Cephalopod-omics: emerging fields and technologies in cephalopod biology

14 pages, 1 figure.-- This is an Open Access article distributed under the terms of the Creative Commons Attribution LicenseFew animal groups can claim the level of wonder that cephalopods instill in the minds of researchers and the general public. Much of cephalopod biology, however, remains unexplored: the largest invertebrate brain, difficult husbandry conditions, and complex (meta-)genomes, among many other things, have hindered progress in addressing key questions. However, recent technological advancements in sequencing, imaging, and genetic manipulation have opened new avenues for exploring the biology of these extraordinary animals. The cephalopod molecular biology community is thus experiencing a large influx of researchers, emerging from different fields, accelerating the pace of research in this clade. In the first post-pandemic event at the Cephalopod International Advisory Council (CIAC) conference in April 2022, over 40 participants from all over the world met and discussed key challenges and perspectives for current cephalopod molecular biology and evolution. Our particular focus was on the fields of comparative and regulatory genomics, gene manipulation, single-cell transcriptomics, metagenomics, and microbial interactions. This article is a result of this joint effort, summarizing the latest insights from these emerging fields, their bottlenecks, and potential solutions. The article highlights the interdisciplinary nature of the cephalopod-omics community and provides an emphasis on continuous consolidation of efforts and collaboration in this rapidly evolving fieldPeer reviewe

Lactobacillus acidophilus counteracts inhibition of NHE3 and DRA expression and alleviates diarrheal phenotype in mice infected with Citrobacter rodentium

Impaired absorption of electrolytes is a hallmark of diarrhea associated with inflammation or enteric infections. Intestinal epithelial luminal membrane NHE3 (Na+/H+ exchanger 3) and DRA (Down-Regulated in Adenoma; Cl−/HCO3− exchanger) play key roles in mediating electroneutral NaCl absorption. We have previously shown decreased NHE3 and DRA function in response to short-term infection with enteropathogenic E. coli (EPEC), a diarrheal pathogen. Recent studies have also shown substantial downregulation of DRA expression in a diarrheal model of infection with Citrobacter rodentium, the mouse counterpart of EPEC. Since our previous studies showed that the probiotic Lactobacillus acidophilus (LA) increased DRA and NHE3 function and expression and conferred protective effects in experimental colitis, we sought to evaluate the efficacy of LA in counteracting NHE3 and DRA inhibition and ameliorating diarrhea in a model of C. rodentium infection. FVB/N mice challenged with C. rodentium [1 × 109 colony-forming units (CFU)] with or without administration of live LA (3 × 109 CFU) were assessed for NHE3 and DRA mRNA and protein expression, mRNA levels of carbonic anhydrase, diarrheal phenotype (assessed by colonic weight-to-length ratio), myeloperoxidase activity, and proinflammatory cytokines. LA counteracted C. rodentium-induced inhibition of colonic DRA, NHE3, and carbonic anhydrase I and IV expression and attenuated diarrheal phenotype and MPO activity. Furthermore, LA completely blocked C. rodentium induction of IL-1β, IFN-γ, and CXCL1 mRNA and C. rodentium-induced STAT3 phosphorylation. In conclusion, our data provide mechanistic insights into antidiarrheal effects of LA in a model of infectious diarrhea and colitis

Activation of Nuclear Factor—κB by Tumor Necrosis Factor in Intestinal Epithelial Cells and Mouse Intestinal Epithelia Reduces Expression of the Chloride Transporter SLC26A3

Background & Aims: Diarrhea associated with inflammatory bowel diseases has been associated with increased levels of inflammatory cytokines, including tumor necrosis factor (TNF). The intestinal mucosa of patients with inflammatory bowel diseases has reduced expression of solute carrier family 26 member 3 (SLC26A3, also called DRA). We investigated whether TNF directly affects expression of DRA in human intestinal epithelial cells (IECs) and in the intestines of mice, and studied the mechanisms of these effects. Methods: We performed quantitative reverse transcription polymerase chain reaction, immunofluorescence, and immunoblot analyses in Caco-2, HT-29, and T-84 cells human IECs cultured in 2 or 3 dimensions with or without TNF (50 ng/mL for 6–24 hours). We purified nuclear extracts and quantified nuclear factor−κB (NF-κB) activation and DNA binding. We isolated intestinal crypts from C57BL/6 mice, cultured enteroids, incubated these with TNF (50 ng/mL, 24 hours), and quantified messenger RNAs. DRA-mediated exchange of Cl− for HCO3− was measured by uptake of 125I. Expression of the NF-κB inhibitor α (IkBa) was knocked down in Caco-2 cells with small interfering RNAs. Activation of NF-κB in response to TNF was measured by luciferase reporter assays; binding of the NF-κB subunit p65 in cells was analyzed in chromatin immunoprecipitation assays. DRA promoter activity was measured in a luciferase reporter assay. C57BL/6 mice were injected with TNF (5 μg/mouse for 3–6 hours) or vehicle (control); intestines were collected and analyzed by immunofluorescence, or RNA and protein were collected from the mucosa. Results: Incubation of IECs with TNF reduced expression of DRA. Knockdown of NF-κB inhibitor α in IECs led to nuclear translocation of the NF-κB subunit p65 and reduced levels of DRA messenger RNA and protein. Expression of a transgene encoding p65 or p50 in IECs led to significant reductions in the promoter activity of DRA and its expression. In chromatin immunoprecipitation assays, p65 bound directly to the promoter of DRA, at the regions of −935 to −629 and −375 to −84. Injection of mice with TNF or incubation of crypt-derived enteroids with TNF reduced their expression of DRA messenger RNA and protein. Conclusions: In human IECs and intestinal tissues from mice, we found TNF to activate NF-κB, which reduced expression of the Cl− / HCO3− exchanger DRA (SLC26A3), via direct binding to the promoter of DRA. This pathway is an important therapeutic target for inflammatory bowel disease−associated diarrhea

Lactobacillus acidophilus

Impaired absorption of electrolytes is a hallmark of diarrhea associated with inflammation or enteric infections. Intestinal epithelial luminal membrane NHE3 (Na(+)/H(+) exchanger 3) and DRA (Down-Regulated in Adenoma; Cl(−)/HCO(3)(−) exchanger) play key roles in mediating electroneutral NaCl absorption. We have previously shown decreased NHE3 and DRA function in response to short-term infection with enteropathogenic E. coli (EPEC), a diarrheal pathogen. Recent studies have also shown substantial downregulation of DRA expression in a diarrheal model of infection with Citrobacter rodentium, the mouse counterpart of EPEC. Since our previous studies showed that the probiotic Lactobacillus acidophilus (LA) increased DRA and NHE3 function and expression and conferred protective effects in experimental colitis, we sought to evaluate the efficacy of LA in counteracting NHE3 and DRA inhibition and ameliorating diarrhea in a model of C. rodentium infection. FVB/N mice challenged with C. rodentium [1 × 10(9) colony-forming units (CFU)] with or without administration of live LA (3 × 10(9) CFU) were assessed for NHE3 and DRA mRNA and protein expression, mRNA levels of carbonic anhydrase, diarrheal phenotype (assessed by colonic weight-to-length ratio), myeloperoxidase activity, and proinflammatory cytokines. LA counteracted C. rodentium-induced inhibition of colonic DRA, NHE3, and carbonic anhydrase I and IV expression and attenuated diarrheal phenotype and MPO activity. Furthermore, LA completely blocked C. rodentium induction of IL-1β, IFN-γ, and CXCL1 mRNA and C. rodentium-induced STAT3 phosphorylation. In conclusion, our data provide mechanistic insights into antidiarrheal effects of LA in a model of infectious diarrhea and colitis