Development of genetically encoded cell type-specific activity markers for the mouse olfactory system

The olfactory system consists of the olfactory epithelium in the nasal cavity, the olfactory bulb, the olfactory cortex and higher brain regions. Olfactory information processing begins at the olfactory sensory neurons that express odorant receptors. These proteins bind odors and transduce a biochemical signal that is converted to action potentials by the neuron. In the olfactory bulb, sensory neurons synapse with projection neurons called the mitral/tufted cells in structures called glomeruli and pass on information to the olfactory cortex. The olfactory bulb is not a simple way station for transmission of odorant information; significant processing of information is carried out by the circuitry in the olfactory bulb. The major neuronal components of the bulb that take part in these interactions are the periglomerular cells, short axon cells and the granule cells. Neural information processing is carried out by ensembles of neurons and it is important to study such populations to reach an understanding of nervous system function. Recent developments in identification of cell type specific promoters and construction of GFP based sensors of Ca2+ influx and voltage changes has made it possible to devise genetic strategies to study them. Another strategy that is frequently employed is studying the expression of Immediate Early Genes, especially C-fos after stimulation. In this study we developed two mice strains, one that can express the short halflife version of EYFP called d2EYFP under the C-fos promoter and another that codes for the calcium sensitive fluorescent protein ratiometric pericam. The expression of both these reporters can be made cell type specific because they are blocked by a floxed stop cassette and can be activated in specific cell types by crossing to mice strains that express the cre recombinase under specific promoters. A Bacterial Artificial Chromosome that carries the Cfos gene was modified by inserting a STOP-IRES-d2EYFP cassette at the C-fos locus after truncating the C-fos-coding region. The loxP site in the BAC's back bone was removed by another round of recombination and circular BAC molecules were used of pronuclear microinjection. One positive founder was identified and is being bred for further analysis. Ratiomeric pericam was cloned under the ubiquitous CAG promoter with a stop cassette in between and transgenic mice were made after testing expression in cell culture. Seven different transgenic lines with different transgene copy numbers were obtained and were subjected to further analysis. No transgene expression could be detected upon breeding to a CMV-Cre mouse strain that should delete in all cells. Analysis of DNA of double transgenic mice showed no deletion of stop cassette. The mice were further crossed to a CamKIIa-cr

Sweet Taste Signaling: The Core Pathways and Regulatory Mechanisms

Sweet taste, a proxy for sugar-derived calories, is an important driver of food intake, and animals have evolved robust molecular and cellular machinery for sweet taste signaling. The overconsumption of sugar-derived calories is a major driver of obesity and other metabolic diseases. A fine-grained appreciation of the dynamic regulation of sweet taste signaling mechanisms will be required for designing novel noncaloric sweeteners with better hedonic and metabolic profiles and improved consumer acceptance. Sweet taste receptor cells express at least two signaling pathways, one mediated by a heterodimeric G-protein coupled receptor encoded by taste 1 receptor members 2 and 3 (TAS1R2 + TAS1R3) genes and another by glucose transporters and the ATP-gated potassium (KATP) channel. Despite these important discoveries, we do not fully understand the mechanisms regulating sweet taste signaling. We will introduce the core components of the above sweet taste signaling pathways and the rationale for having multiple pathways for detecting sweet tastants. We will then highlight the roles of key regulators of the sweet taste signaling pathways, including downstream signal transduction pathway components expressed in sweet taste receptor cells and hormones and other signaling molecules such as leptin and endocannabinoids

Type II taste cells participate in mucosal immune surveillance

The oral microbiome is second only to its intestinal counterpart in diversity and abundance but its effects on taste cells remains largely unexplored. Using single-cell RNASeq, we found that mouse taste cells, in particular, sweet and umami receptor cells that express taste 1 receptor member 3 (Tas1r3), have a gene expression signature reminiscent of Microfold (M) cells, a central player in immune surveillance in the mucosa-associated lymphoid tissue (MALT) such as those in the Peyer’s patch and tonsils. Administration of tumor necrosis factor ligand superfamily member 11 (TNFSF11; also known as RANKL), a growth factor required for differentiation of M cells, dramatically increased M cell proliferation and marker gene expression in the taste papillae and in cultured taste organoids from wild-type (WT) mice. Taste papillae and organoids from knockout mice lacking Spib (SpibKO), a RANKL-regulated transcription factor required for M cell development and regeneration on the other hand, failed to respond to RANKL. Taste papillae from SpibKO mice also showed reduced expression of NF-κB signaling pathway components and proinflammatory cytokines and attracted fewer immune cells. However, lipopolysaccharide-induced expression of cytokines was strongly up-regulated in SpibKO mice compared to their WT counterparts. Like M cells, taste cells from WT but not SpibKO mice readily took up fluorescently labeled microbeads, a proxy for microbial transcytosis. The proportion of taste cell subtypes are unaltered in SpibKO mice; however, they displayed increased attraction to sweet and umami taste stimuli. We propose that taste cells are involved in immune surveillance and may tune their taste responses to microbial signaling and infection

Fcγ Receptor I Alpha Chain (CD64) Expression in Macrophages Is Critical for the Onset of Meningitis by Escherichia coli K1

Neonatal meningitis due to Escherichia coli K1 is a serious illness with unchanged morbidity and mortality rates for the last few decades. The lack of a comprehensive understanding of the mechanisms involved in the development of meningitis contributes to this poor outcome. Here, we demonstrate that depletion of macrophages in newborn mice renders the animals resistant to E. coli K1 induced meningitis. The entry of E. coli K1 into macrophages requires the interaction of outer membrane protein A (OmpA) of E. coli K1 with the alpha chain of Fcγ receptor I (FcγRIa, CD64) for which IgG opsonization is not necessary. Overexpression of full-length but not C-terminal truncated FcγRIa in COS-1 cells permits E. coli K1 to enter the cells. Moreover, OmpA binding to FcγRIa prevents the recruitment of the γ-chain and induces a different pattern of tyrosine phosphorylation of macrophage proteins compared to IgG2a induced phosphorylation. Of note, FcγRIa−/− mice are resistant to E. coli infection due to accelerated clearance of bacteria from circulation, which in turn was the result of increased expression of CR3 on macrophages. Reintroduction of human FcγRIa in mouse FcγRIa−/− macrophages in vitro increased bacterial survival by suppressing the expression of CR3. Adoptive transfer of wild type macrophages into FcγRIa−/− mice restored susceptibility to E. coli infection. Together, these results show that the interaction of FcγRI alpha chain with OmpA plays a key role in the development of neonatal meningitis by E. coli K1

Sweet Taste Signaling: The Core Pathways and Regulatory Mechanisms

Sweet taste, a proxy for sugar-derived calories, is an important driver of food intake, and animals have evolved robust molecular and cellular machinery for sweet taste signaling. The overconsumption of sugar-derived calories is a major driver of obesity and other metabolic diseases. A fine-grained appreciation of the dynamic regulation of sweet taste signaling mechanisms will be required for designing novel noncaloric sweeteners with better hedonic and metabolic profiles and improved consumer acceptance. Sweet taste receptor cells express at least two signaling pathways, one mediated by a heterodimeric G-protein coupled receptor encoded by taste 1 receptor members 2 and 3 (TAS1R2 + TAS1R3) genes and another by glucose transporters and the ATP-gated potassium (KATP) channel. Despite these important discoveries, we do not fully understand the mechanisms regulating sweet taste signaling. We will introduce the core components of the above sweet taste signaling pathways and the rationale for having multiple pathways for detecting sweet tastants. We will then highlight the roles of key regulators of the sweet taste signaling pathways, including downstream signal transduction pathway components expressed in sweet taste receptor cells and hormones and other signaling molecules such as leptin and endocannabinoids

Preface new horizons in biotechnology – NHBT 2019

Producción CientíficaRelevance of Biotechnology for a sustainable living has increased over the past few decades by addressing the different domains including agriculture, food, health care, livestock management, energy, environment, climate change, waste management and a multitude of other areas. Today, the subject not only encompasses life sciences and engineering, but spans across most of the human activities, affecting and influencing every one. The subject is poised to tackle emerging challenges like rapidly spreading global pandemics by providing solutions in the form of vaccines or drugs, immune boosters and diagnostics. Green processes, renewable clean energy, super foods and nutraceuticals, biodegradable polymers and materials are but a few of the outcomes of development in this field. In wake of the rapid deterioration of land and water resources and environment catalysed by anthropogenic activities, urgent calls have to be made on addressing the issues of solid, liquid and chemical waste management, atmospheric pollution, and moving towards greener manufacturing processes and deriving energy through renewable routes for a sustainable development and improved quality of life. It may not be incorrect to say that the world needs to move towards a sustainable bio-economy for human race to persist on this planet

Chelerythrine Induces Apoptosis through a Bax/Bak-independent Mitochondrial Mechanism*S⃞

Although murine embryonic fibroblasts (MEFs) with Bax or Bak deleted displayed no defect in apoptosis signaling, MEFs with Bax and Bak double knock-out (DKO) showed dramatic resistance to diverse apoptotic stimuli, suggesting that Bax and Bak are redundant but essential regulators for apoptosis signaling. Chelerythrine has recently been identified as a Bcl-xL inhibitor that is capable of triggering apoptosis via direct action on mitochondria. Here we report that in contrast to classic apoptotic stimuli, chelerythrine is fully competent in inducing apoptosis in the DKO MEFs. Wild-type and DKO MEFs are equally sensitive to chelerythrine-induced morphological and biochemical changes associated with apoptosis phenotype. Interestingly, chelerythrine-mediated release of cytochrome c is rapid and precedes Bax translocation and integration. Although the BH3 peptide of Bim is totally inactive in releasing cytochrome c from isolated mitochondria of DKO MEFs, chelerythrine maintains its potency and efficacy in inducing direct release of cytochrome c from these mitochondria. Furthermore, chelerythrine-mediated mitochondrial swelling and loss in mitochondrial membrane potential (ΔΨm) are inhibited by cyclosporine A, suggesting that mitochondrial permeability transition pore is involved in chelerythrine-induced apoptosis. Although certain apoptotic stimuli have been shown to elicit cytotoxic effect in the DKO MEFs through alternate death mechanisms, chelerythrine does not appear to engage necrotic or autophagic death mechanism to trigger cell death in the DKO MEFs. These results, thus, argue for the existence of an alternative Bax/Bak-independent apoptotic mechanism that involves cyclosporine A-sensitive mitochondrial membrane permeability

Norspermidine and Novel Pd(II) and Pt(II) Polynuclear Complexes of Norspermidine as Potential Antineoplastic Agents Against Breast Cancer

New strategies are needed for breast cancer treatment and one initial step is to test new chemotherapeutic drugs in breast cancer cell lines, to choose candidates for further studies towards clinical use

The Hepatitis C Virus Core Protein Contains a BH3 Domain That Regulates Apoptosis through Specific Interaction with Human Mcl-1 ▿ †

The hepatitis C virus (HCV) core protein is known to modulate apoptosis and contribute to viral replication and pathogenesis. In this study, we have identified a Bcl-2 homology 3 (BH3) domain in the core protein that is essential for its proapoptotic property. Coimmunoprecipitation experiments showed that the core protein interacts specifically with the human myeloid cell factor 1 (Mcl-1), a prosurvival member of the Bcl-2 family, but not with other prosurvival members (Bcl-XL and Bcl-w). Moreover, the overexpression of Mcl-1 protects against core-induced apoptosis. By using peptide mimetics, core was found to release cytochrome c from isolated mitochondria when complemented with Bad. Thus, core is a bona fide BH3-only protein having properties similar to those of Noxa, a BH3-only member of the Bcl-2 family that binds preferentially to Mcl-1. There are three critical hydrophobic residues in the BH3 domain of the core protein, and they are essential for the proapoptotic property of the core protein. Furthermore, the genotype 1b core protein is more effective than the genotype 2a core protein in inducing apoptosis due to a single-amino-acid difference at one of these hydrophobic residues (residue 119). Replacing this residue in the J6/JFH-1 infectious clone (genotype 2a) with the corresponding amino acid in the genotype 1b core protein produced a mutant virus, J6/JFH-1(V119L), which induced significantly higher levels of apoptosis in the infected cells than the parental J6/JFH-1 virus. Furthermore, the core protein of J6/JFH-1(V119L), but not that of J6/JFH-1, interacted with Mcl-1 in virus-infected cells. Taken together, the core protein is a novel BH3-only viral homologue that contributes to the induction of apoptosis during HCV infection

Effects of NSpd, Pd-NSpd or Pt-NSpd on the uptake of ³H-spermidine in JIMT-1, L56Br-C1, MCF-7 and MCF-10A cells.

<p>The cells were seeded in 12 well plates and incubated for 48 h, whereupon the polyamine analogue or its complexes were added to give the final concentrations shown in the figure. The concentration of ³H-spermidine used was 1 µM. The results are presented as mean values (n = 4 samples from two independent experiments) and bars represent ± SD.</p