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Abstract
Acute high fat diet (HFD) exposure induces a brief period of hyperphagia before caloric balance is restored. Previous studies have demonstrated this period of regulation is associated with activation of synaptic NMDA receptors (NMDARs) on dorsal motor nucleus of the vagus (DMV) neurons, which increases vagal control of gastric functions. Our aim was to test the hypothesis that activation of DMV NMDARs occurs subsequent to activation of extrasynaptic NMDA receptors (NMDARex). Sprague-Dawley rats were fed control or HFD for 3-5 days prior to experimentation. Whole cell patch clamp recordings from gastric-projecting DMV neurons, in vivo recordings of gastric motility, tone, compliance, and emptying, as well as food intake studies were used to assess the effects of NMDAR antagonism on caloric regulation. Following acute HFD exposure, inhibition of NMDARex prevented the NMDARs-mediated increase in glutamatergic transmission to DMV neurons, as well as the increase in gastric tone and motility, while chronic NMDARex inhibition attenuated the regulation of caloric intake. Following acute HFD exposure, the regulation of food intake involves NMDARs-mediated currents, which occur in response to NMDARex activation. Understanding these events may provide a mechanistic basis for hyperphagia and identify potential novel therapeutic targets for the treatment of obesity.
Authors
Courtney Clyburn, R. Alberto Travagli, Amy C. Arnold, Kirsteen N. Browning
Abstract
Sphingosine-1-phosphate lyase insufficiency syndrome (SPLIS) is a rare metabolic disorder caused by inactivating mutations in SGPL1, which is required for the final step of sphingolipid metabolism. SPLIS features include steroid-resistant nephrotic syndrome (SRNS) and impairment of neurological, endocrine, and hematopoietic systems. Many affected individuals die within the first two years. No targeted therapy for SPLIS is available. We hypothesized that SGPL1 gene replacement would address the root cause of SPLIS, thereby serving as a universal treatment for the condition. As proof of concept, we evaluated the efficacy of adeno-associated virus 9-mediated transfer of human SGPL1 (AAV-SPL) given to newborn Sgpl1 KO mice that model SPLIS and die in the first weeks of life. Treatment dramatically prolonged survival and prevented nephrosis, neurodevelopmental delay, anemia, and hypercholesterolemia. STAT3 pathway activation and elevated pro-inflammatory and fibrogenic cytokines observed in KO kidneys were attenuated by treatment. Plasma and tissue sphingolipids were reduced in treated compared to untreated KO pups. SGPL1 expression and activity were measurable for at least 40 weeks. In summary, early AAV-SPL treatment prevents nephrosis, lipidosis and neurological impairment in a mouse model of SPLIS. Our results suggest that SGPL1 gene replacement holds promise as a durable and universal targeted treatment for SPLIS.
Authors
Piming Zhao, Gizachew B. Tassew, Joanna Y. Lee, Babak Oskouian, Denise P. Muñoz, Jeffrey B. Hodgin, Gordon L. Watson, Felicia Tang, Jen-Yeu Wang, Jinghui Luo, Yingbao Yang, Sarah M. King, Ronald M. Krauss, Nancy Keller, Julie D. Saba
Abstract
Patients with colorectal cancers (CRCs) generally exhibit improved survival through intensive lymph node (LN) dissection. However, recent progress in cancer immunotherapy revisits the potential importance of regional LNs, where T cells are primed to attack tumor cells. To elucidate the role of regional LN, we investigated the immunological status of non-metastatic regional LN lymphocytes (LNLs) in comparison with those in the tumor microenvironment (tumor-infiltrating lymphocytes; TILs) using flow cytometry and next-generation sequencing. LNLs comprised an intermediate level of the effector T cell population between peripheral blood lymphocytes (PBLs) and TILs. Significant overlap of the T-cell receptor (TCR) repertoire was observed in microsatellite instability-high (MSI-H)/mismatch repair deficient (dMMR) CRCs with high tumor mutation burden (TMB), although limited TCRs were shared between non-metastatic LNs and primary tumors in microsatellite stable (MSS)/MMR proficient (pMMR) CRC patients with low TMB. In line with the overlap of the TCR repertoire, an excessive LN dissection did not provide a positive impact on long-term prognosis in our MSI-H/dMMR CRC cohort (n =130). We propose that regional LNs play an important role in antitumor immunity, particularly in MSI-H/dMMR CRCs with high TMB, requiring to be careful of excessive non-metastatic LN dissection in MSI-H/dMMR CRC patients.
Authors
Koji Inamori, Yosuke Togashi, Shota Fukuoka, Kiwamu Akagi, Kouetsu Ogasawara, Takuma Irie, Daisuke Motooka, Yoichi Kobayashi, Daisuke Sugiyama, Motohiro Kojima, Norihiko Shiiya, Shota Nakamura, Shoichi Maruyama, Yutaka Suzuki, Masaaki Ito, Hiroyoshi Nishikawa
Abstract
Most colorectal cancers (CRCs) are moderately-differentiated or well-differentiated, a status that is preserved even in metastatic tumors. However, the molecular mechanisms underlying CRC differentiation remain to be elucidated. Herein, we unravel a novel post-transcriptional regulatory mechanism via a previously unappreciated LIN28B-CDX2 signaling axis that plays a critical role in mediating CRC differentiation. Owing to a large number of mRNA targets, the mRNA-binding protein LIN28B has diverse functions in development, metabolism, tissue regeneration and tumorigenesis. Our RNA-binding protein immunoprecipitation (RIP) assay revealed LIN28B directly binds CDX2 mRNA, which is a pivotal homeobox transcription factor in normal intestinal epithelial cell identity and differentiation. Furthermore, LIN28B overexpression results in enhanced CDX2 expression to promote both differentiation in subcutaneous xenograft tumors generated from CRC cells and metastatic tumor colonization through mesenchymal-epithelial transition in CRC liver metastasis mouse models. Chromatin immunoprecipitation (ChIP) sequence for CDX2 identified Alpha-Methylacyl-CoA racemase (AMACR) as a novel transcriptional target of CDX2 in the context of LIN28B overexpression. We also found AMACR enhances intestinal alkaline phosphatase (ALPi) activity, which is known as a key component of intestinal differentiation, through the upregulation of butyric acid. Overall, we demonstrate that LIN28B promotes CRC differentiation through CDX2-AMACR axis.
Authors
Kensuke Suzuki, Yasunori Masuike, Rei Mizuno, Uma M Sachdeva, Priya Chatterji, Sarah F. Andres, Wenping Sun, Andres J Klein-Szanto, Sepideh Besharati, Helen E Remotti, Michael P Verzi, Anil K. Rustgi
Abstract
Choroideremia (CHM) is a X-linked recessive chorioretinal dystrophy caused by mutations in CHM, encoding for Rab escort protein 1 (REP1). Loss of functional REP1 leads to the accumulation of unprenylated Rab proteins and defective intracellular protein trafficking, the putative cause for photoreceptor, retinal pigment epithelium (RPE) and choroidal degeneration. CHM is ubiquitously expressed, but adequate prenylation is considered to be achieved, outside the retina, through the isoform REP2. Recently, the possibility of systemic features in CHM has been debated, hence, in this study whole metabolomic analysis of plasma samples from 25 CHM patients versus age and gender matched controls was performed. Results showed plasma alterations in oxidative stress-related metabolites, coupled with alterations in tryptophan metabolism leading to significantly raised serotonin levels. Lipid metabolism was disrupted with decreased branched fatty acids and acylcarnitines, suggestive of dysfunctional lipid oxidation, and imbalances of several sphingolipids and glycerophospholipids. Targeted lipidomics of the chmru848 zebrafish provided further evidence for dysfunction, with the use of Fenofibrates over Simvastatin circumventing the prenylation pathway to improve the lipid profile and increase survival. This study provides strong evidence for systemic manifestations of CHM and proposes novel pathomechanisms and targets for therapeutic consideration.
Authors
Dulce Lima Cunha, Rose Richardson, Dhani Tracey-White, Alessandro Abbouda, Andreas Mitsios, Verena Horneffer-van der Sluis, Panteleimon Takis, Nicholas Owen, Jane Skinner, Ailsa A. Welch, Mariya Moosajee
Abstract
Background: Estimates of seroprevalence to SARS-CoV-2 vary widely and may influence vaccination response. We ascertained IgG levels across a single US metropolitan site, Chicago, from June 2020 through December 2020. Methods: Participants (n=7935) were recruited through electronic advertising and received materials for a self-sampled dried blood spot assay through the mail or a minimal contact in person method. IgG to the receptor binding domain of SARS-CoV-2 was measured using an established highly sensitive and highly specific assay. Results: Overall seroprevalence was 17.9%, with no significant difference between method of contact. Only 2.5% of participants reported having had a diagnosis of COVID-19 based on virus detection, consistent with a 7-fold greater exposure to SARS-CoV-2 measured by serology than detected by viral testing. The range of IgG level observed in seropositive participants from this community survey overlapped with the range of IgG levels associated with COVID-19 cases having a documented positive PCR positive test. From a subset of those who participated in repeat testing, half of seropositive individuals retained detectable antibodies for 3-4 months. Conclusions: Quantitative IgG measurements with a highly specific and sensitive assay indicate more widespread exposure to SARS-CoV-2 than observed by viral testing. The range of IgG concentration produced from these asymptomatic exposures is similar to IgG levels occurring after documented non-hospitalized COVID-19, which is considerably lower than that produced from hospitalized COVID-19 cases. The differing ranges of IgG response, coupled with the rate of decay of antibodies, may influence response to subsequent viral exposure and vaccine.
Authors
Alexis R. Demonbreun, Thomas W. McDade, Lorenzo L. Pesce, Lauren A. Vaught, Nina L. Reiser, Elena Bogdanovic, Matthew P. Velez, Ryan R. Hsieh, Lacy M. Simons, Rana Saber, Daniel T. Ryan, Michael G. Ison, Judd F. Hultquist, John T. Wilkins, Richard T. D'Aquila, Brian Mustanski, Elizabeth M. McNally
Abstract
To identify small molecules that shield mammalian sensory hair cells from the ototoxic side effects of aminoglycoside antibiotics we screened 10,240 compounds, selecting those that protected against neomycin and gentamicin in zebrafish lateral-line hair cells and, when retested in mouse cochlear cultures, prevented gentamicin-induced death of outer hair cells (OHCs). Of 64 compounds that protected zebrafish hair cells, 8 protect OHCs from gentamicin in vitro. These hits share structural features and all block, to varying degrees, the OHC’s mechano-electrical transducer (MET) channel, a known route of aminoglycoside entry into hair cells. Further characterisation of one of the strongest MET-channel blockers, UoS-7692, revealed it additionally protects against kanamycin and tobramycin, and does not abrogate the bactericidal activity of gentamicin. UoS-7692 behaves, like the aminoglycosides, as a permeant blocker of the MET channel, significantly reduces gentamicin-Texas Red loading into OHCs, and preserves lateral-line function in neomycin-treated zebrafish. Trans-tympanic injection of UoS-7692 protects mouse OHCs from furosemide-kanamycin exposure in vivo and partially preserves hearing. The results confirm the hair-cell MET channel as a viable target for the identification of compounds that protect the cochlea from aminoglycosides, and provide a series of hit compounds that will inform the design of future otoprotectants.
Authors
Emma J. Kenyon, Nerissa K. Kirkwood, Siân R. Kitcher, Richard J. Goodyear, Marco Derudas, Daire M. Cantillon, Sarah Baxendale, Antonio de la Vega de León, Virginia N. Mahieu, Richard T. Osgood, Charlotte Donald Wilson, James C. Bull, Simon J. Waddell, Tanya T. Whitfield, Simon E. Ward, Corné J. Kros, Guy P. Richardson.
Abstract
Autoimmune diseases are characterized by a breakdown of immune tolerance partly due to environmental factors. The short-chain fatty acid acetate, derived mostly from gut microbial fermentation of dietary fiber, promotes anti-inflammatory regulatory T cells and protects mice from type 1 diabetes, colitis and allergies. Here, we show that the effects of acetate extend to another important immune subset involved in tolerance, the IL-10 producing regulatory B cells (B10 cells). Acetate directly promoted B10 cell differentiation from mouse B1a cells both in vivo and in vitro. These effects were linked to metabolic changes through the increased production of acetyl-CoA, which fueled the tricarboxylic acid cycle and promoted post-translational lysine acetylation. Acetate also promoted B10 cells from human blood cell through similar mechanisms. Finally, we identified that dietary fiber supplementation in healthy individuals was associated with increased blood B10 cells. Direct delivery of acetate or indirectly via acetate-producing diets or -bacteria might be a promising approach to restore B10 cells in non-communicable diseases.
Authors
Claire I. Daïen, Jian Tan, Rachel Audo, Julie Mielle, Lake-Ee Quek, James R. Krycer, Alexandra S. Angelatos, Martha Duares, Gabriela V. Pinget, Duan Ni, Remy Robert, Md Jahangir Alam, Carmen Balguerías Amián, Frederic Sierro, Arvind Parmar, Gary J. Perkins, Sumaiya Hoque, Alison K. Gosby, Stephen J. Simpson, Rosilene V. Ribeiro, Charles R. Mackay, Laurence Macia
Abstract
Though low circulating levels of the vitamin A metabolite, all-trans retinoic acid (ATRA), are associated with increased risk of cardiovascular events and all-cause mortality, few studies have addressed whether cardiac retinoid levels are altered in the failing heart. Here, we show that proteomic analyses of human and guinea pig heart failure (HF) are consistent a decline in resident cardiac ATRA. Quantitation of the retinoids in ventricular myocardium by mass spectrometry reveals 32 and 39% ATRA decreases in guinea pig HF and in patients with idiopathic dilated cardiomyopathy (IDCM), respectively, despite ample reserves of cardiac vitamin A. ATRA (2mg/kg/day) is sufficient to mitigate cardiac remodeling and prevent functional decline in guinea pig HF. Though cardiac ATRA declines in both guinea pig HF human IDCM, levels certain retinoid metabolic enzymes diverge. Specifically, high expression of the ATRA-catabolizing enzyme, CYP26A1, in human IDCM could dampen prospects for an ATRA-based therapy. Pertinently, a pan-CYP26 inhibitor, talarozole, abrogates the impact of phenylephrine on ATRA decline and hypertrophy in neonatal rat ventricular myocytes. Taken together, we submit that low cardiac ATRA attenuates the expression of critical ATRA-dependent gene programs in HF and that strategies to normalize ATRA metabolism, like CYP26 inhibition, may have therapeutic potential.
Authors
Ni Yang, Lauren Parker, Jianshi Yu, Jace W. Jones, Ting Liu, Kyriakos N. Papanicolaou, C. Conover Talbot Jr., Kenneth B. Margulies, Brian O'Rourke, Maureen Kane, D. Brian Foster
Abstract
Nephrogenic diabetes insipidus (NDI) patients produce large amounts of dilute urine. NDI can be congenital, resulting from mutations in the type-2 vasopressin receptor (V2R), or acquired, resulting from medications such as lithium. There are no good treatment options for NDI. Activation of PKA is disrupted in both congenital and acquired NDI, resulting in decreased aquaporin-2 phosphorylation and water reabsorption. We showed that adenosine monophosphate-activated protein kinase (AMPK) also phosphorylates aquaporin-2. We identified an activator of AMPK, NDI-5033, and tested its ability to increase urine concentration in animal models of NDI. NDI-5033 increased AMPK phosphorylation by 2.5 fold, confirming activation. It increased urine osmolality in tolvaptan-treated NDI rats by 30-50%, and in V2R knockout mice by 50%. Metformin, another AMPK activator, can cause hypoglycemia. It would be risky to treat NDI patients, especially children, with NDI-5033 if it caused hypoglycemia. Rats with NDI receiving NDI-5033 showed no hypoglycemia in a calorie-restricted, exercise protocol. Congenital NDI therapy needs to be effective long-term. We administered NDI-5033 for 3 weeks and saw no reduction in efficacy. We conclude that NDI-5033 can improve urine concentration in animals with NDI and holds promise as a potential therapy for patients with congenital NDI due to V2R mutations.
Authors
Janet D. Klein, Ish Khanna, Sivaram Pillarisetti, Rachael A. Hagan, Lauren M. LaRocque, Eva L. Rodriguez, Jeff M. Sands
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