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I acknowledge that I can unsubscribe any time. Protocols and Documentation. Resources and Publications. Related Products. cAMP is an important second messenger involved in many signal transduction pathways, including activation of protein kinase A PKA; Awad et al.

Cell Type. View More View Less. Protocols and Documentation Find supporting information and directions for use in the Product Information Sheet or explore additional protocols below.

Document Type. Applications This product is designed for use in the following research area s as part of the highlighted workflow stage s. Research Area. Workflow Stages for Human Pluripotent Stem Cell Research.

Workflow Stages for Mouse Pluripotent Stem Cell Research. Workflow Stages for Neural Stem and Progenitor Cell Research. Tissue Dissociation. Resources and Publications Educational Materials 4. Small Molecules, Big Impact in Pluripotent Stem Cell Research. Small Molecules, Big Impact in Cancer Research.

ISSCR Innovation Showcase - Induction of a Native State in Human Pluripotent Stem Cells. Small Molecules. Publications 9 Small-Molecule-Driven Direct Reprogramming of Mouse Fibroblasts into Functional Neurons. Li X et al. Cell stem cell AUG. Abstract Recently, direct reprogramming between divergent lineages has been achieved by the introduction of regulatory transcription factors.

This approach may provide alternative cell resources for drug discovery and regenerative medicine, but applications could be limited by the genetic manipulation involved.

After a further maturation stage, these chemically induced neurons CiNs possessed neuron-specific expression patterns, generated action potentials, and formed functional synapses. Mechanistically, we found that a BET family bromodomain inhibitor, I-BET, disrupted the fibroblast-specific program, while the neurogenesis inducer ISX9 was necessary to activate neuron-specific genes.

Overall, our findings provide a proof of principle" for chemically induced direct reprogramming of somatic cell fates across germ layers without genetic manipulation� View publication. Direct Conversion of Normal and Alzheimer's Disease Human Fibroblasts into Neuronal Cells by Small Molecules.

Hu W et al. Abstract Neuronal conversion from human fibroblasts can be induced by lineage-specific transcription factors; however, the introduction of ectopic genes limits the therapeutic applications of such induced neurons iNs.

Here, we report that human fibroblasts can be directly converted into neuronal cells by a chemical cocktail of seven small molecules, bypassing a neural progenitor stage.

These human chemical-induced neuronal cells hciNs resembled hiPSC-derived neurons and human iNs hiNs with respect to morphology, gene expression profiles, and electrophysiological properties.

This approach was further applied to generate hciNs from familial Alzheimer's disease patients. Taken together, our transgene-free and chemical-only approach for direct reprogramming of human fibroblasts into neurons provides an alternative strategy for modeling neurological diseases and for regenerative medicine.

HDPs, also known as antimicrobial peptides, are ubiquitously expressed in phagocytes and epithelial cells lining the digestive, respiratory, urinary, and reproductive tracts, and constitute an important component of host immune defense [ 4 , 5 , 6 ]. Defensins and cathelicidins are 2 major families of HDPs in vertebrates, and 14 β-defensins AvBD1—14 and 4 cathelicidins CATH1—3 and CATHB1 have been reported in chickens [ 5 ].

Besides the ability to directly kill a broad spectrum of microbes through membrane disruption, HDPs possess a variety of immunomodulatory activities such as chemotaxis, endotoxin neutralization, suppression of inflammation, and activation of adaptive immunity [ 6 ]. Because of membrane-lytic antimicrobial and immune regulatory activities, HDPs are less likely to trigger microbial resistance than conventional antibiotics [ 7 ].

Insufficient HDP production has been implicated in increased susceptibility to infectious diseases, while enhancing endogenous HDP synthesis is capable of strengthening host immunity and conferring protection [ 6 , 8 ]. Besides infection and inflammation, HDP synthesis can be modulated by a variety of small-molecule compounds such as histone deacetylase inhibitors, fatty acids, and phytochemicals [ 3 , 9 , 10 ].

For example, butyrate, a major short-chain fatty acid SCFA produced by intestinal bacterial fermentation of undigested dietary carbohydrates, is essential to intestinal health and homeostasis by regulating energy metabolism, inflammation, immunity, and barrier integrity of the intestinal epithelial cells [ 11 , 12 ].

Recently, butyrate was found to be a potent HDP inducer in humans and several other animal species [ 3 ]. Most of the butyrate functions including its HDP-inducing activity are mediated through inhibiting histone deacetylases or interacting with G-protein coupled receptors such as GPR41, GPR43, and GPRA [ 11 ].

Forskolin FSK , a labdane diterpene isolated from the roots of an Indian plant Coleus forskohlii CF , is also capable of stimulating HDP synthesis in humans [ 13 ] and chickens [ 14 ]. FSK modulates a variety of physiological functions such as promoting lipolysis and thermogenesis, appetite regulation, and anti-inflammation by acting as a direct agonist of adenylyl cyclase, which in turn activates cyclic adenosine monophosphate cAMP signaling to influence gene transcription [ 15 , 16 ].

We previously showed a strong synergy between butyrate and FSK in inducing AvBD9 gene expression in chickens [ 14 ]. However, the synergy between butyrate and FSK in barrier integrity and disease resistance has remained unknown.

Therefore, this study was aimed at investigating the role of butyrate and FSK in modulating innate immunity, inflammation, and barrier function. We also studied the efficacy of the two natural compounds in the resistance of necrotic enteritis NE in broiler chickens, which is among the most economically significant diseases in chickens caused by a Gram-positive bacterium, Clostridium perfringens [ 17 ].

The impact of the two compounds on growth performance, carcass traits, and meat quality was also evaluated to further explore their potential as novel alternatives to antibiotics for use in broilers. Three independent experiments were conducted to ensure the reproducibility of the results.

Relative fold changes of gene expression levels were calculated using the 2 -ΔΔCt method normalized against GAPDH. All animal procedures were approved by the Institutional Animal Care and Use Committee of Oklahoma State University under protocol number AG A total of newly-hatched male Cobb broilers were obtained from a commercial hatchery Cobb-Vantress Hatchery, Siloam Springs, AR, USA and reared in an environmentally controlled room under standard management as recommended by Cobb-Vantress.

Chickens in 5 treatments were subjected to daily challenge with an overnight culture of C. A netB- and tpeL- positive C. perfringens strain Brenda B kindly provided by Dr. Lisa Bielke at the Ohio State University, Columbus, OH [ 20 ] was sequentially passaged in chopped cooked meat medium and fluid thioglycollate medium prior to inoculation of chickens.

On d 18, chickens were weighed individually and euthanized by CO 2 asphyxiation prior to sample collection. Gross lesions of NE in the duodenum and jejunum of each chicken were graded separately in a blind manner using a 0—6 scoring system as described [ 17 ]. The jejunal and cecal C.

perfringens were quantified using a standard curve-based qPCR method as described [ 21 ]. Briefly, bacterial genomic DNA from pure C.

perfringens culture or intestinal digesta samples was extracted using the ZR Fecal DNA MicroPrep Kit Zymo Research, Irvine, CA and quantified using Nanodrop. The C. perfringens genomic DNA.

Two separate feeding trials were conducted to evaluate the effect of butyrate and FSK on the growth performance of healthy broilers reared under standard management. Average daily gain ADG , average daily feed intake ADFI , and feed conversion ratio FCR were calculated and compared among all treatments.

The starter diet Broilers were euthanized by CO 2 asphyxiation after h feed withdrawal, followed by ventral neck cutting, bleeding, de-feathering, and evisceration. The carcass yield was calculated as the percentage of eviscerated carcass weight, relative to live weight.

Moreover, both left and right sides of the breast muscle pectoral major and minor were removed and weighed. The abdominal fat pad was collected and weighed as described [ 15 ]. The yields of the breast meat and abdominal fat were calculated as percentages of eviscerated carcass weight.

The color of the right pectoral major muscle was determined on each sample in duplicate with 1 reading in the anterior and the other in the posterior portion of the muscle using MiniScan XE Plus Spectrophotometer 2.

All readings were taken on the skin side surface in an area free of obvious color defects over scald, bruises, and blood accumulation [ 23 ]. Data analysis and visualization were performed using Prism GraphPad Software Inc. The mRNA expression levels of representative HDP, barrier function, and inflammatory cytokine genes were evaluated using RT-qPCR.

Moreover, AvBD9 induction was maintained in the context of LPS stimulation. Similarly, butyrate synergized with FSK in enhancing the expressions of AvBD10 Fig. Additionally, butyrate augmented the expressions of CLDN1 and TJP1 in HD11 cells, while FSK was largely ineffective Fig.

Furthermore, butyrate, FSK, or the combination had little effect on the expression of IL-1β in HD11 cells, indicating that the 2 compounds are not proinflammatory Fig. Taken together, butyrate synergized with FSK to suppress inflammation, while enhancing the expressions of HDP and MUC2 genes, with no obvious synergy in regulating tight junction genes.

Regulation of host defense peptide, barrier function, and inflammatory cytokine gene expressions in macrophages by butyrate and forskolin FSK.

Quantitative reverse transcription PCR RT-qPCR was performed to measure the expressions of avian β-defensin 9 AvBD9 A , AvBD10 B , mucin 2 MUC2 C , claudin 1 CLDN1 D , tight junction protein 1 TJP1 E , and interleukin 1β IL-1β F.

Results were presented as means ± SEM of 3 independent experiments. perfringens to induce NE as described [ 19 ]. Among C. While there was no lethality, C. Alleviation of necrotic enteritis by butyrate and forskolin FSK.

Chickens were individually weighted on d 13 A and d 18 B , and gross lesions of necrotic enteritis in the duodenum C and jejunum D were scored on d The CP titers in the digesta of the jejunum E and cecum F were quantified using qPCR.

perfringens colonization in the jejunum and cecum was also quantified among different groups using qPCR. perfringens colonization by approximately fold in the jejunum relative to the non-medication group Fig. In the cecum, butyrate or FSK alone had a negligible effect on C.

perfringens numerically by approximately 3-fold Fig. perfringens colonization in the intestinal tract of C. perfringens -challenged chickens.

To understand the synergistic effect of butyrate and FSK on NE alleviation, the mRNA expressions of representative chicken HDP AvBD9 and AvBD10 , barrier function MUC2 , CLDN1 , and TJP1 , and inflammatory cytokine IL-1β genes were measured in the jejunum of C.

perfringens- challenged chickens on d S1 A and AvBD10 Supplementary Fig. perfringens infection reduced the expressions of CLDN1 and TJP1 , whereas the CF extract with or without butyrate tended to reverse the trend and enhanced both gene expressions Supplementary Fig.

To examine the effect of butyrate and FSK on the growth performance of healthy broilers, 2 feeding trials were conducted with Cobb chicks. A second feeding trial was further conducted for an entire growth cycle of 42 d to examine the influence of butyrate and FSK on growth performance of broilers.

Both feeding trials collectively suggested that a combination of butyrate and CF extract had no negative influence on the growth of broilers, but with a strong tendency to reduce feed intake and thus improve feed efficiency.

Influence of butyrate and forskolin FSK on growth performance of broilers. Average daily gain ADG , average daily feed intake ADFI , and feed conversion ratio FCR were calculated for the entire period.

To ensure butyrate and FSK have no negative impact on carcass traits or meat quality, broilers were randomly selected from the second trial on d 28 and 48 and processed for the measurement of a series of carcass and meat quality traits. Such a reduction failed to be observed with butyrate or FSK alone.

Overall, butyrate and FSK had little impact on either carcass traits or meat quality, except for a significant influence on reducing abdominal fat. Antibiotic alternatives are needed in animal agriculture, given the restricted use of in-feed antibiotics in a growing number of countries [ 2 , 3 ].

Host-directed strategies such as the induction of endogenous HDP synthesis are being actively explored against infectious diseases [ 3 , 9 , 10 ].

We previously showed that butyrate stimulates HDP synthesis and enhances the clearance of Salmonella Enteritidis in chickens [ 18 ] and that butyrate and FSK synergize with each other in inducing AvBD9 gene expression both in vitro and in vivo [ 14 ]. In this study, we further demonstrated that dietary supplementation of butyrate and FSK synergizes to improve the expressions of AvBD9 and AvBD10 as well as barrier function genes such as MUC2 , while suppressing inflammation in cell culture.

Importantly, butyrate and FSK alleviates experimentally-induced NE in broilers in a synergistic manner. Furthermore, feeding butyrate and FSK has a strong tendency to improve feed efficiency and reduce abdominal fat with no negative impact on growth performance, carcass yield, or meat quality of broilers.

Larger-scale animal trials are needed to confirm these promising results and provide a stronger statistical conclusion on some of the parameters measured in this study.

HDPs are critically important in animal immunity and disease resistance [ 4 , 6 ]. Aberrant expression or deletion of HDP genes is often associated with increased susceptibility to infectious diseases [ 6 , 8 ]. Downregulating HDP expression is specifically employed by certain bacteria to evade host innate immunity and establish infection [ 24 ].

In this study, C. perfringens infection also suppresses the expressions of AvBD9 and AvBD10 in the chicken jejunum; however, supplementation of butyrate and FSK reverses C. perfringens -mediated HDP suppression and, unsurprisingly, alleviates NE.

Similarly, C. perfringens downregulates the expression of an antimicrobial peptide, LEAP-2 , in the jejunum of chickens, and dietary supplementation of butyrate enhances LEAP-2 expression and ameliorated NE [ 25 ].

These results collectively demonstrate that induction of HDPs represents a feasible approach to mitigate infectious diseases. However, the mechanisms underlying butyrate- and FSK-mediated mitigation of NE appear to go beyond HDP induction. Mucins secreted by goblet cells and tight junctions connecting adjacent epithelial cells form important physiological and biochemical barriers between hosts and the external environment to maintain intestinal homeostasis [ 26 , 27 ].

Butyrate is capable of maintaining intestinal barrier integrity by upregulating mucins and tight junction proteins [ 11 ], while we showed in this study that FSK enhances MUC2 gene expression both in vitro and in vivo.

Importantly, butyrate synergizes with FSK in promoting the transcription of MUC2 , CLDN1 , and TJP1 in the intestinal tract. perfringens -infected chicken jejunum. Consistent with well-known anti-inflammatory properties of butyrate [ 11 , 28 ] and FSK [ 29 , 30 ], we have shown that butyrate and FSK suppress IL-1β induction in LPS-stimulated HD11 cells and C.

perfringens -infected jejunum individually and also in combination. Although the mechanism of action is not directly addressed in this study, the synergy in suppressing inflammatory response between butyrate and FSK is likely to act by inhibiting the activation of NF-κB and the NLRP3 inflammasome, 2 major targets for both butyrate and FSK [ 11 , 28 , 29 , 30 ].

However, FSK alone and particularly in combination with butyrate has a strong tendency to reduce feed intake and improve feed efficiency without affecting the growth rate of broilers in 2 feeding trials.

Consistently, supplementation of FSK-containing CF extract has been shown to significantly reduce food intake and appetite in both rats [ 35 ] and humans [ 36 ]. This is perhaps unsurprising, given that FSK is a natural agonist of adenylyl cyclase and activates cAMP signaling [ 37 , 38 ], which subsequently promotes lipolysis, thermogenesis, and loss of body fat without muscle mass loss [ 39 , 40 , 41 ], although the evidence on fat mass reduction is not entirely consistent [ 36 ].

Nevertheless, FSK is currently being explored to manage overweight and obesity in humans [ 42 , 43 , 44 ]. We observed that broilers fed a combination of butyrate and FSK show reduced abdominal fat deposition without affecting the carcass or breast muscle mass.

However, FSK alone fails to reduce abdominal fat of broilers, perhaps due to the level of supplementation or a species difference. Apparently, optimal levels of dietary inclusion of FSK or FSK-containing CF extract remain to be investigated.

It is noted that, similar to FSK, butyrate is also known to reduce appetite and lipogenesis [ 12 , 45 ], which perhaps helps explain a synergistic effect of butyrate and FSK in reducing feed intake and abdominal fat deposition, although butyrate or FSK alone at the levels used in this study fails to achieve such an effect.

Sodium butyrate or butyric acid supplemented between 0. Optimal concentrations of butyrate and its derivatives warrant further investigation for their effectiveness in regulating fat deposition. Taken together, these results highlight a need to continue to explore the potential of combining butyrate with FSK as a promising antibiotic alternative to improve disease prevention, feed efficiency, and carcass composition in poultry and possibly other livestock species.

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