bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification and Quantification of Free Amino Acids Using Gas Chromatography Coupled with Vacuum Ultraviolet Absorption Spectroscopy
Authors: Harrison Michelle L. , Baars Ben
Published: November 2023

Amino acids serve a variety of important biological functions so their identification and quantification is integral to a number of different fields including the diagnosis of inborn errors of metabolism, food science, metabolomics and biomedical research. There are several existing methodologies for their identification and quantification; however, there remains a need for improvement in areas such as speed of analysis, robustness, and reproducibility. GC‐VUV, a novel technology, provides a new 2D‐detection technique in a field that has been dominated by mass spectrometry for decades. The GC‐VUV detector is unique in its ability to probe molecular electronic transitions. Virtually all molecules absorb in the 125–240 nm energy range and thus provide unique absorbance spectra that can be used for identification. The distinctiveness of the spectra enable closely related structural isomers and isobaric compounds to be readily distinguished. Subsequent quantitative analysis follows well established Beer‐Lambert Law principles allowing for simple, predictable and accurate quantitation via spectral deconvolution without the addition of internal standards. The objective of this work was to develop a method for the detection and quantification of free amino acids as their chloroformate derivatives using gas chromatography (GC) coupled with vacuum ultraviolet (VUV) absorption spectroscopy. In a procedure requiring less than 5 minutes, stock concentrations of individual amino acids were derivatized using isobutanol and isobutylchloroformate, with pyridine as a catalyst. The derivatized amino acids were then extracted into chloroform and injected into the GC. Initially, the derivatized amino acids were run individually so their resultant spectra could be ‘banked’ in the VUV spectral library. Subsequently, the individual amino acids were pooled and the resulting amino acid mixture was derivatized and injected into the GC. All 19 of the amino acids in the mixture were identified and quantified, including the isomers leucine and isoleucine and the nearly isobaric molecules lysine and glutamine. The simplicity, speed, robustness and reproducibility of this approach has the potential to overcome current methodological limitations and, thus, to provide a novel analytic method for use in amino acid applications.
AT1A Receptors in AgRP Neurons Disinhibit αMSH Neurotransmission
Authors: Claflin Kristin E , Morgan Donald A , Rahmouni Kamal , Grobe Justin L
Published: November 2023

The brain renin‐angiotensin system (RAS) and leptin contribute to the control of resting metabolic rate (RMR) and their receptors are co‐expressed in areas of the brain critical for metabolic control, supporting the concept that the central interaction between angiotensin and leptin may be critical to regulate RMR and thus obesity. We have previously demonstrated that angiotensin AT1A receptors co‐localize to leptin‐sensitive cells in the arcuate nucleus (ARC), which also express agouti‐related peptide (AgRP). Moreover, mice lacking angiotensin AT1A receptors in AgRP neurons (AT1AAgRP‐KO) exhibit attenuated brown adipose tissue sympathetic nerve activity in response to leptin (4 hrs post‐leptin, control n=4, 221±18 vs AT1AAgRP‐KO n=5, 150±10 % baseline, p<0.05). AT1AAgRP‐KO mice also exhibit suppressed stimulation of RMR in response to exogenous alpha‐melanocyte stimulating hormone (αMSH; control n=13, +0.032±0.005 vs AT1AAgRP‐KO n=9, +0.012±0.005 kcal/hr, p<0.05), leading us to hypothesize that the activation of AT1A receptors in leptin‐sensitive AgRP neurons disinhibits αMSH neurotransmission and subsequent stimulation of RMR via suppression of inhibitory signaling within the ARC. We examined brain punches from the ARC of AT1AAgRP‐KO mice for the expression of AgRP, neuropeptide Y (NPY), and genes involved in gamma‐aminobutyric acid (GABA) signaling, including the glutamate decarboxylase enzymes, GAD1 and GAD2, and the vesicular GABA transporter VGAT, in response to selective ablation of the AT1A receptor. AT1AAgRP‐KO mice (n=5) exhibited significant (p<0.05) inductions in AgRP (10.3‐fold), NPY (4.8‐fold), GAD1 (1.9‐fold), GAD2 (1.5‐fold) and VGAT (6.9‐fold) in the ARC compared to control littermates (n=10). Taken together, these data support a role for AT1A receptors within AgRP neurons of the ARC in the control of BAT SNA and RMR through suppression of inhibitory AgRP/NPY/GABA signaling and thus disinhibition of stimulatory αMSH neurotransmission.
Diet and Exercise Training Improve Insulin‐Glucose Metabolism and Sympathetic Hyperactivation in Obstructive Sleep Apnea and Metabolic Syndrome
Authors: Cepeda Felipe X. , Rodrigues Sara , Dutra‐Marques Akothirene C.B. , Carvalho Jefferson C. , Toschi‐Dias Edgar , Rondon Maria Urbana P.B. , Alves Maria Janieire N.N. , Lorenzi‐Filho Geraldo , Trombetta Ivani C.
Published: November 2023

Obstructive sleep apnea (OSA) impairs insulin‐glucose metabolism and muscle sympathetic nervous activity (MSNA). Metabolic syndrome (MetS) frequently is common in OSA patients, exacerbating these alterations. In this context, the effect of hypocaloric diet and exercise training (D+ET) in patients with OSA+MetS is not clear. We tested the hypotheses that: 1) D+ET would improve insulin‐glucose metabolism and MSNA; and 2) The reduction in MSNA is associated with the improvement insulin‐glucose metabolism. Never treated OSA patients (apnea‐hypopnea index‐AHI>15 events/h by polysomnography, PSG) and MetS (ATP‐III), were divided into: Control (C, n=10, 46±2y) and D+ET (n=11, 55±3y). D (−500kcal/day) and ET (40min, 3times/week of cycle exercise for 4‐months). We evaluated MSNA (microneurography) and HOMA‐IR and QUICK by fasting insulin/glucose. Both groups were similar in MetS risk factors, PSG parameters, MSNA and fasting insulin/glucose at pre‐intervention. Compared with C, D+ET improved VO2 peak, body weight, waist circumference, systolic and diastolic blood pressure, AHI (P<0,05). D+ET decreased MSNA (31±2 vs. 25±2 bursts/min, P=0.01), fasting insulin (13±1.3 vs. 9±1.0 μUI/mL, P=0.02), HOMA‐IR (3.4±0.4 vs. 2.2±0.3, P=0.03) and increased QUICK (0.32±0.01 vs. 0.35±0.01, P=0.02). No changes were found in C group. Further analysis showed a significant association between change in fasting insulin (R=0.49, P=0.03), in HOMA‐IR (R=0.54, P=0.02) and in QUICK (R=−0.55, P=0.02) with the change in MSNA. D+ET improved insulin‐glucose metabolism and MSNA in patients with OSA+MetS. The effect of D+ET in insulin‐glucose metabolism, can be a candidate to explain, at least in part, the improvement in sympathetic activity.

Support or Funding Information

Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) #2011/17533‐6, #2015/17533‐6 and #2016/16831‐7
Neutral Sphingomyelinase: structure and function
Authors: Hannun Yusuf
Published: November 2023

Sphingolipid metabolism constitutes a network of connected bioactive metabolites that include sphingosine, sphingosine 1‐phosphate, ceramide, ceramide 1‐phosphate, and several others. A compendium of enzymes control the metabolism, regulation, and interconversion of these molecules. These enzymes show very specific sub cellular localization, distinct mechanisms of regulation, and specific cellular functions. Sphingomyelinases (SMases) are positioned at the apex of regulated hydrolytic pathways involved in the regulation of ceramide formation and function. There are three families of SMases distinguished the pH optimum of their action, and the neutral SMases include the largest number of members of which nSMase2 is the best studied. nSMase2 is activated by multiple extracellular agents, including cytokines, ROS, and specific chemotherapeutic agents. Basic mechanistic and structural results will be presented based on the recent crystal structure of the enzyme. Results will be discussed on the regulation of this enzyme by doxorubicin, and a role for the enzyme in the DNA damage response. The yeast counterpart of this enzyme, Isc1p also defines a novel pathway involved in the DNA damage response that recapitulates the functions of the mammalian enzyme. Together, these results define novel and compartment specific functions of neutral sphingomyelinases in the DNA damage response.

Support or Funding Information

National Institutes of Health R37GM043825
Mild Whole Body Heating Decreases Muscle Sympathetic Nerve Activity in Middle Aged and Older Individuals
Authors: Cui Jian , Gao Zhaohui , Blaha Cheryl , Sinoway Lawrence I.
Published: November 2023

A number of reports suggest that whole body heat stress (i.e. increase in core temperature (Tcore) > 0.5 °C) induces a significant increase in muscle sympathetic nerve activity (MSNA) in young and older individuals. However, the effects of mild whole body heating (MWBH) on MSNA have not been specifically examined. A prior report from this laboratory suggests that MSNA is lower in the summer than it is in the winter. In this study, we hypothesized that MWBH would decrease the resting MSNA in middle aged and older individuals. MSNA from a peroneal nerve, ECG and heart rate (HR), blood pressure (BP), Tcore with telemetry temperature pill, mean skin temperature (Tsk), forearm skin blood flow (SkBF) and sweat rate (SR) were measured throughout the study in 8 male healthy subjects (59.6 ± 2.4 years, 177 ± 3 cm, 88 ± 6 kg). After the normothermic (Tsk at ~34 °C) data collection, passive whole body heating was applied via water‐perfused suits with ~46 °C water. After Tsk reached ~36 °C, the water temperature was slightly decreased. The MWBH data were collected during a period (1522 ± 61 sec) with Tsk > 36 °C and the peak increase in Tcore < 0.35 °C. Cardiac output was measured with echocardiography during baseline and MWBH. During a separate visit, a time control trial (non‐heating trial) was performed when the Tsk was controlled at ~34 °C throughout the study. The data were collected during the same time windows as those in the heating trial. Over the MWBH period, the averaged Tsk was 36.8 ± 0.3 °C, and the averaged increase in Tcore was 0.09 ± 0.05 °C. From normothermic baseline, SkBF (100 to 174 ± 31%) and cutaneous vascular conductance (CVC, 100 to 190 ± 43%) increased. Systolic BP (117.5 ± 1.3 to 113.5 ± 2.2 mmHg) did not significantly change, while diastolic BP (75.3 ± 1.9 to 70.7 ± 2.5 mmHg) and mean BP (89.4 ± 1.3 to 85.0 ± 2.3 mmHg, both P < 0.05) fell. MSNA burst rate (38.9 ± 3.0 to 28.9 ± 3.4 bursts/min), burst incidence (60.4 ± 4.5 to 42.4 ± 5.2 bursts/100 beats) and total activity (847 ± 47 to 583 ± 66 units/min, all P < 0.05) decreased, while HR (64.7 ± 2.9 to 69.1 ± 3.0 beats/min) increased. Cardiac output increased (P < 0.05). In the non‐heating time control trial, none of these variables changed. These data suggest that the MWBH may decrease resting MSNA and BP in middle aged and older individuals. Further studies are necessary to examine the mechanisms.

Support or Funding Information

Supported by American Heart Association Grant 15GRNT24480051 (Cui), National Institutes of Health Grants P01 HL096570 (Sinoway) and UL1 TR000127 (Sinoway).
Reflex effects of drinking on thermoregulatory responses in hyperthermic and hyperosmotic men.
Authors: Kamijo Yoshi‐ichiro , Okada Yoshiyuki , Okazaki Kazunobu , Ikegawa Shigeki , Ogawa Yu , Nose Hiroshi
Published: November 2023

Oropharyngeal stimulation by drinking released dehydration‐induced suppression of cutaneous vasodilation and sweating in hyperthermic humans. However, association of skin sympathetic nervous activity (SSNA) with the mechanisms remained unknown. Recently, we have reported that SSNA involved components synchronized and non‐synchronized with cardiac cycle, the components increased with an increase in esophageal temperature (Tes) and the increase in synchronized component was suppressed by hypovolemia and head‐up tilt, but that in non‐synchronized component was not. These results suggested that the synchronized and non‐synchronized components were related with cutaneous vasodilation and sweating, respectively. We hypothesized that hyperosmolality would suppress both SSNA components in hyperthermia, and oropharyngeal stimulation by drinking would release the hyperosmotic suppressions of both components, simultaneously with releasing suppressions of cutaneous vasodilation and sweating. Fourteen young males were divided into isosmolality (Iso; N = 7) and isovolume and hyperosmolality groups (Hos; N = 7) with no treatments and taking diuretics and hypertonic saline infusion before experiment, respectively. Subjects wore water‐perfused suit and took a semi‐recumbent position while 34‐°C water was perfused into the suit. After 10‐min thermoneutral data collection; mean skin temperature (Tsk), Tes, skin blood flow (SkBF; laser‐Doppler velocimetry) and sweat rate (SR; capacitance hygrometry) on the dorsal foot, and mean arterial pressure (MAP), 47‐°C water perfused into the suit for 45 min, then subjects drank a 200‐ml water, warmed at 37 °C. Data collection was continued until 10 min after drinking. Plasma osmolality were 298 and 290 mOsm/kgH2O before the onset of passive warming in Hos and Iso, respectively. Tsk increased by ~2 °C in both groups then Tes increased by 0.4 and 0.5°C at 45min of passive warming in Iso and Hos, respectively (Group × Time; P<0.05). Increases in cutaneous vascular conductance (CVC=SkBF/MAP) and SR at a given increase in Tes tended to be lower in Hos than Iso, simultaneously with suppressed increases in both SSNA components (both P < 0.02). After drinking, both SSNA components increased in Hos (P < 0.05) and an increase in CVC was faster in Hos than Iso and that in SR was greater in Hos than Iso (Group × Time; P < 0.05), while Tsk and Tes remained unchanged before and after drinking. In summary, both SSNA components would be associated with the release of hyperosmolality‐induced suppression of cutaneous vasodilation and sweating by oropharyngeal stimulation in hyperthermic men.

Support or Funding Information

This work was supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (Grant‐in‐Aid for Young Scientists (B) to Y.K., 16790143, Grant‐in‐Aid for Scientific Research (A) to H.N., 17209007) and the Uehara Memorial Fundation in 2008 to Y.K.
IMP1 plays a protective role in LIN28B‐associated intestinal tumorigenesis by regulating ER stress and cell junction assembly
Authors: Chatterji Priya , Hamilton Kathryn , Andres Sarah , Foley Shawn , Mizuno Rei , Castells Antoni , Cuatrecasas Miriam , Simon Lauren , Gregory Brian , Madison Blair , Rustgi Anil
Published: November 2023

Introduction

RNA binding proteins and miRNAs have emerged as crucial regulators of intestinal homeostasis by controlling the stability and translation of target mRNAs. LIN28B, an mRNA binding protein, plays a critical role in regulating growth and proliferation in the intestinal epithelium. Previous work in our lab revealed that LIN28B promotes growth and tumorigenesis of the intestinal epithelium via suppression of mature let‐7 miRNAs (Genes and Dev., 2013). LIN28B suppression of let‐7 promotes upregulation of let‐7 targets, including IMP1 (Insulin‐like growth factor II mRNA‐binding protein‐1). Previous studies from our lab have shown that transgenic mice expressing LIN28B from the mouse Vil1 promoter (Vil‐Lin28b mice) have an increase in IMP1 protein levels that is increased further with conditional knockout of let‐7. Mechanistically, Let‐7 isoforms have been known to physically and functionally interact with IMP1; however, the specific role of IMP1 in Lin28b‐let 7‐mediated tumorigenesis remains unknown.

Methods

We evaluated LIN28B and IMP1 expression and localization in colorectal cancer (CRC) patient samples using tissue microarrays with clinical outcomes. We used intestinal epithelial cell lines with LIN28B overexpression and CRISPR‐Cas9 mediated knockout of IMP1 to study the functional consequences on migration, invasion and proliferation. In parallel, we used RNA sequencing to understand the role of IMP1 deletion in LIN28B over‐expressing cell lines. Additionally, we used mice with intestinal epithelium specific LIN28B overexpression combined with IMP1 knockout to evaluate effect of IMP1 loss on tumor initiation and progression in an aging model as well as through AOM‐DSS induced tumorigenesis.

Results

LIN28B expression correlates positively with expression of IMP1 in CRC. Individually, LIN28B and IMP1 expression associated significantly with worse prognosis in stage II colon cancer that worsens when both were co‐expressed. Knockout of IMP1 in LIN28B overexpressing cells decreased migration of CRC cell lines and altered tumorigenesis in vivo. RNA‐seq revealed a potential role for IMP1 in ER stress and cell junction assembly proteins in the context of LIN28B overexpression.

Conclusions

These data suggest a requirement for IMP1 in LIN28B‐mediated colorectal tumor progression, potentially via ER stress modulation, and support an emerging paradigm for a critical and cooperative role of RNA‐binding proteins in intestinal homeostasis and cancer.

Support or Funding Information

NIH R01DK056645 (AKR), NIH U01DK085551 (AKR), NIH K01DK100485 (KEH),

NIH F32DK107052‐01 (SFA), NIH T32CA1152999 (SFA)
The Effect of a Western Diet on Hepatic Autophagy in Age Accelerated SAMP8 Mice
Authors: Harris Matthew P , Schaller Megan L , Umbaugh David , Sandoval Karin , Witt Ken A , Wooten Joshua S
Published: November 2023

Non‐alcoholic steatohepatitis (NASH) is characterized as a dysregulation of hepatic lipid metabolism and a chronic inflammatory state. It is hypothesized the link between lipid dysregulation and inflammation may be due in part to defective hepatic autophagy and reduced mitochondrial capacity to oxidize fatty acids. It remains to be determined; however, the effects of a Western diet on hepatic autophagy and mitochondrial function during aging.

PURPOSE

The purpose of this study was to determine the effect of a high‐fat high‐fructose diet (HFF) on markers of hepatic autophagy and mitochondrial function in an age‐accelerated mouse model.

METHODS

Twenty‐week old, male and female, SAMP8 mice (n=49) were randomly assigned, matching for gender, to either a standard chow (SC) or HFF (45% fat, 24% fructose) diet for 32 weeks. Liver tissue was analyzed for mRNA expression of autophagic (BNIP3, Beclin 1, p62, and Atg7) and mitochondrial (PGC‐1α and COX‐IV) genes. Differences between gender and dietary groups were identified by a 2 × 2 ANOVA and statistical significance was set at p<0.05.

RESULTS

Following 32 weeks of feeding, male mice fed the HFF diet were significantly heavier than male mice in the SC group (31.64 vs 26.45; p=0.001); however, no difference was observed between diet groups for female mice. The HFF diet resulted in higher autophagic activity as observed by Beclin 1 (+36%; p=0.001) and BNIP3 (+40%; P=0.003) expression. Despite the higher autophagic activity, p62 (+31%; p<0.001) was higher in the HFF compared to the SC group, suggesting impaired autophagic flux. In addition, mitochondrial COX‐IV expression was elevated (+43%; P<0.001) in the HFF group compared to the SC group suggesting increased β‐oxidation. Overall, the expression of all autophagic and mitochondrial markers were higher in males compared to females; however both male and female mice responded similarly to the HFF diet.

CONCLUSION

Despite the higher expression of autophagic and mitochondrial genes, elevated expression of p62 suggests an impaired autophagic flux in age‐accelerated mice following a Western diet.
EPEC Perturbation of Crb3 and Pals1 Localization Precedes Tight Junction Disruption
Authors: Tapia Rocio , Kralicek Sarah , Hecht Gail
Published: November 2023

Enteropathogenic Escherichia coli (EPEC) alters both the architecture and barrier function of intestinal tight junctions (TJ). EPEC also perturbs the apical‐basal polarity of intestinal cells, demonstrated by redistribution of the basolateral proteins Na+/K+ATPase and β1‐integrin to the apical membrane domain and the formation of aberrant TJ strands throughout the lateral membrane. TJ are crucial for the establishment and maintenance of epithelial apical‐basal polarity. The apical Crumbs (Crb) complex (Crb3/Pals1/Patj) is essential for epithelial cell polarization and TJ assembly. We hypothesized that EPEC disrupts intestinal barrier function via perturbation of Crb polarity complex. The aim of this study was to determine the impact of EPEC on Crb polarity complex in vivo and in vitro. Our results indicate that EPEC redistributes Crb3 and Pals1, but not Patj, from the membrane to the cytoplasm of colonic epithelial cells. Depletion of espF, but not map, ablates the cytoplasmic accumulation of Crb3. Complementation of espF (ΔespF/pespF) restores the wild‐type EPEC phenotype of Crb3 internalization. Depletion of both espF and map, attenuates the redistribution of Pals1, while complemented strains (ΔespF/pespF and Δmap/pmap) reverted this phenotype. Immunofluoresce intensity of murine colonic tissues showed that EPEC significantly reduces the level of membrane‐associated Crb3 and Pals1 (−36.0±4.0% and −38.0±3.0%; p<0.001; n=3, respectively) and increases cytoplasmic accumulation (82.0±5.0% and 106.0±6.0%; p<0.001; n=3, respectively) compared to uninfected mice. To determine the temporal sequence of Crb3 and TJ disruption, SKCO‐15 monolayers were infected and Crb3 and occludin localization and transepithelial resistance (TER) were analyzed over time. Crb3 internalization occurred as early as 1 hr yet occludin was not disrupted until 3 hr post‐infection. Decrease in TER occurred as early as ‐‐2hr (−28.0±7.0% vs +11.0±6.0% change from baseline, respectively; n=3, ***p<0.0001) and continued to decline over the course of infection dropping 41.0±8.0% from baseline vs 14±9.0% increase of controls from baseline at 3 hr (n=3, ***p<0.0001). These results show that EPEC‐induced perturbation of Crb3 and Pals1 precedes alteration of TJ structure and function and may contribute to TJ disruption.
The phenotype and wound healing property of IL‐4‐treated human macrophages is not affected by co‐treatment with antigen from the tapeworm, Hymenolepis diminuta
Authors: Jayme Timothy Santiago , Luis Jose , Wang Arthur , Thomson Emma T. , Beck Paul , McKay Derek
Published: November 2023

Background

Infection with parasitic helminths can reduce inflammation in animal models of colitis. A component of this anti‐inflammatory effect may be induction of alternatively‐activated IL‐4 driven anti‐inflammatory/pro‐resolution macrophages (M(IL4)) that have been implicated in wound repair. Infection with helminth parasites or exposure to worm extracts may elicit unique macrophage phenotypes. We hypothesized that macrophages co‐treated with IL‐4 and a crude extract Hymenolepis diminuta (HdE), would have an enhanced ability to promote repair in wounded epithelial monolayers.

Aim

To assess the impact of HdE on the development of human and murine M(IL4)s using canonical markers and assess the ability of these cells to promote epithelial wound repair.

Methods

Human (blood‐derived) and murine (bone marrow‐derived) macrophages were exposed to IL‐4 (10 ng/ml; 48h), HdE (100 mg/ml; 48h), IL4+HdE, or left unstimulated (controls). Expression of M(IL4) markers were assessed by qPCR, immunoblotting or ELISA (human: CD206, CCL18, CD14; murine: arginase‐1, Ym1). Confluent Caco2 epithelial cell monolayers were wounded with a razor blade and treated with supernatants from the different human macrophage sub‐types populations; protein denatured (trypsin‐treated + boiling) were also used. The total area of epithelial migration was measured (in mm2) 24h later. TGFb levels in the macrophage supernatants were measured by ELISA.

Results

The development of human M(IL4)s was not affected by HdE co‐treatment as assessed by any of the markers examined, whereas HdE did reduce arginase‐1 and Ym1 in murine M(IL4)s (n=4–6). Supernatants from M(IL4)s increased epithelial migration by 36% ± 14% (n=8, p<0.05) compared to baseline, and this was not affected by HdE co‐treatment of the macrophages. Protein denaturing ablated the ability of M(IL4) supernatant to enhance epithelial wound repair (n=6). TGFb levels were increased in M(IL4) supernatants (353 ± 78*) compared to those from non‐stimulated macrophages (204 ± 71 pg/ml) (n=11; *p<0.05 (paired t test)).

Conclusion

HdE has negligible effects on the induction of a human M(IL4) and does not inhibit its ability to promote epithelial wound repair, that could be TGFb dependent. Thus, (1) M(IL4)s elicited in humans following infection with helminths will not be adversely affected by the release of worm antigen, and (2) if an anti‐colitic effect of systemic delivery of helminth antigens to humans results in mobilization of an IL‐4‐type alternatively activated macrophage, this would be an indirect effect of the worm antigen.

Support or Funding Information

Supported by Crohn's Colitis Canada grant to DMM and an NSERC CREATE Host‐Parasite Interactions scholarship to Timothy Jayme
Colonic inflammation and cancer are driven by elevated D‐2‐hydroxyglutarate levels in an EglN1‐dependent mechanism
Authors: Theiss Arianne L. , Han Jie , Turner Kevin O. , Genta Robert M. , Sweetman Lawrence
Published: November 2023

Emerging evidence suggests a crucial role for altered expression of enzymes with functions in cellular metabolism in the pathogenesis of inflammation and cancer. But the detailed molecular mechanisms that link these alterations to carcinogenesis remain elusive. Here, we identify a novel role for altered metabolism of D‐2‐hydroxyglutarate (D2HG) in colitis and colitis‐associated colon cancer. We found that the levels of D2HG were elevated in the urine of mice during dextran sodium sulfate (DSS)‐induced colitis. In a mouse model of colitis‐associated cancer, intraperitoneal injection of D2HG resulted in delayed recovery from colitis and severe tumorigenesis. The colonic expression of D‐2‐hydroxyglutarate dehydrogenase (D2HGDH), an enzyme that eliminates D2HG, was decreased during DSS colitis and human ulcerative colitis (UC) but unaltered in mouse or UC tumors. Mechanistically, we demonstrate that elevated expression of Egg‐laying defective Nine 1 (EglN1) inhibited hypoxia inducible factor (Hif)‐1a, which in turn attenuated D2HGDH transcription, thereby resulting in elevated D2HG. Furthermore, pharmacological inhibition of EglN1 ameliorated colitis‐associated cancer in mice. Thus, we have identified a novel regulatory mechanism where D2HG promotes colonic inflammation and carcinogenesis, which may provide functional therapeutic targets to inhibit the progression from colitis to cancer.

Support or Funding Information

Cancer Prevention & Research Institute of Texas grant RP150638 (A.L.T) and Charles A. Sammons Cancer Center, Baylor University Medical Center grant (A.L.T).
Formononetin Relaxes Cholecystokinin‐ and KCl‐induced Tension in Guinea Pig Gallbladder Strips
Authors: Kline Loren
Published: November 2023

Formononetin, a phytoestrogen and isoflavone, relaxed cholecystokinin octapeptide‐ (CCK) and KCl‐induced tension in a concentration dependent manner. An in vitro technique was used to determine which system(s) mediated the relaxation. Paired t‐tests were used; differences between mean values of p<0.05 were considered significant. Adding formononetin prior to CCK or KCl produced a significant decrease in the amount of tension {1.2±0.07 vs. 0.81±0.05 g CCK, (p<0.001); 0.84±0.04 vs. 0.74±0.05 g KCl, p<0.01}. When the PKC inhibitors bisindolymaleimide IV and chelerythrine Cl− were used together, a significant (p<0.01) reduction in the formononetin‐induced relaxation (78.1±4.5 vs. 69.7±3.4%) was observed. Genistein, a protein tyrosine kinase inhibitor, significantly (p<0.01) decreased the amount of formononetin‐induced relaxation (72.2±6.5 vs. 59.3±6.5%). To determine if PKA mediated the formononetin‐induced relaxation, PKA inhibitor 14–22 amide myristolated (PKA‐IM) was used. PKA‐IM had no significant effect on the amount of formononetin‐induced relaxation. Neither the use of 2‐APB, a blocker of intracellular Ca2+ release; KT5823, a blocker of PKG; or L‐NMMA, a nitric oxide synthase inhibitor, had a significant effect on the amount of formononetin‐induced relaxation. The formononetin‐induced relaxation of CCK‐ or KCl‐induced tension was mediated by blocking extracellular Ca2+ entry which then affected downstream events such as activation of PKC and protein tyrosine kinase.
Immunohistochemical Identification of Purinergic Neurons in the Enteric Nervous System
Authors: Perez‐Medina Alberto L , Galligan James J.
Published: November 2023

Propulsive gastrointestinal (GI) motility patterns depend on coordinated contraction and relaxation of GI smooth muscles. The enteric nervous system (ENS) controls GI motility. Currently, it is accepted that smooth muscle relaxation is mainly mediated by the co‐release of ATP and nitric oxide (NO) from one class of inhibitory motor neuron (IMN). ATP and NO mediate fast and slow inhibitory junction potentials, respectively. Neuronal ATP and NO release depend on activation of voltage‐gated calcium channels (VGCC) and different VGCC may drive ATP and NO release independently. Functional data also suggest that descending interneurons communicate with other interneurons and IMNs via ATP release. However, direct identification of purinergic nerves in the ENS is lacking. Our study aim was to directly identify purinergic nerves in the ENS using an antibody against the vesicular nucleotide transporter (VNUT, SLC17A9), a purinergic nerve marker to distinguish the purinergic and non‐purinergic neurons in mice and guinea pig colon and ileal myenteric plexus. We used immunohistochemical methods to localize nitric oxide synthase (NOS), VNUT, choline acetyl transferase (ChAT) and the a1E subunit of the R‐type Ca2+ channels. . In addition, longitudinal muscle myenteric plexus (LMMP) preps were placed in tissue culture with colchicine for 48 h to facilitate staining of purinergic and non‐purinergic nerve cell bodies. Our results show VNUT+/NOS‐‐, NOS+/VNUT‐, ChAT+/VNUT‐, and ChAT‐/VNUT+ nerve fibers supplying the muscle layers. Moreover, multiple populations of cell bodies were identified (VNUT+ neurons, NOS+ neurons, and NOS‐/VNUT+ neurons), NOS+/VNUT+ neurons being the smallest population observed. In addition, VNUT and ChAT did not co‐localize in myenteric neurons. Finally NOS and the a1E subunit localize in the same axon bundles, but not in the same axons, that innervate the smooth muscle. These results suggest that there may be two types of inhibitory motorneuron supplying the muscle layers in the gut (purinergic and nitrergic). There may also be two types of excitatory interneurons: cholinergic and puringergic. Further studies of neuromuscular and synaptic transmission are required to establish the function of the puringeric, nitrergic and cholinergic neurons.

(Supported by 1R01DK094932)

Support or Funding Information

Supported by 1R01DK094932
Epithelial Interleukin‐15 Functions as a Chemoattractant for γΔ IEL Migration
Authors: Edelblum Karen L , Ethridge Alexander D , Hu Madeleine
Published: November 2023

Positioned between intestinal epithelial cells in the lateral intercellular space, intraepithelial lymphocytes (IEL) expressing the γΔ T cell receptor exhibit a surveillance‐like migratory behavior, which is required to limit microbial invasion. Epithelial interleukin (IL)‐15 is critical for the development, proliferation and survival of γΔ IELs; however, the role of IL‐15 in γΔ IEL migration remains unclear. Transpresentation of membrane bound IL‐15 requires cell‐cell contact between activating and responder cells, thus based on the direct interactions between IELs and epithelial cells we tested the hypothesis that IL‐15 exposure promotes γΔ IEL migration into the lateral intercellular space. To this end, in vitro migration assays were performed by co‐culturing γΔ IELs with Caco2 epithelial cells in the presence of recombinant IL‐15. We found that IL‐15 induced γΔ IEL migration in a dose‐dependent manner (P<0.001). In contrast, the addition of IL‐15 neutralizing antibody reduced basal migration by nearly 25% (P<0.05), indicating that basal epithelial IL‐15 expression contributes to γΔ IEL migration in vitro. Since IL‐15 has been reported to function as a NK cell chemoattractant, we performed 3D chemotaxis assays in which sorted GFP+ γΔ IELs were embedded in Matrigel, exposed to an IL‐15 diffusion gradient and timelapse microscopy was performed. Over the course of 6 hours, IL‐15 significantly enhanced both the track displacement and velocity of γΔ IEL migration (P<0.0001), demonstrating that IL‐15 alone is sufficient to promote γΔ IEL motility. Lastly, to assess the requirement for epithelial IL‐15 in γΔ IEL migration into the lateral intercellular space, γΔ IELs were co‐cultured with enteroids derived from the jejunum of wildtype mice or those overexpressing murine IL‐15 in the intestinal epithelium (vil‐IL‐15). Morphometric analysis of γΔ IEL number and location within the co‐cultures showed that vil‐IL‐15 expression increased the number of γΔ IELs associated with enteroids by 38±0.2% (P<0.05) and migration into the lateral intercellular space by 17±7.1% (P<0.05). Taken together, these data indicate that epithelial IL‐15 expression functions as a γΔ T chemoattractant and demonstrates a novel role for IL‐15 in maintaining basal γΔ IEL surveillance of the villous epithelium.

Support or Funding Information

NIH, RBHS Chancellor Scholar Award
RGS10 Mediates Changes in Gut Permeability and Inflammation After an Intestinal Inflammatory Challenge
Authors: Khosravian Kiana , Houser Madelyn , Tansey Malu
Published: November 2023

Parkinson's disease (PD) pathology is marked by degeneration of dopamine neurons and the accumulation of the protein alpha‐synuclein in Lewy bodies in the Substantia Nigra pars Compacta (SNPC). A lesser known aspect of PD pathology is chronic intestinal inflammation which may promote the progression of the disease by dysregulating alpha‐synuclein in the gut. Regulator of G‐protein signaling 10 (RGS10) is a GTPase‐activating protein that is expressed in intestinal immune cells and acts as an anti‐inflammatory regulator by inhibiting NFkB. Previous studies have also shown that RGS10 knock‐out mice developed dopamine neuron loss in the SNPC after systemic lipopolysaccharide exposure. We have also found that mice deficient in RGS10 have higher levels of NFkB in the intestine, significantly shorter intestine lengths, increased fecal water content, and changes in tight junction protein expression. We predicted that reductions in RGS10 expression would increase inflammation and permeability in the intestine to a greater extent in the context of a chemical inflammatory challenge, modeling, perhaps, the contribution of pronounced intestinal inflammation to Parkinsonian neuropathology. To evaluate this, we subjected RGS10 knockout, heterozygous, and wild‐type mice to a dextran sodium sulfate (DSS) colitis model. We took baseline measurements of weight as well as peripheral blood immune cell populations by flow cytometry. For a total of 14 days, mice were monitored for weight loss and bloody stool. At the end of the experiment, a FITC‐dextran gut permeability assay was performed. The mice were then sacrificed, and small intestine and colon length were measured, along with cecum weight. We collected blood, brain, and gut tissue for flow cytometry, qPCR, and Western blots. We found that RGS10 knockout mice have significant changes in immune cell population. We will compare the effect of RGS10 deficiency on levels of intestinal inflammation at baseline and after DSS. Our work will demonstrate that RGS10 deficiency leads to changes in intestinal structure and immunity, which can increase the risk for Parkinsonian‐like pathology in knockout mice.

Support or Funding Information

Acknowledgments: This work was supported by NSF Graduate Research Fellowship DGE‐1444932 and the American Physiological Society's Undergraduate Summer Research Fellowship. A special thank you to the Tansey Lab and the Emory University Department of Physiology.
Inhibition of PTK6 Improves Intestinal Barrier Function and Claudin‐3 Expression in an Experimental Mouse Model for Ulcerative Colitis
Authors: Haines Ricci J , Eitnier Rebecca A , Zhang Shimin , Wang Fang , Wu Mack H
Published: November 2023

Ulcerative colitis (UC) is a debilitating disease characterized by chronic inflammation in the gut. It is thought that increased intestinal permeability is an initial physiological mechanism that gives rise to the persistent state of inflammation. In fact, it has been demonstrated that targeting epithelial cells in a manner that increases intestinal barrier function provides patients with significant relief and fewer subsequent flare‐ups. Since protein tyrosine kinase 6 has recently been shown to play a critical role in intestinal epithelial barrier function, and PTK6 is most highly expressed in intestinal epithelial cells, we hypothesized that targeting PTK6 would improve intestinal barrier function as well as reduce clinical signs of UC. Gut mucosal barrier function was evaluated by measuring gut lumen‐to‐blood transport of a fluorescent tracer using a mouse model for experimental UC in both wild type (WT) and transgenic null mice for PTK6 expression. We found that genetic ablation of PTK6 improved gut barrier function by approximately two‐fold in the animal with experimentally induced UC. Similarly, we observed three‐fold improvement of gut barrier function in the UC mice treated with a pharmacological PTK6 inhibitor. In addition, H&E staining of colonic tissues in UC mice showed enhanced crypt structure in mice with genetic interruption of PTK6 or those treated with PTK6 inhibitor. Western blot analysis of colonic tissues showed UC mice had elevated levels of active PTK6, decreased detection of the tight junction protein claudin‐3, increased detection of pY925 focal adhesion kinase (FAK), and elevated expression of matrix metalloproteinase 9 (MMP9). Fecal supernatants from UC WT mice showed enhanced detection of cleaved E‐cadherin consistent with the size generated by MMP9 cleavage. Since PTK6 is known to phosphorylate FAK at Y925, and this phosphorylation event is important for MMP9 expression, we explored whether PTK6 interruption could enhance claudin‐3 expression and reduce the level of FAK pY925, active MMP9 and cleaved E‐cadherin in fecal supernatants. Indeed, colonic lysates from UC PTK6 null mice showed significant reductions of pY925 FAK, active MMP9 and E‐cadherin cleavage when compared to the lysates obtained from WT UC mice. Immunostaining of histological samples also showed improved tight junction organization and claudin‐3 localization at epithelial tight junctions. Taken together, these results suggest that PTK6 plays a critical role in mediating gut mucosal barrier injury in UC and may potentially regulate FAK‐dependent MMP9 activity.

Support or Funding Information

IBX000799, NIH HL‐120954
Steroid Treatment Impairs Intestinal Stem Cell (ISC) Activation and Contributes to Delayed Ulcer Healing in Inflammatory Bowel Disease (IBD)
Authors: Lynch Evan Brady , Goretsky Tatiana , Bradford Emily , Barrett Terrence
Published: November 2023

In cases of moderate to severe inflammatory bowel disease (IBD), exogenous glucocorticoid steroids (GC) are the preferred treatment due to their safety and efficacy in alleviating symptoms. Despite the clinical benefits, endoscopic data show that GCs delay ulcer healing through mechanisms that are poorly understood. Intestinal stem cells (ISC) require Wnt/β‐catenin signaling for maintenance of the crypt proliferative compartment, and Wnt responsiveness is an important component of a functional mucosal healing response. The objective of our study was to determine whether GCs impair ISC activation in ulcer re‐epithelialization. Specifically, we hypothesized that that steroids impair canonical Wnt/β‐catenin signaling required for ISC activation needed for mucosal repair.

Methods

We examined the effects of dexamethasone, a GC steroid, on cell signaling pathways involved in Wnt/β‐catenin, PI3 Kinase, and NFkB signaling. We utilized NCM460 (NCM) cells, a normal human colonic intestinal epithelial cell (IEC) line, mouse epithelial cells isolated after DSS‐mediated colitis induction, and epithelial isolations from patient biopsies obtained from University of Kentucky and VA Lexington Hospitals. IEC from untreated and steroid‐treated (7 days) UC patients with equivalent levels of colitis were compared.

Results

In steroid‐treated NCMs, we saw a significant decrease in transcript levels of Wnt target gene Axin2, a marker of Wnt‐responsive multipotent IECs that participate directly in ulcer healing, and the β‐catenin transcriptional co‐activator CREB Binding Protein (CBP). After transfecting NCM460s with lentivirus containing a TCF/LEF luciferase construct (a marker of Wnt transcriptional activation), we saw that steroid treatment diminished TNF‐mediated increase in TCF/LEF signaling back to control levels. In both murine and IECs isolated from patient biopsies, GC inhibited colitis‐induced Axin2, p‐LRP6 (marker of Wnt signaling) and nuclear β‐catenin protein levels. Together, these data indicate strongly that GCs impair Wnt signaling.

Discussion

Our results indicate that GCs impair Wnt/β‐catenin signaling during ulcer healing in in vitro and in vivo models of colitis. While steroids play a significant role in regulating the basal immune response to IBD, our data suggest steroids inhibit epithelial signaling required for ulcer healing. Given the recent data implicating NFkB in Wnt signaling activation, our studies suggest a potential explanation for GC‐induced impairment of stem cell activation needed for mucosal repair in IBD.

Support or Funding Information

1I01CX001353‐01A1 VA Merit 5TL1TR000115‐05 TL1 Pre‐doctoral CTSA Funding
Infection with the tapeworm parasite Hymenolepis diminuta in early life protects against experimental colitis in mice
Authors: Arai Toshio , Lopes Fernando , Wang Arthur , McKay Derek
Published: November 2023

Background

The hygiene hypothesis asserts that lack of exposure to infectious disease in childhood contributes to the development of auto‐inflammatory disease. We showed that infection with the rat tapeworm Hymenolepis diminuta evoked a Th2 response, including IL‐10, in adult mice and protected the mice from di‐nitrobenzene‐sulfonic acid (DNBS) induced colitis. Within North America there has been a startling increase in pediatric (early)‐onset inflammatory bowel disease. We tested the hypothesis that infection of mice immediately post‐weaning with H. diminuta would protect them from colitis.

Aim

To determine if three‐week old mice effectively expel H. diminuta and receive protection from chemically‐induced colitis by infection with this helminth parasite.

Methods

First, helminth infectivity was compared in three‐ and eight‐week old Balb/c mice (bred at Univ. Calgary). Second, three‐week old Balb/c mice were infected with 5 cysticercoids of H. diminuta by oral gavage and 8 or 10 days later received 1.5 mg of DNBS intra‐rectally. Colitis was assessed 72h post‐DNBS by: 1) loss of body weight, 2) colon length, 3) disease activity score, 4) histopathology, and 5) concanavalin‐A stimulated cytokine production from spleen cells. Third, the ability of re‐challenge with H. diminuta antigen to protect mice from DNBS‐induced colitis in later life was tested.

Results

Juvenile mice expelled H. diminuta although this was delayed by ~2–3 days compared to infected adult mice. Mice infected with H. diminuta 8 days DNBS were not protected against colitis, whereas those infected 10‐days prior to ir. DNBS displayed substantially severe colitis The anti‐colitic effect in the infected mice was paralleled by increased splenic production of IL‐4 and IL‐10, and increased mRNA IL‐4, IL‐10 and FOXP3 mRNA in extracts of colon. Infection also lead to immunological memory in the young mice allowing triggering of an anti‐colitic effect by systemic delivery of worm antigen to previously infected mice.

Conclusions

Juvenile mice expel H. diminuta (indicating a functional immune response) and infected mice can be protected from DNBS‐induced colitis. We provide these preliminary data in support of the consideration of helminth therapy in early‐onset of IBD and speculate that exploiting immunological memory for H. diminuta could be a new an

Support or Funding Information

Supported by Yamanashi Scholarship and NSERC CREATE Host‐Parasite Interactions scholarship to TA and NSERC operating grant to DMM.
Emu Oil Reduces Colonic Damage Severity in a Mouse Model of Chronic Ulcerative Colitis
Authors: Safaeian Romina , Howarth Gordon S , Lawrance Ian C , Trinder Debbie , Mashtoub Suzanne
Published: November 2023

Background/Aims

Ulcerative colitis (UC) is characterised by large intestinal inflammation and ulceration. Previously, Emu Oil (EO) has been reported to protect the intestine in models of NSAID‐enteropathy, mucositis and acute UC. It was hypothesized that EO would reduce disease and colonic damage severity in a mouse model of dextran sulphate sodium (DSS)‐induced chronic UC.

Methods

Mice (n=10/group) were gavaged with water or EO (80microL [low dose] or 160microL [high dose]) thrice weekly. Mice were subjected to two cycles each consisting of water or DSS (2% w/v) ad libitum for one week and two weeks water recovery. Mice were euthanized two days after a third week of DSS. Disease activity index (DAI), burrowing activity and histologically‐assessed damage were quantified. Data were statistically analysed using ANOVA with LSD and Kruskal Wallis with Mann Whitney U tests. P<0.05 was considered significant.

Results

DAI was significantly greater in DSS controls compared to controls (P<0.05). EO‐low dose (days 6–9, 26–35) and EO‐high dose (days 6–16, 25–26, 33–35, 45) reduced DAI compared to DSS controls (P<0.05). DSS reduced burrowing activity in mice compared to controls; an effect significantly improved by both doses of EO (days 27, 40; P<0.05). In DSS‐treated mice, colonic damage severity increased compared to controls (P<0.001). Importantly, both doses of EO significantly decreased damage severity in the distal colon compared to DSS controls (P<0.001).

Conclusions

Emu Oil reduced colonic damage severity and disease activity and increased burrowing activity in experimentally‐induced chronic UC, suggesting potential as a treatment adjunct for UC.

Support or Funding Information

N/A
Novel G protein‐coupled receptors in Pancreatic Beta cells
Authors: Saunders Fiona , Iyinikkel Jean , Lima Maria , Thompson Dawn , Delibegovic Mirela , Docherty Kevin , Murray Fiona
Published: November 2023

Type 2 diabetes is a widespread metabolic disorder that is on the rise worldwide. Type 2 diabetes is a significant economic burden and increases the frequency of other diseases, such as hypertension and stroke. Reduced secretion of the hormone insulin by pancreatic beta cells, and resistance of the body to insulin contribute to the development of the disease. Insulin secretion can be influenced by circulating mediators, such as hormones, many of which act though G protein‐coupled receptors (GPCR). GPCRs are the largest receptor family in the human genome and are the targets for >30% of approved drugs. GPR75, an orphan GPCR, has been proposed to regulate insulin secretion, thus we aimed to determine its expression in pancreatic beta cells and changes with type 2 diabetes. Using real‐time PCR we found GPR75 is expressed in primary human pancreatic beta cells and the pancreas from control C57Bl/6 mice. Immunofluorescence staining of MIN‐6 cells (a pancreatic beta cell line) showed GPR75 expression on the cell membrane and punctate intracellular staining (indicative of constitutive activity). We found GPR75 to be upregulated in diabetic mice, either with a spontaneous knock‐out of the leptin gene or in LDL receptor knock‐out mice. Our data show expression of GPR75 in pancreatic beta cells and increased expression with diabetes. Uncovering the function of GPR75 in pancreatic beta cells may advance the understanding of type 2 diabetes and even uncover novel treatments for the disease.

Support or Funding Information

Wellcome Trust