High Fat and Diet Induced Obesity

i-Fect^TM Delivers Again!

Research shows that rats and humans on a high-fat diet (HFD) are less sensitive to satiety signals known to act via vagal afferent pathways. Impaired vagal afferent responsiveness to both gastric satiety hormones (CCK and leptin) and mechanical stimulation raises the possibility that changes in electrophysiological properties may be the underlying mechanism responsible for impaired vagal responsiveness to a wide variety of satiety signals.

Potassium channels play a central role. To demonstrate this researchers used our i-Fect siRNA Transfection Kit to silence TRESK and TASK1 to understand there impact on HFD on vagal responsiveness. Gintautas Grabauskas, Xiaoyin Wu, ShiYi Zhou, JiYao Li, Jun Gao, and Chung Owyang. (2019). High-fat diet–induced vagal afferent dysfunction via upregulation of 2-pore domain potassium TRESK channel. JCI Insight. https://doi.org/10.1172/jci.insight.130402.

Images: (A) Representative recordings of NG neuron responses to intra–superior pancreaticoduodenal artery infusions of CCK-8 (60 pmol/kg) and leptin (60 pmol/kg) in LFD-fed or HFD-fed rats and transfected with control siRNA or TRESK siRNA. Note that CCK-8 generated significantly fewer action potentials in HFD-fed rats compared with those fed an LFD. (B) Summary histograms showing single-unit discharges in response to CCK-8 in rats given an LFD and transfected with control siRNA (n = 11) or TRESK siRNA (n = 6), HFD + control siRNA (n = 12), and HFD treated with TRESK siRNA (n = 10). Data are represented as mean ± SEM. One-way ANOVA with Bonferroni’s test, *P < 0.05 vs. LFD + control siRNA; #P < 0.05 vs. HFD + control siRNA. (C) Summary histogram showing single-unit discharges in response to leptin in rats given an LFD and transfected with control siRNA (n = 11) and TRESK siRNA (n = 5), HFD (n = 12), and HFD treated with TRESK siRNA (n = 10). Data are represented as mean ± SEM. One-way ANOVA with Bonferroni’s test, *P less than 0.05 vs. LFD + control siRNA; #P less than 0.05 vs. HFD + control siRNA. (D) Summary histogram showing CCK-AR and ObR expression in vagal sensory ganglia from LFD- and HFD-fed rats were not significantly different. HPRT was used as a loading control. Data are represented as mean ± SEM. CCK-8, cholecystokinin-8.

Following 2 weeks of high-fat feeding, there was a significant upregulation of TRESK and a modest increase in TASK1 channels in the NG. Silencing studies indicate that the upregulation by TRESK channels is mainly responsible for a global decrease in excitability of vagal sensory neurons, which in turn dampens the response to satiety signals, such as CCK and leptin. 

This make TRESK a potential therapeutic target for treating Obesity.

i-Fect Delivers circRNA and miRNA

Blocks Bone Cancer Pain
Altered expression of circular RNA (circRNA) is recognized as a contributor to malignant pain where microRNA (miRNA) exerts an essential effect. Researchers used our i-Fect^TMTransfection Kit to knock them down. Zhongqi Zhang, Xiaoxia Zhang, Yanjing Zhang, Jiyuan Li, Zumin Xing. Yiwen Zhang. Spinal circRNA-9119 Suppresses Nociception by Mediating the miR-26a-TLR3 Axis in a Bone Cancer Pain Mouse Model. Spinal circRNA-9119 Suppresses Nociception by Mediating the miR-26a-TLR3 Axis in a Bone Cancer Pain Mouse Model. Journal of Molecular Neuroscience. pp 1–10

Intrathecal Administration of miRNA and circRNA Pre-miRNA sequence of miR-26a and circ9119 were cloned into a plasmid. The i-Fect transfection reagent (10 μL; Neuromics, Edina, USA) was used to resuspend plasmids for injection.

Sample Data

Image: siRNA-mediated suppression of target gene expression in Schwann cells.

i-Fect Kits sell for 399 USD. They are widely used and frequently published. Check the out today.

i-Fect Delivers Plasmids!

Important for Gene Expression Studies.
I have posted many examples of how our customers use i-Fect^TM  and other Transfection Solutions for Gene Manipulation Studies. There are also many publications.

Here we feature how i-Fect was used to delivery plasmids to the CNS: Sara Elramah, María José López-González, Matthieu Bastide, Florence Dixmérias, Olivier Roca-Lapirot, Anne-Cécile Wielanek-Bachelet, Anne Vital, Thierry Leste-Lasserre, Alexandre Brochard, Marc Landry & Alexandre Favereaux. Spinal miRNA-124 regulates synaptopodin and nociception in an animal model of bone cancer pain. Scientific Reports 7, Article number: 10949 (2017) doi:10.1038/s41598-017-10224-1...Intrathecal administration of miRNAs and ShRNA To over-express miR-124, we cloned the pre-miRNA sequence of miR-124 into a plasmid. To determine cells expressing this miR-124 encoding plasmid, we added a GFP-coding sequence to the construct under the control of an IRES. Thus, miR-124 over-expressing cells also express GFP. To inhibit synaptopodin expression, we cloned a ShRNA sequence directed against synaptopodin into a plasmid. To determine cells expressing this ShRNA, we added a GFP-coding sequence to the construct under the control of an IRES. Thus, ShRNA expressing cells also expressed GFP. Two micrograms of these plasmids or the corresponding controls, were solubilized in 10 µl of i-Fect reagent (Neuromics, Edina, USA), and injected intrathecally between the L5 and L6 lumbar vertebrae every two days for a total of 3 injections, according to the manufacturer’s instructions and previously published experiments...

Figures: (C and D) Immunostaining of synpo in spinal cord after miR-124 intrathecal injections: only the dorsal horn which receive nociceptive information was quantified (white dash area). Measurement of synaptopodin stained area reveals ability of miR-124 to inhibit endogenous Synpo expression (20/3 and 17/3 denotes number of sections/animals for control and miR-124-injected mice, respectively.

I am confident there will be many more positive reports regarding our Transfection Reagents.

More on Epigenetics and Pain

Great Research Tools!

Our Markers and i-Fect^TM  Transection Kit are  used to study Epigenetics and pain. Here's yet another example: M. Leinders, b, N. Üçeyler, R.A. Pritchard, C. Sommer, L.S. Sorkin. Increased miR-132-3p expression is associated with chronic neuropathic pain. Experimental Neurology. Volume 283, Part A, September 2016, Pages 276–286...The inhibitor and mimetic were administered to awake rats via the it catheters. Prior to injection, active or mismatch inhibitors were mixed with (1:5 w/v) i-Fect™ in vivo transfection reagent(Neuromics, Edina, USA) to final doses of 5, 2 and 1 μg in 10 μl...

Figure: Inhibition of MOR. Stained with Dorsal Horn Neuromics' Rabbit Polyclonal Antbody.

Spinal administration of miR-132-3p antagonists via intrathecal (i.t.) catheters dose dependently reversed mechanical allodyina  and eliminated pain behavior in the place escape avoidance paradigm (p < 0.001). Intrathecal administration of miR-132-3p mimetic dose-dependently induced pain behavior in naïve rats (p < 0.001). Taken together these results indicate a pro-nociceptive effect of miR-132-3p in chronic neuropathic pain.

Finding like these could pave the way for an miRNA like therapy for pain.