Keratitis

Purpose: To investigate the antimicrobial efficacy of a novel amphibian antimicrobial peptide, Esculentin1-21 (Esc1-21), in vitro and in a murine model of Pseudomonas aeruginosa (PA) keratitis. Methods: Standard cfu assays were used to determine the MIC of Esc1-21 against PA strains ATCC 27853 and 19660. The effects of physiological salt concentrations and tears (basal and reflex) on antipseudomonal activity were also tested. MTT assays were performed to determine if Esc1-21 was toxic to a human corneal epithelial cell line (HCEC). For in vivo studies corneas of C57BL/6 mice were scratched; then 105-106 cfu PA ATCC19660 applied topically. Esc1-21 (40 μM) or PBS was applied topically three times/day for up to 5 days (pi) post-infection. At 1, 3 and 5 days pi, severity of infection was graded by slit-lamp, neutrophil infiltration was assessed by MPO assay and viable bacterial counts were determined. Results: The MIC for Esc1-21 was 4 μM and 32 μM for ATCC 27853 and 19660 (n=3) respectiv

Simultaneous submission of seven CTSA proposals: UM1, K12, R25, T32-predoctoral, T32-postdoctoral, and RC2: strategies, evaluation, and lessons learned

Translation is the process of turning observations in the research laboratory, clinic, and community into interventions that improve people’s health. The Clinical and Translational Science Awards (CTSA) program is a National Center for Advancing Translational Sciences (NCATS) initiative to advance translational science and research. Currently, 64 “CTSA hubs” exist across the nation. Since 2006, the Houston-based Center for Clinical Translational Sciences (CCTS) has assembled a well-integrated, high-impact hub in Texas that includes six partner institutions within the state, encompassing ∼23,000 sq. miles and over 16 million residents. To achieve the NCATS goal of “more treatments for all people more quickly,” the CCTS promotes diversity and inclusion by integrating underrepresented populations into clinical studies, workforce training, and career development. In May 2023, we submitted the UM1 application and six “companion” proposals: K12, R25, T32-Predoctoral, T32-Postdoctoral, and RC2 (two applications). In October 2023, we received priority scores for the UM1 (22), K12 (25), T32-Predoctoral (20), and T32-Postdoctoral (23), which historically fall within the NCATS funding range. This report describes the grant preparation and submission approach, coupled with data from an internal survey designed to assimilate feedback from principal investigators, writers, reviewers, and administrative specialists. Herein, we share the challenges faced, the approaches developed, and the lessons learned

Protective Role of Murine -Defensins 3 and 4 and Cathelin-Related Antimicrobial Peptide in Fusarium solani Keratitis

Antimicrobial peptides (AMPs), such as β-defensins and cathelicidins, are essential components of innate and adaptive immunity owing to their extensive multifunctional activities. However, their role in fungal infection in vivo remains elusive. In this study, we investigated the protective effect of murine β-defensin 3 (mBD3), mBD4, and the cathelicidin cathelin-related antimicrobial peptide (CRAMP) in a murine model of Fusarium solani keratitis. C57BL/6 mice showed significant corneal disease 1 and 3 days after infection, which was accompanied by enhanced expression of β-defensins and CRAMP. Disease severity was significantly improved 7 days after infection, at which time AMP expression was returning to baseline. Mice deficient in mBD3 (genetic knockout), mBD4 (short interfering RNA knockdown), or CRAMP (genetic knockout) exhibited enhanced disease severity and progression, increased neutrophil recruitment, and delayed pathogen elimination compared to controls. Taken together, these data suggest a vital role for AMPs in defense against F. solani keratitis, a potentially blinding corneal disease