The role of protein phosphatase 2A (PP2A) in the unfolded protein response (UPR) of plants.

Protein phosphatase 2A (PP2A) is a key regulator of plant growth and development, but its role in the endoplasmic reticulum (ER) stress response remains elusive. In this study, we investigated the function of PP2A under ER stress using loss-of-function mutants of ROOTS CURL of NAPHTHYLPHTHALAMIC ACID1 (RCN1), a regulatory A1 subunit isoform of Arabidopsis PP2A. RCN1 mutants (rcn1-1 and rcn1-2) exhibited reduced sensitivity to tunicamycin (TM), an inhibitor of N-linked glycosylation and inducer of unfolded protein response (UPR) gene expression, resulting in less severe effects compared to wild-type plants (Ws-2 and Col-0). TM negatively impacted PP2A activity in Col-0 plants but did not significantly affect rcn1-2 plants. Additionally, TM treatment did not influence the transcription levels of the PP2AA1(RCN1), 2, and 3 genes in Col-0 plants. Cantharidin, a PP2A inhibitor, exacerbated growth defects in rcn1 plants and alleviated TM-induced growth inhibition in Ws-2 and Col-0 plants. Furthermore, cantharidin treatment mitigated TM hypersensitivity in ire1a&b and bzip28&60 mutants. These findings suggest that PP2A activity is essential for an efficient UPR in Arabidopsis.

CRISPR/Cas9-Mediated HY5 Gene Editing Reduces Growth Inhibition in Chinese Cabbage (Brassica rapa) under ER Stress.

Various stresses can affect the quality and yield of crops, including vegetables. In this study, CRISPR/Cas9 technology was employed to examine the role of the ELONGATED HYPOCOTYL 5 (HY5) gene in influencing the growth of Chinese cabbage (Brassica rapa). Single guide RNAs (sgRNAs) were designed to target the HY5 gene, and deep-sequencing analysis confirmed the induction of mutations in the bZIP domain of the gene. To investigate the response of Chinese cabbage to endoplasmic reticulum (ER) stress, plants were treated with tunicamycin (TM). Both wild-type and hy5 mutant plants showed increased growth inhibition with increasing TM concentration. However, the hy5 mutant plants displayed less severe growth inhibition compared to the wild type. Using nitroblue tetrazolium (NBT) and 3,3'-diaminobenzidine (DAB) staining methods, we determined the amount of reactive oxygen species (ROS) produced under ER stress conditions, and found that the hy5 mutant plants generated lower levels of ROS compared to the wild type. Under ER stress conditions, the hy5 mutant plants exhibited lower expression levels of UPR- and cell death-related genes than the wild type. These results indicate that CRISPR/Cas9-mediated editing of the HY5 gene can mitigate growth inhibition in Chinese cabbage under stresses, improving the quality and yield of crops.

GABBR2 mutations determine phenotype in rett syndrome and epileptic encephalopathy.

OBJECTIVE: Rett syndrome (RTT) and epileptic encephalopathy (EE) are devastating neurodevelopmental disorders with distinct diagnostic criteria. However, highly heterogeneous and overlapping clinical features often allocate patients into the boundary of the two conditions, complicating accurate diagnosis and appropriate medical interventions. Therefore, we investigated the specific molecular mechanism that allows an understanding of the pathogenesis and relationship of these two conditions. METHODS: We screened novel genetic factors from 34 RTT-like patients without MECP2 mutations, which account for ∼90% of RTT cases, by whole-exome sequencing. The biological function of the discovered variants was assessed in cell culture and Xenopus tropicalis models. RESULTS: We identified a recurring de novo variant in GABAB receptor R2 (GABBR2) that reduces the receptor function, whereas different GABBR2 variants in EE patients possess a more profound effect in reducing receptor activity and are more responsive to agonist rescue in an animal model. INTERPRETATION: GABBR2 is a genetic factor that determines RTT- or EE-like phenotype expression depending on the variant positions. GABBR2-mediated γ-aminobutyric acid signaling is a crucial factor in determining the severity and nature of neurodevelopmental phenotypes. Ann Neurol 2017;82:466-478.

Ligand binding pocket formed by evolutionarily conserved residues in the glucagon-like peptide-1 (GLP-1) receptor core domain.

Glucagon-like peptide-1 (GLP-1) plays a pivotal role in glucose homeostasis through its receptor GLP1R. Due to its multiple beneficial effects, GLP-1 has gained great attention for treatment of type 2 diabetes and obesity. However, little is known about the molecular mechanism underlying the interaction of GLP-1 with the heptahelical core domain of GLP1R conferring high affinity ligand binding and ligand-induced receptor activation. Here, using chimeric and point-mutated GLP1R, we determined that the evolutionarily conserved amino acid residue Arg(380) flanked by hydrophobic Leu(379) and Phe(381) in extracellular loop 3 (ECL3) may have an interaction with Asp(9) and Gly(4) of the GLP-1 peptide. The molecular modeling study showed that Ile(196) at transmembrane helix 2, Met(233) at ECL1, and Asn(302) at ECL2 of GLP1R have contacts with His(1) and Thr(7) of GLP-1. This study may shed light on the mechanism underlying high affinity interaction between the ligand and the binding pocket that is formed by these conserved residues in the GLP1R core domain.

Clinical Outcomes of Surgery After Neoadjuvant Chemotherapy in Locally Advanced Pancreatic Ductal Adenocarcinoma.

Purpose: Clinical outcomes of surgery after neoadjuvant chemotherapy have not been investigated for locally advanced pancreatic cancer (LAPC), despite well-established outcomes in borderline resectable pancreatic cancer (BRPC). This study aimed to investigate the clinical outcomes of patients with LAPC who underwent curative resection following neoadjuvant chemotherapy. Materials and Methods: We retrospectively reviewed the records of patients diagnosed with pancreatic adenocarcinoma between January 2017 and December 2020. Results: Among 1,358 patients, 260 underwent surgery following neoadjuvant chemotherapy. Among 356 LAPC patients, 98 (27.5%) and 147 (35.1%) of 418 BRPC patients underwent surgery after neoadjuvant chemotherapy. Compared to resectable pancreatic cancer (resectable PC) with upfront surgery, both LAPC and BRPC exhibited higher rates of venous resection (28.6% vs. 49.0% vs. 4.0%), arterial resection (30.6% vs. 6.8% vs. 0.5%) and greater estimated blood loss (260.5 vs. 213.1 vs. 70.4 mL). However, hospital stay, readmission rates and postoperative pancreatic fistula rates (Grade B or C) did not differ significantly between LAPC, BRPC, and resectable PC. Overall and relapse-free survival did not differ significantly between LAPC and BRPC patients. The median overall survival was 37.3 months for LAPC and 37.0 months for BRPC. The median relapse-free survival was 22.7 months for LAPC and 26.0 months for BRPC. Conclusion: Overall survival time and postoperative complications in LAPC patients who underwent curative resection following neoadjuvant chemotherapy showed similar results to those of BRPC patients. Further research is needed to identify specific sub-populations of LAPC patients who benefit most from conversion surgery and to minimize postoperative complications.

Computed tomography-based vascular burden index as a predictor of vascular resection and pathological vascular invasion in pancreatic cancer with neo-adjuvant chemotherapy.

BACKGROUND: Determination of vessel resection in patients with pancreatectomy after neo-adjuvant chemotherapy remains controversial. The recently introduced computed tomography-based vascular burden index presents a potential solution to this challenge. This study aimed to evaluate the model performance for the prediction of vascular resection and pathological invasion. METHODS: Patients who underwent surgery after neo-adjuvant chemotherapy were included. Two independent reviewers measured the vascular tumour burden index around the adjacent artery (AVBI), and vein (VVBI). The area under the curve was compared to assess the predictive capacity of vascular burden index values and their changes for vascular resection and pathological vascular invasion. RESULTS: Among 252 patients, 179 and 73 had borderline resectable and locally advanced pancreatic cancer, respectively. Concurrent vessel resection and pathological vascular invasion were observed in 121 (48.0 %) and 42 (16.6 %) patients, respectively. In all patients, the VVBI (area under the curve: 0.872) and AVBI (0.911) after neo-adjuvant therapy significantly predicted vessel resection. In patients with vascular resection, the VVBI after neo-adjuvant chemotherapy (0.752) and delta value of the AVBI (0.706) demonstrated better performance for predicting pathological invasion of the resected vein. The regression of the AVBI and VVBI was an independent prognostic factor for survival (hazard ratio: 0.54, 95 % confidence interval: 0.34-0.85; P = 0.009) CONCLUSIONS: Regressed VVBI on serial computed tomography scans is useful for predicting vein resection and pathological venous invasion before surgery. The delta value of the AVBI may therefore be helpful for predicting pathological arterial invasion after neo-adjuvant chemotherapy.

IRE1 is implicated in protein synthesis regulation under ER stress conditions in plants.

The unfolded protein response (UPR) is a crucial cellular mechanism for maintaining protein folding homeostasis during endoplasmic reticulum (ER) stress. In this study, the role of IRE1, a key component of the UPR, was investigated in protein translation regulation under ER stress conditions in Arabidopsis. We discovered that the loss of IRE1A and IRE1B leads to diminished protein translation, indicating a significant role for IRE1 in this process. However, this regulation was not solely dependent on the interaction with bZIP60, a key transcription factor in the UPR. Interestingly, while chemical chaperones TUDCA and PBA effectively alleviated the translation inhibition observed in ire1a ire1b mutants, this effect was more pronounced than the mitigation observed from suppressing GCN2 expression or introducing a non-phosphorylatable eIF2α variant. Additionally, the kinase and ribonuclease activities of IRE1B were demonstrated to be crucial for plant adaptation and protein synthesis regulation under ER stress conditions. Overall, this study not only highlights the complex regulatory mechanisms of IRE1 in plant ER stress responses but also provides insights into its multifaceted roles in protein translation regulation.

Microbial changes in stool, saliva, serum, and urine before and after anti-TNF-α therapy in patients with inflammatory bowel diseases.

Inflammatory bowel diseases (IBD), including Crohn's disease and ulcerative colitis, are chronic immune-mediated intestinal inflammatory disorders associated with microbial dysbiosis at multiple sites, particularly the gut. Anti-tumor necrosis factor-α (TNF-α) agents are important treatments for IBD. We investigated whether microbiome changes at multiple sites can predict the effectiveness of such treatment in IBD. Stool, saliva, serum, and urine biosamples were collected from 19 IBD patients before (V1) and 3 months after (V2) anti-TNF-α treatment, and 19 healthy subjects (control). Microbiota analysis was performed using extracellular vesicles (EVs; all four sample types) and next-generation sequencing (NGS; stool and saliva). The stool, using NGS analysis, was the only sample type in which α-diversity differed significantly between the IBD and control groups at V1 and V2. Relative to non-responders, responders to anti-TNF-α treatment had significantly higher levels of Firmicutes (phylum), Clostridia (class), and Ruminococcaceae (family) in V1 stool, and Prevotella in V1 saliva. Non-responders had significantly higher V2 serum and urine levels of Lachnospiraceae than responders. Finally, Acidovorax caeni was detected in all V1 sample types in responders, but was not detected in non-responders. Microbiome changes at multiple sites may predict the effectiveness of anti-TNF-α treatment in IBD, warranting further research.

Clinicopathological Characteristics and Surgical Outcomes of Crohn Disease-Associated Colorectal Malignancy.

PURPOSE: Carcinoma arising from Crohn disease (CD) is rare, and there is no clear guidance on how to properly screen for at-risk patients and choose appropriate care. This study aimed to evaluate the clinicopathological characteristics, treatment, and oncologic outcomes of CD patients diagnosed with colorectal cancer (CRC). METHODS: Using medical records, we retrospectively enrolled a single-center cohort of 823 patients who underwent abdominal surgery for CD between January 2006 and December 2015. CD-associated CRC patients included those with adenocarcinoma, lymphoma, or neuroendocrine tumors of the colon and rectum. RESULTS: Nineteen patients (2.3%) underwent abdominal surgery to treat CD-associated CRC. The mean duration of CD in the CD-associated CRC group was significantly longer than that in the benign CD group (124.7 ± 77.7 months vs. 68.9 ± 60.2 months, P = 0.006). The CD-associated CRC group included a higher proportion of patients with a history of perianal disease (73.7% vs. 50.2%, P = 0.035) and colonic location (47.4% vs. 6.5%, P = 0.001). Among 19 CD-associated CRC patients, 17 (89.5%) were diagnosed with adenocarcinoma, and of the 17 cases, 15 (88.2%) were rectal adenocarcinoma. On multivariable analyses for developing CRC, only colonic location was a risk factor (relative risk, 7.735; 95% confidence interval, 2.862-20.903; P = 0.001). CONCLUSION: Colorectal malignancy is rare among CD patients, even among patients who undergo abdominal surgery. Rectal adenocarcinoma accounted for most of the CRC, and colonic location was a risk factor for developing CRC.

Exploring the molecular structures that confer ligand selectivity for galanin type II and III receptors.

Galanin receptors (GALRs) belong to the superfamily of G-protein coupled receptors. The three GALR subtypes (GALR1, GALR2, and GALR3) are activated by their endogenous ligands: spexin (SPX) and galanin (GAL). The synthetic SPX-based GALR2-specific agonist, SG2A, plays a dual role in the regulation of appetite and depression-like behaviors. Little is known, however, about the molecular interaction between GALR2 and SG2A. Using site-directed mutagenesis and domain swapping between GALR2 and GALR3, we identified residues in GALR2 that promote interaction with SG2A and residues in GALR3 that inhibit interaction with SG2A. In particular, Phe103, Phe106, and His110 in the transmembrane helix 3 (TM3) domain; Val193, Phe194, and Ser195 in the TM5 domain; and Leu273 in the extracellular loop 3 (ECL3) domain of GALR2 provide favorable interactions with the Asn5, Ala7, Phe11, and Pro13 residues of SG2A. Our results explain how SG2A achieves selective interaction with GALR2 and inhibits interaction with GALR3. The results described here can be used broadly for in silico virtual screening of small molecules for the development of GALR subtype-specific agonists and/or antagonists.

Establishment of a NanoBiT-Based Cytosolic Ca2+ Sensor by Optimizing Calmodulin-Binding Motif and Protein Expression Levels.

Cytosolic Ca2+ levels ([Ca2+]c) change dynamically in response to inducers, repressors, and physiological conditions, and aberrant [Ca2+]c concentration regulation is associated with cancer, heart failure, and diabetes. Therefore, [Ca2+]c is considered as a good indicator of physiological and pathological cellular responses, and is a crucial biomarker for drug discovery. A genetically encoded calcium indicator (GECI) was recently developed to measure [Ca2+]c in single cells and animal models. GECI have some advantages over chemically synthesized indicators, although they also have some drawbacks such as poor signal-to-noise ratio (SNR), low positive signal, delayed response, artifactual responses due to protein overexpression, and expensive detection equipment. Here, we developed an indicator based on interactions between Ca2+-loaded calmodulin and target proteins, and generated an innovative GECI sensor using split nano-luciferase (Nluc) fragments to detect changes in [Ca2+]c. Stimulation-dependent luciferase activities were optimized by combining large and small subunits of Nluc binary technology (NanoBiT, LgBiT:SmBiT) fusion proteins and regulating the receptor expression levels. We constructed the binary [Ca2+]c sensors using a multicistronic expression system in a single vector linked via the internal ribosome entry site (IRES), and examined the detection efficiencies. Promoter optimization studies indicated that promoter-dependent protein expression levels were crucial to optimize SNR and sensitivity. This novel [Ca2+]c assay has high SNR and sensitivity, is easy to use, suitable for high-throughput assays, and may be useful to detect [Ca2+]c in single cells and animal models.

Investigation on the Intrinsic Potential of Using Potassium Chloride for Partially Replacing NaCl in Kimchi and the Effect of Information on Consumer Acceptance of Sodium-Reduced Kimchi According to Age.

The high sodium content of kimchi is a contradicting factor from its fame as a healthy food. With the aim of reducing the sodium content of kimchi, the objective of this study was to understand the effect of providing "sodium-reduced" information on the acceptance of kimchi according to the age of consumption. Six sodium-reduced kimchi samples, prepared with different percentages of sodium reduction (25% and 50%) and potassium chloride concentration (none, 0.47%, and 0.93%), were compared to control kimchi (2.0% w/v NaCl). Sensory characterization of the samples was obtained using descriptive analysis. A total of 167 kimchi consumers with balanced proportion of the young (below 40) and the old (above 40) evaluated seven kimchi samples in either of the two conditions: blind testing condition or informed testing condition where each of the samples was provided with a label that informed about "sodium reduction percentage" and "whether a salt replacer was used or not." The results showed that in terms of healthiness perception, Korean female consumers believed that kimchi with a high sodium reduction rate would contribute to health in general, though an unfavorable notion of using a salt replacer was also observed. Also, the results suggested that promoting information about sodium reduction in kimchi would generally increase consumer acceptance. However, this phenomenon was influenced not only by the sample for which the information was provided, but also by the age of consumers with different health interests and kimchi experience. PRACTICAL APPLICATION: The findings of this study showed simply reducing sodium and promoting it with a health claim showed limitation in achieving a high level of sodium reduction, such as a 50% reduction rate, which implied the importance of using supplementary material such as potassium chloride that can fulfill the missing saltiness and flavors of the original product. Promotion of "sodium-reduced" claims in kimchi generally results in increased consumer acceptance. However, the effectiveness of the information was dependent on which sample was provided and the age of the consumers, among whom health interests and kimchi experience differ.

Monitoring GPCR-ß-arrestin1/2 Interactions in Real Time Living Systems to Accelerate Drug Discovery.

Interactions between G-protein coupled receptors (GPCRs) and ß-arrestins are vital processes with physiological implications of great importance. Currently, the characterization of novel drugs towards their interactions with ß-arrestins and other cytosolic proteins is extremely valuable in the field of GPCR drug discovery particularly during the study of GPCR biased agonism. Here, we show the application of a novel structural complementation assay to accurately monitor receptor-ß-arrestin interactions in real time living systems. This method is simple, accurate and can be easily extended to any GPCR of interest and also it has the advantage that it overcomes unspecific interactions due to the presence of a low expression promoter present in each vector system. This structural complementation assay provides key features that allow an accurate and precise monitoring of receptor-ß-arrestin interactions, making it suitable in the study of biased agonism of any GPCR system as well as GPCR c-terminus 'phosphorylation codes' written by different GPCR-kinases (GRKs) and post-translational modifications of arrestins that stabilize or destabilize the receptor-ß-arrestin complex.

Spexin-Based Galanin Receptor Type 2 Agonist for Comorbid Mood Disorders and Abnormal Body Weight.

Despite the established comorbidity between mood disorders and abnormal eating behaviors, the underlying molecular mechanism and therapeutics remain to be resolved. Here, we show that a spexin-based galanin receptor type 2 agonist (SG2A) simultaneously normalized mood behaviors and body weight in corticosterone pellet-implanted (CORTI) mice, which are underweight and exhibit signs of anhedonia, increased anxiety, and depression. Administration of SG2A into the lateral ventricle produced antidepressive and anxiolytic effects in CORTI mice. Additionally, SG2A led to a recovery of body weight in CORTI mice while it induced significant weight loss in normal mice. In Pavlovian fear-conditioned mice, SG2A decreased contextual and auditory fear memory consolidation but accelerated the extinction of acquired fear memory without altering innate fear and recognition memory. The main action sites of SG2A in the brain may include serotonergic neurons in the dorsal raphe nucleus for mood control, and proopiomelanocortin/corticotropin-releasing hormone neurons in the hypothalamus for appetite and body weight control. Furthermore, intranasal administration of SG2A exerted the same anxiolytic and antidepressant-like effects and decreased food intake and body weight in a dose-dependent manner. Altogether, these results indicate that SG2A holds promise as a clinical treatment for patients with comorbid mood disorders and abnormal appetite/body weight.

Conformational signatures in ß-arrestin2 reveal natural biased agonism at a G-protein-coupled receptor.

Discovery of biased ligands and receptor mutants allows characterization of G-protein- and ß-arrestin-mediated signaling mechanisms of G-protein-coupled receptors (GPCRs). However, the structural mechanisms underlying biased agonism remain unclear for many GPCRs. We show that while Galanin induces the activation of the galanin receptor 2 (Galr2) that leads to a robust stimulation toward Gαq-protein and ß-arrestin1/2, an alternative ligand Spexin and its analog have biased agonism toward G-protein signaling relative to Galanin. We used intramolecular fluorescein arsenical hairpin bioluminescence resonance energy transfer-based biosensors of ß-arrestin2 combined with NanoBit technology to measure ß-arrestin2-Galr2 interactions in real-time living systems. We found that Spexin and Galanin induce specific active conformations of Galr2, which may lead to different internalization rates of the receptor as well as different signaling outputs. This work represents an additional pharmacological evidence of endogenous G-protein-biased agonism at a GPCR.

Development of Spexin-based Human Galanin Receptor Type II-Specific Agonists with Increased Stability in Serum and Anxiolytic Effect in Mice.

The novel neuropeptide spexin (SPX) was discovered to activate galanin receptor 2 (GALR2) and 3 (GALR3) but not galanin receptor 1 (GALR1). Although GALR2 is known to display a function, particularly in anxiety, depression, and appetite regulation, the further determination of its function would benefit from a more stable and selective agonist that acts only at GALR2. In the present study, we developed a GALR2-specific agonist with increased stability in serum. As galanin (GAL) showed a low affinity to GALR3, the residues in SPX were replaced with those in GAL, revealing that particular mutations such as Gln5 → Asn, Met7 → Ala, Lys11 → Phe, and Ala13 → Pro significantly decreased potencies toward GALR3 but not toward GALR2. Quadruple (Qu) mutation of these residues still retained potency to GALR2 but totally abolished the potency to both GALR3 and GALR1. The first amino acid modifications or D-Asn1 substitution significantly increased the stability when they are incubated in 100% fetal bovine serum. Intracerebroventricular administration of the mutant peptide with D-Asn1 and quadruple substitution (dN1-Qu) exhibited an anxiolytic effect in mice. Taken together, the GALR2-specific agonist with increased stability can greatly help delineation of GALR2-mediated functions and be very useful for treatments of anxiety disorder.