ANKRD26 and Its Interacting Partners TRIO, GPS2, HMMR and DIPA Regulate Adipogenesis in 3T3-L1 Cells

Partial inactivation of the Ankyrin repeat domain 26 (Ankrd26) gene causes obesity and diabetes in mice and increases spontaneous and induced adipogenesis in mouse embryonic fibroblasts. However, it is not yet known how the Ankrd26 protein carries out its biological functions. We identified by yeast two-hybrid and immunoprecipitation assays the triple functional domain protein (TRIO), the G protein pathway suppressor 2 (GPS2), the delta-interacting protein A (DIPA) and the hyaluronan-mediated motility receptor (HMMR) as ANKRD26 interacting partners. Adipogenesis of 3T3-L1 cells was increased by selective down-regulation of Ankrd26, Trio, Gps2, Hmmr and Dipa. Furthermore, GPS2 and DIPA, which are normally located in the nucleus, were translocated to the cytoplasm, when the C-terminus of ANKRD26 was introduced into these cells. These findings provide biochemical evidence that ANKRD26, TRIO, GPS2 and HMMR are novel and important regulators of adipogenisis and identify new targets for the modulation of adipogenesis

Expanding the Capacity of Otolaryngologists in Kenya through Mobile Technology

Objective To determine if reliable, objective audiologic data can be obtained by nonotolaryngology and nonaudiology practitioners using novel mobile technology in an effort to expand the capacity for early identification and treatment of disabling hearing loss in the developing world. Study Design Cross-sectional, proof-of-concept pilot study. Setting Screenings took place during an annual 2-week otolaryngology surgical mission in October 2016 in semirural Malindi, Kenya. Subject and Methods Eighty-seven patients (174 total ears) were included from 2 deaf schools (n = 12 and 9), a nondeaf school (n = 9), a tuberculosis ward (n = 8), and a walk-in otology clinic at a local hospital (n = 49). An automated, tablet-based, language-independent, clinically validated, play audiometry system and wireless otoscopic endoscopy via an iPhone or laptop platform was administered by Kenyan community health workers (CHWs) and nursing staff. Results Various degrees of hearing loss and otologic pathology were identified, including 1 child presumed to be deaf who was found to have unilaterally normal hearing. Other pathology included 2 active perforations, 2 healed perforations, 2 middle ear effusions, and 1 cholesteatoma. CHWs and nursing staff demonstrated proficiency performing audiograms and endoscopy. Patients screened in a deaf school were more likely to complete an unreliable audiogram than patients screened in other settings ( P < .01). Conclusion This study demonstrates the feasibility of a non–otolaryngology-based hearing screening program. This may become an important tool in reducing the impact of hearing loss and otologic pathology in areas bereft of otolaryngologists and audiologists by allowing CHWs to gather important patient data prior to otolaryngologic evaluation

Head and neck surgery global outreach: Ethics, planning, and impact

Head and neck surgical oncology and reconstruction are uniquely suited to address burdens of disease in underserved areas. Since these efforts are not well known in our specialty, we sought to understand global outreach throughout our society of surgeons. Survey distributed to members of the American Head and Neck Surgery involved in international humanitarian head and neck surgical outreach trips. Thirty surgeons reported an average of seven trips to over 70 destinations. Identification of candidates, finances, on-site patient care, complications, long-term post-surgical care, ethics, and educational goals are reported. We report a success rate of 90% on 125 free flaps performed in these settings. The effort to answer the call for alleviating the global burden of surgical disease is strong within our specialty. There is a shared focus on humanitarian effort and teaching. Ethics of high resource surgeries such as free flap reconstruction remains controversial

Real Time Quantitative PCR primer sequences and primer efficiencies.

<p>Real Time Quantitative PCR primer sequences and primer efficiencies.</p

shRNA knockdown of <i>Ankrd26</i>, <i>Trio</i>, <i>Gps2, HMMR</i> and <i>Dipa</i> induces adipocyte differentiation in 3T3-L1 cells.

<p>To down-regulate <i>Ankrd26</i>, <i>Trio</i>, <i>Gps2, HMMR</i> and <i>Dipa</i> expression cells were transfected with specific shRNA for <i>Ankrd26</i> (Ankrd-sh), <i>Trio</i> (Trio-sh), <i>Gps2</i> (Gps2-sh), <i>HMMR</i> (HMMR-sh), <i>Dipa</i> (Dipa-sh) or with non-targeting control shRNA (Co-sh). <b>A</b>) <i>Ankrd26</i> mRNA expression determined by Real-Time Quantitative PCR analysis of total RNA isolated from 3T3-L1 cells transfected with Ankrd-sh or Co-sh. mRNA levels in Ankrd-sh treated cells are relative expression units to those in control (Co-sh; mean ± SD; n = 3). **<i>p</i><0.01. Macroscopic images (<b>B</b>) and lipid quantification (<b>C</b>) in Ankrd-sh and Co-sh transfected cells stained with oil-red-O upon differentiation. Bars are expressed as means ± SEM of oil-red-O values measured at 490 nm. Ankrd-sh vs. Co-sh, **<i>p</i><0.01. mRNA expression (<b>D, G, J</b> and <b>M</b>), macroscopic images (<b>E, H, K</b> and <b>N</b>) and lipid quantification (<b>F, I, L</b> and <b>O</b>) of 3T3-L1 cells transduced with Trio-sh (<b>D–F</b>), Gps2-sh (<b>G–I</b>), HMMR-sh (<b>J–L</b>) and Dipa-sh (<b>M–O</b>) were evaluated as <b>A</b>, <b>B</b> and <b>C</b>, respectively.</p

ANKRD26 yeast two-hybrid strategy.

<p>The position of ANKRD26's ankyrin and spectrin coiled-coil repeats are depicted on a schematic diagram of the full-length, 1710 amino acid, protein. The line below indicates the relative locations of ANKRD26 bait clones used in the <i>GAL4-</i>based yeast two-hybrid cDNA library screens. DIPA interacts with baits 7, 8, 9 and 17; GPS2 interacts with bait 17; HMMR interacts with baits 9 and 17; and TRIO interacts with bait 10.</p

Analysis of <i>Ankrd26</i>, <i>Trio</i>, <i>Gps2, HMMR</i> and <i>Dipa</i> RNA by rq-pcr during adipogenesis induction.

<p>3T3-L1 cells plated 6-well plates (1.8×10⁵/well) and harvested at various time points as: day 1 (pre-confluence), day 2 (confluence), day 4 (2 days after confluence), day 5 (IDM induction day 1), day 6 (IDM induction day 2), day 8 (IDM induction day 4), day12 (IDM induction day 8). RNA made by Trizol regent and subjected to Real Time Quantitative PCR using SYBR method. Bar represents triple for each sample.</p