Image-based consensus molecular subtyping in rectal cancer biopsies and response to neoadjuvant chemoradiotherapy.

The development of deep learning (DL) models to predict the consensus molecular subtypes (CMS) from histopathology images (imCMS) is a promising and cost-effective strategy to support patient stratification. Here, we investigate whether imCMS calls generated from whole slide histopathology images (WSIs) of rectal cancer (RC) pre-treatment biopsies are associated with pathological complete response (pCR) to neoadjuvant long course chemoradiotherapy (LCRT) with single agent fluoropyrimidine. DL models were trained to classify WSIs of colorectal cancers stained with hematoxylin and eosin into one of the four CMS classes using a multi-centric dataset of resection and biopsy specimens (n = 1057 WSIs) with paired transcriptional data. Classifiers were tested on a held out RC biopsy cohort (ARISTOTLE) and correlated with pCR to LCRT in an independent dataset merging two RC cohorts (ARISTOTLE, n = 114 and SALZBURG, n = 55 patients). DL models predicted CMS with high classification performance in multiple comparative analyses. In the independent cohorts (ARISTOTLE, SALZBURG), cases with WSIs classified as imCMS1 had a significantly higher likelihood of achieving pCR (OR = 2.69, 95% CI 1.01-7.17, p = 0.048). Conversely, imCMS4 was associated with lack of pCR (OR = 0.25, 95% CI 0.07-0.88, p = 0.031). Classification maps demonstrated pathologist-interpretable associations with high stromal content in imCMS4 cases, associated with poor outcome. No significant association was found in imCMS2 or imCMS3. imCMS classification of pre-treatment biopsies is a fast and inexpensive solution to identify patient groups that could benefit from neoadjuvant LCRT. The significant associations between imCMS1/imCMS4 with pCR suggest the existence of predictive morphological features that could enhance standard pathological assessment.

S100Z is expressed in a lateral subpopulation of olfactory receptor neurons in the main olfactory system of Xenopus laevis.

In contrast to other S100 protein members, the function of S100 calcium-binding protein Z (S100Z) remains largely uncharacterized. It is expressed in the olfactory epithelium of fish, and it is closely associated with the vomeronasal organ (VNO) in mammals. In this study, we analyzed the expression pattern of S100Z in the olfactory system of the anuran amphibian Xenopus laevis. Using immunohistochemistry in whole mount and slice preparations of the larval olfactory system, we found exclusive S100Z expression in a subpopulation of olfactory receptor neurons (ORNs) of the main olfactory epithelium (MOE). S100Z expression was not co-localized with TP63 and cytokeratin type II, ruling out basal cell and supporting cell identity. The distribution of S100Z-expressing ORNs was laterally biased, and their average number was significantly increased in the lateral half of the olfactory epithelium. The axons of S100Z-positive neurons projected exclusively into the lateral and intermediate glomerular clusters of the main olfactory bulb (OB). Even after metamorphic restructuring of the olfactory system, S100Z expression was restricted to a neuronal subpopulation of the MOE, which was then located in the newly formed middle cavity. An axonal projection into the ventro-lateral OB persisted also in postmetamorphic frogs. In summary, S100Z is exclusively associated with the main olfactory system in the amphibian Xenopus and not with the VNO as in mammals, despite the presence of a separate accessory olfactory system in both classes.

Unraveling Resistance to Immunotherapy in MSI-High Colorectal Cancer.

Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer-related deaths. Incidences of early CRC cases are increasing annually in high-income countries, necessitating effective treatment strategies. Immune checkpoint inhibitors (ICIs) have shown significant clinical efficacy in various cancers, including CRC. However, their effectiveness in CRC is limited to patients with mismatch-repair-deficient (dMMR)/microsatellite instability high (MSI-H) disease, which accounts for about 15% of all localized CRC cases and only 3% to 5% of metastatic CRC cases. However, the varied response among patients, with some showing resistance or primary tumor progression, highlights the need for a deeper understanding of the underlying mechanisms. Elements involved in shaping the response to ICIs, such as tumor microenvironment, immune cells, genetic changes, and the influence of gut microbiota, are not fully understood thus far. This review aims to explore potential resistance or immune-evasion mechanisms to ICIs in dMMR/MSI-H CRC and the cell types involved, as well as possible pitfalls in the diagnosis of this particular subtype.

Systemic treatment of patients with locally advanced or metastatic cholangiocarcinoma - an Austrian expert consensus statement.

Locally advanced or metastatic cholangiocarcinoma is an aggressive carcinoma with a dismal prognosis. For the first-line treatment of locally advanced or metastatic cholangiocarcinoma, cisplatin/gemcitabine has been the standard of care for more than 10 years. Its combination with the immune checkpoint inhibitor durvalumab resulted in an efficiency improvement in the phase III setting. Regarding the use of chemotherapy in the second line, positive phase III data could only be generated for FOLFOX. The evidence base for nanoliposomal irinotecan (Nal-IRI) plus 5-fluorouracil (5-FU) and leucovorin (LV) is contradictory. After the failure of first-line treatment, targeted therapies can be offered if the molecular targets microsatellite instability-high (MSI-H), IDH1, FGFR2, BRAF V600E, and NTRK are detected. These targeted agents are generally preferable to second-line chemotherapy. Broad molecular testing should be performed, preferably from tumor tissue, at the initiation of first-line therapy to timely identify potential molecular targets.

Management of metastatic colorectal cancer in patients ≥70 years - a single center experience.

Background: Age-standardized mortality rates for metastatic colorectal cancer (mCRC) are highest among elderly patients. In current clinical guidelines, treatment recommendations for this patient population are based on a limited number of clinical trials. Patients and methods: In this monocentric, retrospective analysis we characterized patients aged ≥70 years undergoing systemic therapy for mCRC and overall survival (OS) was investigated. Results: We included 117 unselected, consecutive mCRC patients aged ≥70 years undergoing systemic therapy for mCRC between February 2009 and July 2022. Median OS was 25.6 months (95% CI: 21.8-29.4). The median age was 78 years (range: 70-90) and 21%, 48%, 26% and 5% had an ECOG performance score of 0, 1, 2, and 3, respectively. The median number of systemic therapy lines was 2 (range: 1-5). The choice of first-line chemotherapy backbone (doublet/triplet versus mono) did not impact OS (HR: 0.83, p=0.50) or the probability of receiving subsequent therapy (p=0.697). Metastasectomy and/or local ablative treatment in the liver, lung, peritoneum and/or other organs were applied in 26 patients (22%) with curative intent. First-line anti-EGFR-based therapy showed a trend towards longer OS compared to anti-VEGF-based therapy or chemotherapy alone in left-sided mCRC (anti-EGFR: 39.3 months versus anti-VEGF: 27.3 months versus chemotherapy alone: 13.8 months, p=0.105). In multivariable analysis, metastasectomy and/or local ablative treatment with curative intent (yes versus no, HR: 0.22, p<0.001), the ECOG performance score (2 versus 0, HR: 3.07, p=0.007; 3 versus 0, HR: 3.66, p=0.053) and the presence of liver metastases (yes versus no, HR: 1.79, p=0.049) were independently associated with OS. Conclusions: Our findings corroborate front-line monochemotherapy in combination with targeted therapy as the treatment of choice for elderly mCRC patients with palliative treatment intent. Metastasectomy and/or local ablative treatment with curative intent are feasible and may improve OS in selected elderly mCRC patients.

Comparison of gemcitabine plus oxaliplatin versus gemcitabine plus nab-paclitaxel as first-line chemotherapy for advanced pancreatic adenocarcinoma: A single-center retrospective analysis.

BACKGROUND: Pancreatic cancer is mostly diagnosed in an advanced stage and treated with systemic therapy with palliative intent. Nowadays, the doublet chemotherapy of Gemcitabine and nab-paclitaxel (Gem-Nab) is one of the most frequently used regimens worldwide, but is not ubiquitarily available or reimbursed. Therefore, we compared the clinical efficacy of Gem-Nab to a historical control of patients treated with gemcitabine and oxaliplatin (Gem-Ox) at our tertiary cancer center, which was the standard treatment prior to the introduction of FOLFIRINOX. METHODS: This single-center retrospective real world study includes 121 patients diagnosed with locally advanced or primary metastatic pancreatic adenocarcinoma who were treated with chemotherapy doublet, with either Gem-Nab or Gem-Ox in palliative first-line. Survival rates were analyzed using the Kaplan-Meier method, and comparisons were made with log-rank tests. Gem-Ox was considered as standard first line therapy at our institution for patients who were deemed fit for doublet chemotherapy between the years 2006 to 2012. These patients were compared to a cohort of patients treated with the new standard first-line therapy of Gem-Nab between 2013 and 2020. RESULTS: A total of 554 patients with pancreatic cancer of all stages were screened, and 73 patients treated with Gem-Nab and 48 patients treated with Gem-Ox in the palliative first-line setting were identified and included in this analysis. Patients receiving Gem-Ox had a statistically significantly better performance score (ECOG PS) when compared to the Gem-Nab group (Odds ratio (OR) 0.28, 95% CI 0.12-0.65, p = 0.005), more often suffered from locally advanced than metastatic disease (OR 3.10, 95% CI 1.27-7.91, p = 0.019) and were younger in age (OR 0.95, 95% CI 0.91-0.99, p = 0.013). Median overall survival (OS) of the whole study cohort was 10.3 months (95% CI 8.5-11.6). No statistically significant difference in OS could be observed between the Gem-Nab and the Gem-Ox cohort (median OS: 8.9 months (95% CI 6.4-13.5) versus 10.9 months (95% CI 9.5-13.87, p = 0.794, HR 1.27, 95% CI 0.85-1.91)). Median progression-free survival (PFS) was 6.8 months in the entire cohort (95% CI 4.9-8.4). No statistically significant difference in PFS could be observed between the Gem-Nab and the Gem-Ox cohort (median PFS: 5.8 months (95% CI 4.3-8.2) versus 7.9 months (95% CI 5.4-9.5) p = 0.536, HR 1.11, 95% CI 0.74-1.67). Zero-truncated negative binomial regressions on OS and PFS adjusting for gender, age, performance status (ECOG PS), and CA19-9 levels yielded no significant difference between Gem-Nab or Gem-Ox. CONCLUSION: From our analysis, we could evidence no difference in outcome parameters in this retrospective analysis despite the worse prognostic pattern for GemOX. Therefore, we suggest Gem-Ox as potential first line treatment option for inoperable locally advanced or metastatic pancreatic cancer, especially if Gem-Nab is not available.

Functional odor map heterogeneity is based on multifaceted glomerular connectivity in larval Xenopus olfactory bulb.

Glomeruli are the functional units of the vertebrate olfactory bulb (OB) connecting olfactory receptor neuron (ORN) axons and mitral/tufted cell (MTC) dendrites. In amphibians, these two circuit elements regularly branch and innervate multiple, spatially distinct glomeruli. Using functional multiphoton-microscopy and single-cell tracing, we investigate the impact of this wiring on glomerular module organization and odor representations on multiple levels of the Xenopus laevis OB network. The glomerular odor map to amino acid odorants is neither stereotypic between animals nor chemotopically organized. Among the morphologically heterogeneous group of uni- and multi-glomerular MTCs, MTCs can selectively innervate glomeruli formed by axonal branches of individual ORNs. We conclude that odor map heterogeneity is caused by the coexistence of different intermingled glomerular modules. This demonstrates that organization of the amphibian main olfactory system is not strictly based on uni-glomerular connectivity.

Treatment Algorithm for Patients With Gastric Adenocarcinoma: Austrian Consensus on Systemic Therapy - An Update.

Over the last decade, therapeutic options for patients with gastric cancer have improved significantly. However, despite these recent advances, mortality is still substantial. Surgery and chemotherapy represent the cornerstones of patient management. Immune checkpoint inhibitors as well as targeted treatments such as HER2-directed therapies and antiangiogenic agents contribute to improved patient prognosis. Herein, we present the updated version of an Austrian consensus on the systemic treatment of patients with gastric adenocarcinoma and adenocarcinoma of the lower gastroesophageal junction, including those with human epidermal growth receptor 2 (HER2) overexpression, microsatellite instability, programmed death-ligand 1 (PD-L1)-positive disease, and claudin 18.2 positivity. The consensus considers the curative setting as well as first-line and later-line systemic treatment options in advanced disease. For HER2-positive disease, HER2 testing is discussed in addition to a review of first-line and later-line therapies. Potential future therapies are also listed, with a focus on targeted [e.g., fibroblast growth factor receptor 2 (FGRF2)-directed] treatments that might provide a further step forward in the management of patients with gastric cancer.

Ontogenesis of functional platelet subpopulations from preterm and term neonates to adulthood: The PLINIUS study.

Erythrocytes undergo a well-defined switch from fetal to postnatal circulation, which is mainly reflected by the stage-specific expression of hemoglobin chains. Perinatal alterations in thrombopoiesis are poorly understood. We assessed the ontogenesis of platelet phenotype and function from early prematurity to adulthood. We recruited 64 subjects comprising 7 extremely preterm (27-31 weeks gestational age), 25 moderately preterm (32-36 weeks), 10 term neonates, 8 infants (<2 years), 5 children (2-13 years), and 9 adults (>13 years). Blood was withdrawn at up to 3 different time points in neonates (t1: 0-2, t2: 3-7, and t3: 8-14 days after birth). We found that the expression levels of the major surface receptors for fibrinogen, collagen, vWF, fibronectin, and laminin were reduced but correlated with decreased platelet size, indicating a normal surface density. Although CD62P and CD63 surface exposure upon stimulation with TRAP-6, ADP, or U46619 was unaltered or only slightly reduced in neonates, GPIIb/IIIa inside-out and outside-in activation was blunted but showed a continuous increase until adulthood, correlating with the expression of the GPIIb/IIIa regulating tetraspanin CD151. Platelet subpopulation analysis using automated clustering revealed that neonates presented with a CD63+/PAC-1- pattern, followed by a continuous increase in CD63+/PAC-1+ platelets until adulthood. Our findings revealed that the number of platelet-monocyte and platelet-neutrophil aggregates, but not platelet-lymphocyte aggregates, is increased in neonates and that neonatal aggregate formation depends in part on CD62P activation. Our PLatelets In Neonatal Infants Study (PLINIUS) provides several lines of evidence that the platelet phenotype and function evolve continuously from neonates to adulthood.

Prognostic value of [18F]FDG PET/CT on treatment response and progression-free survival of gastroesophageal cancer patients undergoing perioperative FLOT chemotherapy.

PURPOSE: To evaluate the prognostic role of [18F]FDG PET/CT metabolic parameters in gastric cancer (GC) and gastroesophageal adenocarcinoma (GEJAC) patients receiving neoadjuvant chemotherapy. METHOD: In this retrospective study, 31 patients with biopsy-proven GC or GEJAC were included between August 2016 and March 2020. [18F]FDG PET/CT was performed before the neoadjuvant chemotherapy. Primary tumours' semi-quantitative metabolic parameters were extracted. All patients received a perioperative FLOT regimen thereafter. Post-chemotherapy [18F]FDG PET/CT was performed in most patients (17/31). All patients underwent surgical resection. Histopathology response to treatment and progression-free survival (PFS) were evaluated. Two-sided p-values < 0.05 were considered statistically significant. RESULTS: Thirty-one patients (mean age = 62 ± 8), including 21 GC and 10 GEJAC patients, were evaluated. 20/31(65%) patients were histopathology responders to neoadjuvant chemotherapy, including twelve complete and eight partial responders. During the median follow-up of 42.0 months, nine patients experienced recurrence. The median PFS was 60(95% CI:32.9-87.1) months. Pre-neoadjuvant chemotherapy SULpeak was significantly correlated with pathological response to treatment (p-value = 0.03;odds ratio = 16.75). In survival analysis, SUVmax (p-value = 0.01;hazard ratio[HR] = 1.55), SUVmean (p-value = 0.04;HR = 2.73), SULpeak (p-value < 0.001;HR = 1.91) and SULmean (p-value = 0.04;HR = 4.22) in the post-neoadjuvant chemotherapy pre-operative [18F]FDG PET/CT showed significant correlation with PFS. Additionally, aspects of staging were significantly correlated with PFS (p-value = 0.01;HR = 2.21). CONCLUSIONS: Pre-neoadjuvant chemotherapy [18F]FDG PET/CT parameters, especially SULpeak, could predict the pathological response to treatment in GC and GEJAC patients. Additionally, in survival analysis, post-chemotherapy metabolic parameters significantly correlated with PFS. Thus, performing [18F]FDG PET/CT before chemotherapy may help to identify patients at risk for inadequate response to perioperative FLOT and, after chemotherapy, may predict clinical outcomes.

Real-world Data of Palliative First-line Checkpoint Inhibitor Therapy for Head and Neck Cancer.

BACKGROUND/AIM: Pembrolizumab alone or combined with chemotherapy is now approved in PD-L1-positive patients with recurrent or metastatic squamous cell carcinoma of the head and neck (SCCHN). Since real-world data are pending, our goal was to evaluate the efficacy and safety of immune checkpoint inhibitor (CPI) therapy in an unselected cohort of patients with SCCHN. PATIENTS AND METHODS: We analyzed 78 patients with recurrent or metastatic SCCHN from three Austrian cancer centers that received CPI therapy alone or with chemotherapy as palliative first-line systemic treatment for this retrospective study. Patient characteristics, details on treatment, and survival were analyzed by a chart-based review. RESULTS: Of the 78 patients analyzed, 55 patients were treated with CPI alone (45 with Pembrolizumab, 10 with Nivolumab) and 23 patients received chemotherapy with a platinum and 5-FU in addition to CPI. With a median follow-up of twelve months, the median PFS of all patients was 4 months [95% confidence interval (CI)=2.2-5.8] and the median OS was 11 months (95% CI=7.1-14.9). The overall response and disease control rates were 20.5% and 46.1%, respectively. There was no statistically significant difference in clinical outcome between patient groups with a different combined positive score (CPS). The rate of reported immune related adverse events was comparable to existing data. CONCLUSION: Our findings confirm the results of the KEYNOTE-048 trial that CPI therapy alone or together with chemotherapy is an effective treatment for patients with recurrent or metastatic CPS-positive SCCHN.

Two-Photon Calcium Imaging in the Brain of Aedes aegypti Mosquitoes.

Understanding the neural basis of mosquito behavior is critical for designing effective vector control strategies and can potentially shed new light on basic nervous system function. Because mosquitoes are a non-model species, however, functional studies of mosquito nervous systems have long been restricted to electrophysiological recording from peripheral sensory organs such as the antenna. This is now changing with the advent of CRISPR-Cas9 gene editing and the development of other powerful new genetic tools. Transgenic mosquitoes that carry genetically encoded calcium sensors, for example, open the door to optical recording of neural activity with two-photon calcium imaging. Compared with electrophysiology, calcium imaging permits continuous monitoring of neural activity from large populations of neurons, even deep in the brain. When combined with selective neural drivers, it also allows targeted recording from specific neuronal types. Here, we describe a calcium imaging protocol we use in our laboratory to study neural activity in the brain of Aedes aegypti mosquitoes.

GCaMP Imaging in Mosquitoes: Central Nervous System.

Mosquitoes spread dengue, Zika, malaria, and other pathogens to hundreds of millions of people every year. A better understanding of mosquito behavior and its underlying neural mechanisms may lead to new control strategies, but such an understanding requires the development of tools and approaches for exploring the nervous system of key vector species. For example, we can now image neural activity in mosquito brains using genetically encoded calcium sensors like GCaMP. Compared with other types of neural recording, GCaMP imaging has the advantage of allowing one to record from many neurons simultaneously and/or to record from specific neuronal types. Successful implementation requires careful consideration of many factors, including the choice of microscope and how to make the brains of experimental animals visible and stable while minimizing damage. Here, we elaborate on these points and provide a concise introduction to GCaMP imaging in the mosquito central nervous system.

Body composition dynamics and impact on clinical outcome in gastric and gastro-esophageal junction cancer patients undergoing perioperative chemotherapy with the FLOT protocol.

PURPOSE: Perioperative chemotherapy with FLOT constitutes a standard of care approach for locally advanced, resectable gastric or gastro-esophageal junction (GEJ) cancer. We aimed at investigating anthropometric, CT-based and FDG-PET-based body composition parameters and dynamics during this multidisciplinary approach and the impact on clinical outcomes. METHODS: This retrospective, single-center study was based on medical records and (FDG-PET)-CT images among gastric/GEJ cancer patients undergoing perioperative FLOT chemotherapy. RESULTS: Between 2016 and 2021, 46 gastric/GEJ cancer patients started perioperative FLOT at our tertiary cancer center (Salzburg, Austria). At a median follow-up of 32 months median PFS was 47.4 months and median OS was not reached. The skeletal muscle index (SMI, cm2/m2) turned out to be the only body composition parameter with a statistically significant decrease during pre-operative FLOT (51.3 versus 48.8 cm2/m2, p = 0.02). Neither pre-FLOT body mass index (BMI), nor SMI had an impact on the duration of pre-operative FLOT, the time interval from pre-operative FLOT initiation to surgery, the necessity of pre-operative or post-operative FLOT de-escalation or the likelihood of the start of postoperative chemotherapy. Pre-FLOT BMI (overweight versus normal, HR: 0.11, 95% CI: 0.02-0.65, p = 0.02) and pre-FLOT SMI (sarcopenia versus no sarcopenia, HR: 5.08, 95% CI: 1.27-20.31, p = 0.02) were statistically significantly associated with PFS in the multivariable analysis. CONCLUSION: The statistically significant SMI loss during pre-operative FLOT and the meaningful impact of baseline SMI and BMI on PFS argue for the implementation of a nutritional screening and support program prior to the initiation of pre-operative FLOT in clinical routine.

Uncoupling of platelet granule release and integrin activation suggests GPIIb/IIIa as a therapeutic target in COVID-19.

Thromboembolic events are frequent and life-threating complications of COVID-19 but are also observed in patients with sepsis. Disseminated thrombosis can occur despite anticoagulation, suggesting that platelets play a direct but incompletely understood role. Several studies demonstrated altered platelet function in COVID-19 with some controversial findings, while underlying disease-specific mechanisms remain ill defined. We performed a comprehensive cohort study with 111 patients, comprising 37 with COVID-19, 46 with sepsis, and 28 with infection, compared with control participants. Platelet phenotype and function were assessed under static and flow conditions, revealing unexpected disease-specific differences. From hospital admission onward, platelets in COVID-19 failed to activate the integrin glycoprotein IIb/IIa (GPIIb/IIIa) in response to multiple agonists. Dense granule release was markedly impaired due to virtually missing granules, also demonstrated by whole-mount electron microscopy. By contrast, α-granule marker CD62P exposure was only mildly affected, revealing a subpopulation of PAC-1-/CD62P+ platelets, independently confirmed by automated clustering. This uncoupling of α-granule release was not observed in patients with sepsis, despite a similar disease severity. We found overall unaltered thrombus formation in COVID-19 and sepsis samples under venous shear rates, which was dependent on the presence of tissue factor. Unexpectedly, under arterial shear rates, thrombus formation was virtually abrogated in sepsis, whereas we detected overall normal-sized and stable thrombi in blood from patients with COVID-19. These thrombi were susceptible to subthreshold levels of GPIIb/IIIa blockers, eptifibatide, or tirofiban that had only a minor effect in control participants' blood. We provide evidence that low-dose GPIIb/IIIa blockade could be a therapeutic approach in COVID-19.

How We Treat Localized Rectal Cancer-An Institutional Paradigm for Total Neoadjuvant Therapy.

Total neoadjuvant therapy (TNT)-the neoadjuvant employment of radiotherapy (RT) or chemoradiation (CRT) as well as chemotherapy (CHT) before surgery-may lead to increased pathological complete response (pCR) rates as well as a reduction in the risk of distant metastases in locally advanced rectal cancer. Furthermore, increased response rates may allow organ-sparing strategies in a growing number of patients with low rectal cancer and upfront immunotherapy has shown very promising early results in patients with microsatellite instability (MSI)-high/mismatch-repair-deficient (dMMR) tumors. Despite the lack of a generally accepted treatment standard, we strongly believe that existing data is sufficient to adopt the concept of TNT and immunotherapy in clinical practice. The treatment algorithm presented in the following is based on our interpretation of the current data and should serve as a practical guide for treating physicians-without any claim to general validity.

Barley guanine nucleotide exchange factor HvGEF14 is an activator of the susceptibility factor HvRACB and supports host cell entry by Blumeria graminis f. sp. hordei.

In barley (Hordeum vulgare), signalling rat sarcoma homolog (RHO) of plants guanosine triphosphate hydrolases (ROP GTPases) support the penetration success of Blumeria graminis f. sp. hordei but little is known about ROP activation. Guanine nucleotide exchange factors (GEFs) facilitate the exchange of ROP-bound GDP for GTP and thereby turn ROPs into a signalling-activated ROP-GTP state. Plants possess a unique class of GEFs harbouring a plant-specific ROP nucleotide exchanger domain (PRONE). Here, we performed phylogenetic analyses and annotated barley PRONE-GEFs. The leaf epidermal-expressed PRONE-GEF HvGEF14 undergoes a transcriptional down-regulation on inoculation with B. graminis f. sp. hordei and directly interacts with the ROP GTPase and susceptibility factor HvRACB in yeast and in planta. Overexpression of activated HvRACB or of HvGEF14 led to the recruitment of ROP downstream interactor HvRIC171 to the cell periphery. HvGEF14 further supported direct interaction of HvRACB with a HvRACB-GTP-binding CRIB (Cdc42/Rac Interactive Binding motif) domain-containing HvRIC171 truncation. Finally, the overexpression of HvGEF14 caused enhanced susceptibility to fungal entry, while HvGEF14 RNAi provoked a trend to more penetration resistance. HvGEF14 might therefore play a role in the activation of HvRACB in barley epidermal cells during fungal penetration.

ESMO 2021-highlights in colorectal cancer.

This short review reflects on a personal selection of three abstracts on colorectal cancer (CRC) presented at the 2021 ESMO Congress: (1) KRASG12C as a new therapeutic target in metastatic CRC, supported by data from the KRYSTAL­1 and CodeBreaK101 trials, (2) positive phase 3 data on the possible role of selective internal radiotherapy (SIRT) in the second-line treatment of liver-limited metastatic CRC, and (3) the impact of the coronavirus disease 2019 (COVID-19) pandemic on CRC screening, management and mortality, now and in the upcoming years.

Posttranslational modification of the RHO of plants protein RACB by phosphorylation and cross-kingdom conserved ubiquitination.

Small RHO-type G-proteins act as signaling hubs and master regulators of polarity in eukaryotic cells. Their activity is tightly controlled, as defective RHO signaling leads to aberrant growth and developmental defects. Two major processes regulate G-protein activity: canonical shuttling between different nucleotide bound states and posttranslational modification (PTM), of which the latter can support or suppress RHO signaling, depending on the individual PTM. In plants, regulation of Rho of plants (ROPs) signaling activity has been shown to act through nucleotide exchange and GTP hydrolysis, as well as through lipid modification, but there is little data available on phosphorylation or ubiquitination of ROPs. Hence, we applied proteomic analyses to identify PTMs of the barley ROP RACB. We observed in vitro phosphorylation by barley ROP binding kinase 1 and in vivo ubiquitination of RACB. Comparative analyses of the newly identified RACB phosphosites and human RHO protein phosphosites revealed conservation of modified amino acid residues, but no overlap of actual phosphorylation patterns. However, the identified RACB ubiquitination site is conserved in all ROPs from Hordeum vulgare, Arabidopsis thaliana and Oryza sativa and in mammalian Rac1 and Rac3. Point mutation of this ubiquitination site leads to stabilization of RACB. Hence, this highly conserved lysine residue may regulate protein stability across different kingdoms.