Transcriptional and post-translational regulation of plant autophagy.

In response to changing environmental conditions, plants activate cellular responses to enable them to adapt. One such response is autophagy, in which cellular components, for example proteins and organelles, are delivered to the vacuole for degradation. Autophagy is activated by a wide range of conditions, and the regulatory pathways controlling this activation are now being elucidated. However, key aspects of how these factors may function together to properly modulate autophagy in response to specific internal or external signals are yet to be discovered. In this review we discuss mechanisms for regulation of autophagy in response to environmental stress and disruptions in cell homeostasis. These pathways include post-translational modification of proteins required for autophagy activation and progression, control of protein stability of the autophagy machinery, and transcriptional regulation, resulting in changes in transcription of genes involved in autophagy. In particular, we highlight potential connections between the roles of key regulators and explore gaps in research, the filling of which can further our understanding of the autophagy regulatory network in plants.

Highly ABA-Induced 1 (HAI1)-Interacting protein HIN1 and drought acclimation-enhanced splicing efficiency at intron retention sites.

The Highly ABA-Induced 1 (HAI1) protein phosphatase is a central component of drought-related signaling. A screen for HAI1-interacting proteins identified HAI1-Interactor 1 (HIN1), a nuclear protein of unknown function which could be dephosphorylated by HAI1 in vitro. HIN1 colocalization and interaction with serine-arginine rich (SR) splicing factors and appearance of nuclear speckle-localized HIN1 during low water potential (ψw) stress suggested a pre-mRNA splicing-related function. RNA sequencing of Arabidopsis Col-0 wild type identified more than 500 introns where moderate severity low ψw altered intron retention (IR) frequency. Surprisingly, nearly 90% of these had increased splicing efficiency (decreased IR) during stress. For one-third of these introns, ectopic HIN1 expression (35S:HIN1) in unstressed plants mimicked the increased splicing efficiency seen in stress-treated wild type. HIN1 bound to a GAA-repeat, Exonic Splicing Enhancer-like RNA motif enriched in flanking sequence around HIN1-regulated introns. Genes with stress and HIN1-affected splicing efficiency were enriched for abiotic stress and signaling-related functions. The 35S:HIN1 plants had enhanced growth maintenance during low ψw, while hin1 mutants had reduced growth, further indicating the role of HIN1 in drought response. HIN1 is annotated as an MYB/SANT domain protein but has limited homology to other MYB/SANT proteins and is not related to known yeast or metazoan RNA-binding proteins or splicing regulators. Together these data identify HIN1 as a plant-specific RNA-binding protein, show a specific effect of drought acclimation to promote splicing efficiency of IR-prone introns, and also discover HAI1-HIN1 interaction and dephosphorylation that connects stress signaling to splicing regulation.

Phosphoproteomics of Arabidopsis Highly ABA-Induced1 identifies AT-Hook-Like10 phosphorylation required for stress growth regulation.

The clade A protein phosphatase 2C Highly ABA-Induced 1 (HAI1) plays an important role in stress signaling, yet little information is available on HAI1-regulated phosphoproteins. Quantitative phosphoproteomics identified phosphopeptides of increased abundance in hai1-2 in unstressed plants and in plants exposed to low-water potential (drought) stress. The identity and localization of the phosphoproteins as well as enrichment of specific phosphorylation motifs indicated that these phosphorylation sites may be regulated directly by HAI1 or by HAI1-regulated kinases including mitogen-activated protein kinases, sucrose non-fermenting-related kinase 2, or casein kinases. One of the phosphosites putatively regulated by HAI1 was S313/S314 of AT-Hook-Like10 (AHL10), a DNA-binding protein of unclear function. HAI1 could directly dephosphorylate AHL10 in vitro, and the level of HAI1 expression affected the abundance of phosphorylated AHL10 in vivo. AHL10 S314 phosphorylation was critical for restriction of plant growth under low-water potential stress and for regulation of jasmonic acid and auxin-related gene expression as well as expression of developmental regulators including Shootmeristemless These genes were also misregulated in hai1-2 AHL10 S314 phosphorylation was required for AHL10 complexes to form foci within the nucleoplasm, suggesting that S314 phosphorylation may control AHL10 association with the nuclear matrix or with other transcriptional regulators. These data identify a set of HAI1-affected phosphorylation sites, show that HAI1-regulated phosphorylation of AHL10 S314 controls AHL10 function and localization, and indicate that HAI1-AHL10 signaling coordinates growth with stress and defense responses.

The flip side of phospho-signalling: Regulation of protein dephosphorylation and the protein phosphatase 2Cs.

Protein phosphorylation is a key signalling mechanism and has myriad effects on protein function. Phosphorylation by protein kinases can be reversed by protein phosphatases, thus allowing dynamic control of protein phosphorylation. Although this may suggest a straightforward kinase-phosphatase relationship, plant genomes contain five times more kinases than phosphatases. Here, we examine phospho-signalling from a protein phosphatase centred perspective and ask how relatively few phosphatases regulate many phosphorylation sites. The most abundant class of plant phosphatases, the protein phosphatase 2Cs (PP2Cs), is surrounded by a web of regulation including inhibitor and activator proteins as well as posttranslational modifications that regulate phosphatase activity, control phosphatase stability, or determine the subcellular locations where the phosphatase is present and active. These mechanisms are best established for the Clade A PP2Cs, which are key components of stress and abscisic acid signalling. We also describe other PP2C clades and illustrate how these phosphatases are highly regulated and involved in a wide range of physiological functions. Together, these examples of multiple layers of phosphatase regulation help explain the unbalanced kinase-phosphatase ratio. Continued use of phosphoproteomics to examine phosphatase targets and phosphatase-kinase relationships will be important for deeper understanding of phosphoproteome regulation.

Quality of life comparison between smokers and non-smokers with chronic pancreatitis.

OBJECTIVES: The aim of this study was to evaluate the impact of smoking on quality of life in patients with chronic pancreatitis. METHODS: This is a cross-sectional study of chronic pancreatitis patients followed at a single institution comparing smokers with non-smokers. The primary outcome was quality of life and secondary outcomes included demographics, drug and alcohol use, anxiety and depression, pain level, nutritional status, and metabolic factors. RESULTS: 48 smokers and 45 non-smokers participated in this study. Smokers had a worse overall quality of life and higher rates of opioid addiction and depression than non-smokers. Smokers also had less racial diversity, lower education levels, and higher amounts of narcotic use than non-smokers. Furthermore, smokers had a lower BMI and a higher proportional use of pancreatic enzyme replacement therapy. Smoking was found to be independently associated with worse quality of life on multivariable regression. CONCLUSIONS: The worse overall quality of life and higher rates of depression and anxiety create cause for concern in chronic pancreatitis patients who smoke. Smoking cessation should be an important target in chronic pancreatitis patients. Multicenter, multiethnic studies are needed to further elucidate this relationship.

Effects of interleukin-6 on the expression of tight junction proteins in isolated cerebral microvessels from yearling and adult sheep.

OBJECTIVES: The blood-brain barrier is a selective diffusion barrier between brain parenchyma and the intravascular compartment. Tight junctions are integral components of the blood-brain barrier. Pro-inflammatory cytokines are important in the pathogenesis of brain injury and could modify the protein constituents of tight junctions. We hypothesized that interleukin-6 (IL-6) downregulates key protein constituents of endothelial tight junctions (e.g. occludin and claudin-5). METHODS: We examined the effects of IL-6 on tight junction protein expression using an in vitro blood-brain barrier model. We isolated microvessels from yearling and adult ovine cerebral cortex and placed them into culture with IL-6 concentrations of 0 (control, phosphate-buffered saline), 1, 10, and 100 ng/ml for 24 h. Cerebral microvessels were harvested, Western immunoblot performed for occludin and claudin-5, densitometry performed, and results expressed as a ratio to control values. RESULTS: Western immunoblot analysis showed that treatment with 100 ng/ml of IL-6, but not the lower concentrations, reduced (p < 0.05) occludin expression in microvessels from yearling and adult sheep and claudin-5 in microvessels from adult sheep. However, treatment with 10 ng/ml of IL-6 increased claudin-5 in microvessels from yearling sheep. The percent of lactate dehydrogenase released from the microvessels into the surrounding media was not increased by IL-6 treatment, suggesting that the reductions in tight junction proteins did not result from cell death. Treatment of adult cerebral cortical microvessels with IL-6 preincubated with anti-IL-6 monoclonal antibodies partially attenuated the reduction in claudin-5. CONCLUSION: We conclude that IL-6 modulates tight junction protein expression in cerebral cortical microvessels from yearling and adult sheep.

Increased Prevalence of Alcohol-Related Gastrointestinal and Liver Diseases During the COVID-19 Pandemic.

BACKGROUND: Higher prevalence of alcohol-related gastrointestinal (GI) and liver diseases (ARGLDs) were anecdotally reported during the COVID-19 pandemic, but little published evidence exists. METHODS: A healthcare system audit of inpatient GI consults was performed during the pandemic's lockdown phase (3/23/2020-5/10/2020, n=558) and reopening phase (6/1/2020-7/19/2020, n=711) with comparison to those timeframes in 2019. RESULTS: Consult volume decreased by 27.7% during the lockdown, but the proportion of ARGLDs increased by 59.6% (p=0.03). This trend continued during reopening, with potentially more severe disease as more patients required endoscopic intervention. Patients with alcoholic hepatitis during reopening were younger compared to the lockdown. CONCLUSIONS: Our study demonstrates increased prevalence and severity of ARGLDs amongst younger individuals during the COVID-19 pandemic. This increase started during the lockdown but worsened despite relaxation of restrictions. Systems to increase screening for and treatment of alcohol use disorder as society recovers from the pandemic remain imperative.

Role of Mucosal Protrusion Angle in Discriminating between True and False Masses of the Small Bowel on Video Capsule Endoscopy.

The diagnosis of small-bowel tumors is challenging due to their low incidence, nonspecific presentation, and limitations of traditional endoscopic techniques. In our study, we examined the utility of the mucosal protrusion angle in differentiating between true submucosal masses and bulges of the small bowel on video capsule endoscopy. We retrospectively reviewed video capsule endoscopies of 34 patients who had suspected small-bowel lesions between 2002 and 2017. Mucosal protrusion angles were defined as the angle between the small-bowel protruding lesion and surrounding mucosa and were measured using a protractor placed on a computer screen. We found that 25 patients were found to have true submucosal masses based on pathology and 9 patients had innocent bulges due to extrinsic compression. True submucosal masses had an average measured protrusion angle of 45.7 degrees ± 20.8 whereas innocent bulges had an average protrusion angle of 108.6 degrees ± 16.3 (p < 0.0001; unpaired t-test). Acute angle of protrusion accurately discriminated between true submucosal masses and extrinsic compression bulges on Fisher's exact test (p = 0.0001). Our findings suggest that mucosal protrusion angle is a simple and useful tool for differentiating between true masses and innocent bulges of the small bowel.

Exocrine Pancreatic Insufficiency and Malnutrition in Chronic Pancreatitis: Identification, Treatment, and Consequences.

OBJECTIVES: The purpose of this study was to examine the impact of exocrine pancreatic insufficiency (EPI) on chronic pancreatitis (CP) patients and to identify challenges with its diagnosis and treatment. METHODS: Ninety-one patients with CP diagnosed with endoscopic ultrasound were identified and assessed for symptoms of EPI, fat-soluble vitamin levels, dual-energy x-ray absorptiometry scan T-scores, and treatment with pancreatic enzyme replacement therapy. All patients were also screened with the Malnutrition Universal Screening Test. RESULTS: Exocrine pancreatic insufficiency was diagnosed in 84.6% (77/91) of patients based on symptoms of bloating, steatorrhea, or weight loss. Of these patients, 35.2% (19/54) had vitamin A deficiency, 62.5% (55/88) had vitamin D deficiency, and 17.7% (9/51) had vitamin E deficiency. Either osteopenia or osteoporosis was found in 68.9% (31/45). A medium or higher risk for malnutrition based on Malnutrition Universal Screening Test score of 1 or higher was found in 31.5% (28/89). Malnutrition Universal Screening Test score of 1 or higher was associated with an increased risk for osteopenia and osteoporosis on Fisher's exact test (P = 0.0037). CONCLUSIONS: There is a high prevalence of fat-soluble vitamin deficiencies, osteopathy, and malnutrition in CP patients, which is underestimated due to a lack of effective diagnosis and suboptimal therapies for EPI.

Approach to the Diagnostic Workup and Management of Small Bowel Lesions at a Tertiary Care Center.

BACKGROUND: Small bowel lesions (SBL) are rare, representing diagnostic and management challenges. The purpose of this cross-sectional study was to evaluate diagnostic modalities used and management practices of patients with SBL at an advanced endoscopic referral center. METHODS: We analyzed patients undergoing surgical management for SBL from 2005 to 2015 at a single tertiary care center. Patients were stratified into gastrointestinal bleed/anemia (GIBA) or obstruction/pain (OP). RESULTS: One hundred and twelve patients underwent surgery after presenting with either GIBA (n = 67) or OP (n = 45). The mean age of our study population was 61.8 years and 45% were women. Patients with GIBA were more likely to have chronic or acute-on-chronic symptoms (100% vs 67%) and more often referred from outside hospitals (82 vs. 44%) (p < 0.01). The most common preoperative imaging modalities were video capsule endoscopy (VCE) (96%) for GIBA and computer tomography CT (78%) for OP. Findings on VCE and CT were most frequently concordant with operative findings in GIBA (67%) and OP (54%) patients, respectively. Intraoperatively, visual inspection or palpation of the bowel successfully identified lesions in 71% of patients. When performed in GIBA (n = 26), intraoperative enteroscopy (IE) confirmed or identified lesions in 69% of patients. Almost all (90%) GIBA patients underwent small bowel resections; most were laparoscopic-assisted (93%). Among patients with OP, 58% had a small bowel resection and the majority (81%) were laparoscopic-assisted. Surgical exploration failed to identify lesions in 10% of GIBA patients and 24% of OP patients. Among patients who underwent resections, 20% of GIBA patients had recurrent symptoms compared with 13% of OP patients. CONCLUSION: Management and identification of SBL is governed by presenting symptomatology. Optimal management includes VCE and IE for GIBA and CT scans for OP patients. Comprehensive evaluation may require referral to specialized centers.

Carcinoids and Capsules: A Case Series Highlighting the Utility of Capsule Endoscopy in Patients With Small Bowel Carcinoids.

BACKGROUND: Neuroendocine tumors (NETs) or carcinoids arise at many different sites of the gastrointestinal tract. The small intestine is the most common site for NETs. Diagnosing small bowel carcinoids remains challenging given their non-specific presentations and the overall low incidence of small bowel tumors. Video capsule endoscopy (VCE) has significanly improved our ability to detect small bowel malignancies. We explore the value of VCE in the initial workup and management of a series of small bowel carcinoid patients. METHODS: We retrospectively analyzed adult patients undergoing surgical management for small bowel lesions from July 2005 to September 2015 at a tertiary care center. Patient characteristics, presenting symptomatology, diagnostic workup and surgical management were analyzed among patients with histologically confirmed small bowel carcinoid tumors. RESULTS: Our study identified 16 patients treated surgically for small bowel carcinoids. The majority of patients (87.5%) presented with either occult gastrointestinal bleeding or anemia. Most patients (87.5%) were initially evaluated with various endoscopic and imaging modalities before all ultimately undergoing surgery. Seventy-five percent of patients had a VCE, with 83.3% (10/12) having positive findings that correlated with intraoperative findings compared to 62.5% (5/8) with computed tomography scan, 21.4% (3/14) with colonoscopy, 44% (4/9) with deep enteroscopy, and 0% (0/9) with esophagogastroduodenoscopy (EGD). CONCLUSIONS: In the absence of any contraindications, VCE is an effective endoscopic modality in the diagnostic workup of small bowel NETs. Furthermore, positive VCE findings appear to highly correlate with surgical findings, thus suggesting a valuable role for VCE in the initial surgical assessment of patients with small bowel NETs.

Epigenetics and RNA Processing: Connections to Drought, Salt, and ABA?

There have been great research advances in epigenetics, RNA splicing, and mRNA processing over recent years. In parallel, there have been many advances in abiotic stress and Abscisic Acid (ABA) signaling. Here we overview studies that have examined stress-induced changes in the epigenome and RNA processing as well as cases where disrupting these processes changes the plant response to abiotic stress. We also highlight some examples where specific connections of stress or ABA signaling to epigenetics or RNA processing have been found. By implication, this also points out cases where such mechanistic connections are likely to exist but are yet to be characterized. In the absence of such specific connections to stress signaling, it should be kept in mind that stress sensitivity phenotypes of some epigenetic or RNA processing mutants maybe the result of indirect, pleiotropic effects and thus may perhaps not indicate a direct function in stress acclimation.