Bioengineering secreted proteases convert divergent Rcr3 orthologs and paralogs into extracellular immune co-receptors.

Secreted immune proteases Rcr3 (Required for Cladosporium resistance-3) and Pip1 (Phytophthora- inhibited protease-1) of tomato (Solanum lycopersicum) are both inhibited by Avr2 from the fungal plant pathogen Cladosporium fulvum. However, only Rcr3 acts as a decoy co-receptor that detects Avr2 in the presence of the Cf-2 immune receptor. Here, we identified crucial residues in tomato Rcr3 that are required for Cf-2-mediated signalling and bioengineered various proteases to trigger Avr2/Cf-2-dependent immunity. Despite substantial divergence in Rcr3 orthologs from eggplant (Solanum melongena) and tobacco (Nicotiana spp.), minimal alterations were sufficient to trigger Avr2/Cf-2-mediated immune signalling. By contrast, tomato Pip1 was bioengineered with 16 Rcr3-specific residues to initiate Avr2/Cf-2-triggered immune signalling. These residues cluster on one side of the protein next to the substrate-binding groove, indicating a potential Cf-2 interaction site. Our findings also revealed that Rcr3 and Pip1 have distinct substrate preferences determined by two variant residues, and that both are suboptimal for binding Avr2. This study advances our understanding of Avr2 perception and opens avenues to bioengineer proteases to broaden pathogen recognition in other crops.

High Incidence of Appendiceal Neoplasms in the Elderly: A Critical Concern for Non-Surgical Treatment.

OBJECTIVE: Appendiceal neoplasms (ANs) are rare tumors that are often discovered incidentally during histopathological examinations. The increasing incidence of ANs is a critical issue in the non-operative management of acute appendicitis. This study aimed to document the temporal trends over a 12-year period by analyzing the clinical presentation, imaging findings, and histopathological features of ANs. SUBJECTS AND METHODS: Health records of patients who underwent appendectomy from 2011 to 2022 were examined. Demographic and clinical data, laboratory results, imaging findings, and histopathological features were documented. The characteristics of both ANs and non-neoplastic cases were evaluated. RESULTS: A total of 22,304 cases were identified, of which 330 (1.5%) were diagnosed with ANs. The odds ratio for ANs increased with age, with the highest odds ratio observed in patients aged 70 or older. Receiver Operating Characteristic analysis showed that age and appendiceal diameter were significant predictors of ANs. An optimal age cut-off point of 28.5 years was determined, yielding a sensitivity of 72% and a specificity of 64%. For appendiceal diameter, the optimal cut-off was found to be 9.5 mm, exhibiting a sensitivity of 77% and a specificity of 56%. CONCLUSION: Although the incidence of ANs remains relatively low, a steady increase has been observed over the past decade. The increasing rate of ANs raises concerns regarding non-surgical management options. The results of this study highlight the importance of considering ANs as a potential diagnosis in older patients and in patients with an appendix diameter greater than 9.5 mm. These findings may have implications for treatment and management.

Phytocystatin 6 is a context-dependent, tight-binding inhibitor of Arabidopsis thaliana legumain isoform ß.

Plant legumains are crucial for processing seed storage proteins and are critical regulators of plant programmed cell death. Although research on legumains boosted recently, little is known about their activity regulation. In our study, we used pull-down experiments to identify AtCYT6 as a natural inhibitor of legumain isoform ß (AtLEGß) in Arabidopsis thaliana. Biochemical analysis revealed that AtCYT6 inhibits both AtLEGß and papain-like cysteine proteases through two separate cystatin domains. The N-terminal domain inhibits papain-like proteases, while the C-terminal domain inhibits AtLEGß. Furthermore, we showed that AtCYT6 interacts with legumain in a substrate-like manner, facilitated by a conserved asparagine residue in its reactive center loop. Complex formation was additionally stabilized by charged exosite interactions, contributing to pH-dependent inhibition. Processing of AtCYT6 by AtLEGß suggests a context-specific regulatory mechanism with implications for plant physiology, development, and programmed cell death. These findings enhance our understanding of AtLEGß regulation and its broader physiological significance.

An integrated organoid omics map extends modeling potential of kidney disease.

Kidney organoids are a promising model to study kidney disease, but their use is constrained by limited knowledge of their functional protein expression profile. Here, we define the organoid proteome and transcriptome trajectories over culture duration and upon exposure to TNFα, a cytokine stressor. Older organoids increase deposition of extracellular matrix but decrease expression of glomerular proteins. Single cell transcriptome integration reveals that most proteome changes localize to podocytes, tubular and stromal cells. TNFα treatment of organoids results in 322 differentially expressed proteins, including cytokines and complement components. Transcript expression of these 322 proteins is significantly higher in individuals with poorer clinical outcomes in proteinuric kidney disease. Key TNFα-associated protein (C3 and VCAM1) expression is increased in both human tubular and organoid kidney cell populations, highlighting the potential for organoids to advance biomarker development. By integrating kidney organoid omic layers, incorporating a disease-relevant cytokine stressor and comparing with human data, we provide crucial evidence for the functional relevance of the kidney organoid model to human kidney disease.

Clinicopathologic Features and Prognostic Significance of Immunohistochemistry and In Situ Hybridization Based Molecular Classification in Gastric Carcinoma.

BACKGROUND/AIM: Gastric carcinoma (GC) is a highly heterogeneous disease with many subtypes that have different morphologic and molecular characteristics. In the current study, we analyzed immunohistochemical (IHC) and in situ hybridization (ISH) features of GCs and evaluated their association with prognosis and clinicopathological features. MATERIALS AND METHODS: Three hundred cases analyzed by IHC and ISH for microsatellite stability, p53, e-cadherin, HER2, PD-L1 expression, and Epstein-Barr virus (EBV) status. Cases were classified into five subgroups based on expression profile. The relationships between subgroups, clinicopathological features, and survival were determined. RESULTS: Ten (3.3%) cases were classified as EBV-associated, 45 (15%) as microsatellite instable (MSI), 73 (24.3%) as EBV-/microsatellite-stable (MSS)/epithelial-mesenchymal-transformation (EMT)-like, 75 (25%) as EBV-/MSS/ non-EMT-like/p53+, and 97 (32.3%) as EBV-/MSS/non-EMT-like/p53-. The MSI subtype had the best overall survival (OS). In contrast, the EBV-/MSS/EMT-like subtype had the poorest OS. The MSI subtype was also related with old age of the patient and antrum-corpus localized tumors, whereas the EBV-/MSS/EMT-like was associated with young age, larger tumor size, and advanced stage presentation. PD-L1 positivity is highly correlated with MSI and EBV-associated subtypes. CONCLUSION: Our data demonstrated a link between IHC/ISH characteristics of GC and clinical outcomes. IHC/ISH based molecular classification may be helpful in predicting the survival.

Retrospective analysis of the appendiceal neoplasms: sampling technique may influence neoplasm detection.

BACKGROUND AND AIMS: Appendiceal neoplasms are uncommon entities that are usually determined incidentally during the histopathological examination. Different techniques used for the macroscopic sampling of appendectomy material may affect the determinating neoplasms. MATERIALS AND METHODS: H&E-stained slides of 1280 cases who underwent appendectomy between 2013 and 2018 were reviewed retrospectively for histopathological features. RESULTS: Neoplasms were determined in 28 cases (3.09%); 1 lesion was observed in the proximal part of the appendix, 1 covering the entire length from proximal to distal and 26 in the distal part. In the 26 cases that observed in the distal part, the lesion was seen on both sides of the longitudinal section of the distal appendix in 20 cases, while it was seen on only one distal longitudinal section in the remaining 6 cases. CONCLUSION: The vast majority of appendiceal neoplasms are seen in the distal part of the appendix, and, in some cases, neoplasms might be seen on only one side of the distal section. Sampling only one-half of the distal part of the appendix, where tumours are most often observed, could result in some neoplasms being missed. Therefore, sampling the whole distal part would be more beneficial to determine small diameter tumours that do not create macroscopic findings.

Safety and effectiveness of a posterior approach alone for surgical treatment of sacral-presacral tumors.

Aim: We aimed to examine the safety and effectiveness of a posterior approach alone in the surgical treatment of sacral-presacral tumors. In addition, we investigate factors that determine the selection of a posterior approach alone. Materials and Methods: Patients with sacral-presacral tumors who underwent surgery in our institution between 2007 and 2019 were examined in this study. Data regarding patient age, gender, tumor size (>6 cm and <6 cm), tumor localization (below or above S1), tumor pathology (benign or malignant), surgical approach (anterior alone, posterior alone, or combined), and extent of resection were recorded. The Spearman's correlation analyses were conducted between surgical approach and tumor size, localization, and pathology. Factors influencing the extent of resection were also examined. Results: Complete tumor resection was achieved in 18 of 20 patients. A posterior approach alone was used in 16. No strong or significant relation was detected between surgical approach and tumor size (r = 0.218; P = 0.355). There was no strong or significant relationship between surgical approach and tumor localization (r = 0.145; P = 0.541) or tumor pathology (r = 0.250; P = 0.288). Tumor size, localization, and pathology were not independent factors that determined surgical approach. The only significant independent determining factor for incomplete resection was tumor pathology (r = 0.688; P = 0.001). Conclusion: A posterior approach is safe and effective in the surgical treatment of sacral-presacral tumors independent of tumor localization, size, or pathology and is a feasible first-line treatment option.

VPS34-dependent control of apical membrane function of proximal tubule cells and nutrient recovery by the kidney.

The lipid kinase VPS34 orchestrates autophagy, endocytosis, and metabolism and is implicated in cancer and metabolic disease. The proximal tubule in the kidney is a key metabolic organ that controls reabsorption of nutrients such as fatty acids, amino acids, sugars, and proteins. Here, by combining metabolomics, proteomics, and phosphoproteomics analyses with functional and superresolution imaging assays of mice with an inducible deficiency in proximal tubular cells, we revealed that VPS34 controlled the metabolome of the proximal tubule. In addition to inhibiting pinocytosis and autophagy, VPS34 depletion induced membrane exocytosis and reduced the abundance of the retromer complex necessary for proper membrane recycling and lipid retention, leading to a loss of fuel and biomass. Integration of omics data into a kidney cell metabolomic model demonstrated that VPS34 deficiency increased ß-oxidation, reduced gluconeogenesis, and enhanced the use of glutamine for energy consumption. Furthermore, the omics datasets revealed that VPS34 depletion triggered an antiviral response that included a decrease in the abundance of apically localized virus receptors such as ACE2. VPS34 inhibition abrogated SARS-CoV-2 infection in human kidney organoids and cultured proximal tubule cells in a glutamine-dependent manner. Thus, our results demonstrate that VPS34 adjusts endocytosis, nutrient transport, autophagy, and antiviral responses in proximal tubule cells in the kidney.

Multiple Endoclip Retraction Technique (MERT) in Laparoscopic Burch.

Background and Objectives: The multiple endoclip retraction technique (MERT) was developed based on our experience with suturing renal parenchyma in laparoscopic partial nephrectomy. In this study we prospectively evaluated the one-year results of cases treated by transperitoneal laparoscopic Burch with the MERT. Methods: The patients were evaluated with stress test, one-hour pad test, and were requested to complete the International Continence Society Incontinence Questionnaire short form (ICIQ-SF) in the postoperative period. The follow-up periods were postoperative 3, 6, and 12 months. Results: The primary outcome of this study was surgical success, defined as being cured of stress urinary incontinence (SUI) (no symptoms), experiencing improved symptoms of SUI in ICIQ-SF and negative stress test, and less than 2g urine leakage in a one-hour pad test.No statistically significant difference was found in terms of age, number of children, and body mass index (BMI) in patients according to the results of the 12 month postoperative stress test. We found statistically significant improvements at all control months in terms of stress test and pad weight. ICIQ-SF results showed a significant decrease at three months in patients who recovered after the operation. This rate has not changed in following control months. However, there was no statistical change in ICIQ-SF values in patients who did not recover after the operation. Conclusion: MERT seems to be one of the safe and effective modifications in the management of SUI with good one year outcomes when performed by suitably trained experienced surgeons.

Sensitive Plant N-Terminome Profiling with HUNTER.

Protein N-termini provide unique and distinguishing information on proteolytically processed or N-terminally modified proteoforms. Also splicing, use of alternative translation initiation sites, and a variety of co- and post-translational N-terminal modifications generate distinct proteoforms that are unambiguously identified by their N-termini. However, N-terminal peptides are only a small fraction among all peptides generated in a shotgun proteome digest, are often of low stoichiometric abundance, and therefore require enrichment. Various protocols for enrichment of N-terminal peptides have been established and successfully been used for protease substrate discovery and profiling of N-terminal modification, but often require large amounts of proteome. We have recently established the High-efficiency Undecanal-based N-Termini EnRichment (HUNTER) as a fast and sensitive method to enable enrichment of protein N-termini from limited sample sources with as little as a few microgram proteome. Here we present our current HUNTER protocol for sensitive plant N-terminome profiling, including sample preparation, enrichment of N-terminal peptides, and mass spectrometry data analysis.

A User Guide to Validation, Annotation, and Evaluation of N-Terminome Datasets with MANTI.

A large variety of enrichment procedures for protein N-termini have been developed to trace protease activity and determine precise cleavage sites, as well as other N-terminal protein modifications. Typically, enriched N-terminal peptides are identified by tandem mass spectrometry using standard database search engines, in many cases the popular MaxQuant software package. MaxQuant Advanced N-termini Interpreter (MANTI) is a software package that helps to validate, annotate, and visualize peptide identifications in N-termini datasets in a rapid and straightforward manner. Usage of MANTI and especially its graphical interface Yogurtlu MANTI in detail are described to enable users to take full advantage of the software package and the multitude of options it has to offer.

Lipoblastoma of scalp in a 4-month-old patient: an extremely rare case report and review of the literature.

Lipoblastoma is a rare benign tumor originating from adipose tissue, usually seen in infancy and early childhood. Here, we present a case of scalp lipoblastoma in a 4-month-old that we treated surgically and review the literature. Although lipoblastomas may be seen in various locations during infancy and early childhood, rarely, they can also develop in the scalp.

Evaluation of BMP-2, VEGF, and Vitamin D Receptor Levels in the Ligamentum Flavum of Patients with Lumbar Spinal Stenosis and Disc Herniation.

AIM: To histopathologically evaluate and compare bone morphogenetic protein (BMP)-2, vascular endothelial growth factor (VEGF), and vitamin D receptor (VDR) levels in the ligamentum flavum (LF) of patients with lumbar spinal stenosis (LSS) and lumbar disc herniation (LDH). MATERIAL AND METHODS: Surgical specimens of the LF in 25 patients who underwent surgery for LDH and 25 patients who underwent surgery for LSS were examined histopathologically. The prevalence and severity of BMP-2, VEGF, and VDR immunoreactivity were evaluated to create histoscores (prevalence × severity), which were compared between groups. RESULTS: The mean BMP-2 histoscore was similar in both groups. In the LSS group, the mean VEGF histoscore was significantly higher and the mean VDR histoscore was significantly lower. CONCLUSION: Elevated VEGF and decreased VDR levels in the LF in LSS are associated with more intense inflammation and chronic process of the disease. The prominent expression of BMP-2 in the LF in both diseases suggests that BMP-2 might be affected by inflammation regardless of chronic pressure and degeneration.

Association of endocan expression with clinicopathological prognostic parameters in breast carcinoma.

BACKGROUND: Breast cancer (BC) is a common malignancy in women. Some molecules, including endocan, are still under investigation as potential prognostic factors in BC. OBJECTIVE: In the present study, we aimed to determine the relationship between endocan expression and clinicopathological prognostic parameters in BC. METHODS: Two hundred and fifty-five patients diagnosed with BC were included in the present study. The immuno-reactivity scoring (IRS) system was used to reveal the tissue endocan expression levels. RESULTS: We found that endocan expression is associated with tumor necrosis, tumor size, and the presence of lobular carcinoma in situ (LCIS) in BC. There was no relationship between endocan expression and survival as well as other clinicopathological prognostic parameters. CONCLUSION: Endocan overexpression in BC is associated with some prognostic parameters, including tumor necrosis, tumor size, and the presence of LCIS. Further studies with larger series are needed to reveal the clinical and therapeutic implications of endocan in BC.

Endoscopic Third Ventriculostomy for Obstructive Hydrocephalus and Ventriculocystostomy for Intraventricular Arachnoid Cysts.

OBJECTIVE: To evaluate and discuss the outcomes of a combination of ventriculocystostomy (VC) and endoscopic third ventriculostomy (ETV) for obstructive hydrocephalus (HCP) due to ventricular/cisternal arachnoid cysts, and only ETV for obstructive HCP due to different etiologies. METHODS: We retrospectively reviewed all 40 symptomatic patients (aged 4 months - 61 years) of obstructive HCP treated by ETV or VC+ETV during October 2014 - April 2019. VC+ETV was performed in 7 patients with intraventricular/cisternal arachnoid cyst and obstructive HCP. Only ETV was performed in 33 patients with obstructive HCP due to other etiologies. RESULTS: Successful ETV or VC+ETV surgery was performed in 35 patients. The procedure failed in 5 patients aged 90 percentile at the time of surgery. Another 5 patients aged 90 percentile).

MANTI: Automated Annotation of Protein N-Termini for Rapid Interpretation of N-Terminome Data Sets.

Site-specific proteolytic processing is an important, irreversible post-translational protein modification with implications in many diseases. Enrichment of protein N-terminal peptides followed by mass spectrometry-based identification and quantification enables proteome-wide characterization of proteolytic processes and protease substrates but is challenged by the lack of specific annotation tools. A common problem is, for example, ambiguous matches of identified peptides to multiple protein entries in the databases used for identification. We developed MaxQuant Advanced N-termini Interpreter (MANTI), a standalone Perl software with an optional graphical user interface that validates and annotates N-terminal peptides identified by database searches with the popular MaxQuant software package by integrating information from multiple data sources. MANTI utilizes diverse annotation information in a multistep decision process to assign a conservative preferred protein entry for each N-terminal peptide, enabling automated classification according to the likely origin and determines significant changes in N-terminal peptide abundance. Auxiliary R scripts included in the software package summarize and visualize key aspects of the data. To showcase the utility of MANTI, we generated two large-scale TAILS N-terminome data sets from two different animal models of chemically and genetically induced kidney disease, puromycin adenonucleoside-treated rats (PAN), and heterozygous Wilms Tumor protein 1 mice (WT1). MANTI enabled rapid validation and autonomous annotation of >10 000 identified terminal peptides, revealing novel proteolytic proteoforms in 905 and 644 proteins, respectively. Quantitative analysis indicated that proteolytic activities with similar sequence specificity are involved in the pathogenesis of kidney injury and proteinuria in both models, whereas coagulation processes and complement activation were specifically induced after chemical injury.

Host apoplastic cysteine protease activity is suppressed during the mutualistic association of Lolium perenne and Epichloë festucae.

Plants secrete various defence-related proteins into the apoplast, including proteases. Papain-like cysteine proteases (PLCPs) are central components of the plant immune system. To overcome plant immunity and successfully colonize their hosts, several plant pathogens secrete effector proteins inhibiting plant PLCPs. We hypothesized that not only pathogens, but also mutualistic microorganisms interfere with PLCP-meditated plant defences to maintain endophytic colonization with their hosts. Epichloë festucae forms mutualistic associations with cool season grasses and produces a range of secondary metabolites that protect the host against herbivores. In this study, we performed a genome-wide identification of Lolium perenne PLCPs, analysed their evolutionary relationship, and classified them into nine PLCP subfamilies. Using activity-based protein profiling, we identified four active PLCPs in the apoplast of L. perenne leaves that are inhibited during endophyte interactions. We characterized the L. perenne cystatin LpCys1 for its inhibitory capacity against ryegrass PLCPs. LpCys1 abundance is not altered during the mutualistic interaction and it mainly inhibits LpCP2. However, since the activity of other L. perenne PLCPs is not sensitive to LpCys1, we propose that additional inhibitors, likely of fungal origin, are involved in the suppression of apoplastic PLCPs during E. festucae infection.

Comparison of the Effects of Four Treatment Techniques Commonly Used in Ureteral Stone Treatment on Patients' Daily Physical Functioning: An Observational Randomized-Controlled Study.

Objective: To investigate the effect of four different techniques used in the treatment of ureteral stones on patients' daily physical functioning (PF) and quality of life (QoL). Materials and Methods: Patients who underwent ureterorenoscopy (URS)-with or without Double-J stenting (DJS)-and extracorporeal shock wave lithotripsy (SWL) were divided into four groups: Group I: SWL (n = 29), Group II: URS (n = 43), Group III: URS +4.8F DJS (n = 39), Group IV: URS +6F DJS (n = 42), and Group V: Control (n = 30). Short Form-36 (SF-36) was administered to each participant both preoperatively and 14 days after operation. Based on the SF-36 results, the changes in patients' PF and QoL were evaluated. Results: Ureteral stone treatment was performed in 202 patients. Of these, 153 patients who underwent an effective SWL or URS procedure in the first attempt were included in the study. Success rates in the first session were 53.7% (29/54) and 83.8% (124/148) for SWL and URS, respectively (p < 0.001). All the four groups were similar with regard to age, gender, body mass index, stone size, preoperative PF, and QoL. However, although postoperative PF, role limitations due to physical health, and energy/fatigue scores were similar in Group I, III, and IV, they were significantly higher in Group II. No major complication associated with SWL or URS occurred in any patient. However, in Group 2, DJS was inserted in three (7.7%) patients in the early postoperative period (within the first 48 hours) due to renal colic attacks secondary to ureterovesical junction mucosal edema. Conclusion: URS without DJS seems to be the most advantageous technique in the treatment of ureteral stones in terms of daily PF and QoL. However, it should be noted that patients undergoing URS may require postoperative emergency stenting, although rarely.

Retrospective analysis of the appendiceal neoplasms: sampling technique may influence neoplasm detection.

BACKGROUND AND AIMS: Appendiceal neoplasms are uncommon entities that are usually determined incidentally during the histopathological examination. Different techniques used for the macroscopic sampling of appendectomy material may affect the determinating neoplasms. MATERIALS AND METHODS: H&E-stained slides of 1280 cases who underwent appendectomy between 2013 and 2018 were reviewed retrospectively for histopathological features. RESULTS: Neoplasms were determined in 28 cases (3.09%); 1 lesion was observed in the proximal part of the appendix, 1 covering the entire length from proximal to distal and 26 in the distal part. In the 26 cases that observed in the distal part, the lesion was seen on both sides of the longitudinal section of the distal appendix in 20 cases, while it was seen on only one distal longitudinal section in the remaining 6 cases. CONCLUSION: The vast majority of appendiceal neoplasms are seen in the distal part of the appendix, and, in some cases, neoplasms might be seen on only one side of the distal section. Sampling only one-half of the distal part of the appendix, where tumours are most often observed, could result in some neoplasms being missed. Therefore, sampling the whole distal part would be more beneficial to determine small diameter tumours that do not create macroscopic findings.

Abundance of metalloprotease FtsH12 modulates chloroplast development in Arabidopsis thaliana.

The ATP-dependent metalloprotease FtsH12 (filamentation temperature sensitive protein H 12) has been suggested to participate in a heteromeric motor complex, driving protein translocation into the chloroplast. FtsH12 was immuno-detected in proplastids, seedlings, leaves, and roots. Expression of Myc-tagged FtsH12 under its native promotor allowed identification of FtsHi1, 2, 4, and 5, and plastidic NAD-malate dehydrogenase, five of the six interaction partners in the suggested import motor complex. Arabidopsis thaliana mutant seedlings with reduced FTSH12 abundance exhibited pale cotyledons and small, deformed chloroplasts with altered thylakoid structure. Mature plants retained these chloroplast defects, resulting in slightly variegated leaves and lower chlorophyll content. Label-free proteomics revealed strong changes in the proteome composition of FTSH12 knock-down seedlings, reflecting impaired plastid development. The composition of the translocon on the inner chloroplast membrane (TIC) protein import complex was altered, with coordinated reduction of the FtsH12-FtsHi complex subunits and accumulation of the 1 MDa TIC complex subunits TIC56, TIC214 and TIC22-III. FTSH12 overexpressor lines showed no obvious phenotype, but still displayed distinct differences in their proteome. N-terminome analyses further demonstrated normal proteolytic maturation of plastid-imported proteins irrespective of FTSH12 abundance. Together, our data suggest that FtsH12 has highest impact during seedling development; its abundance alters the plastid import machinery and impairs chloroplast development.