Ammonium treatment inhibits cell cycle activity and induces nuclei endopolyploidization in Arabidopsis thaliana.

MAIN CONCLUSION: This study determined the effect of ammonium supply on the cell division process and showed that ammonium-dependent elevated reactive oxygen species production could mediate the downregulation of the cell cycle-related gene expression. Plants grown under high-ammonium conditions show stunted growth and other toxicity symptoms, including oxidative stress. However, how ammonium regulates the development of plants remains unknown. Growth is defined as an increase in cell volume or proliferation. In the present study, ammonium-related changes in cell cycle activity were analyzed in seedlings, apical buds, and young leaves of Arabidopsis thaliana plants. In all experimental ammonium treatments, the genes responsible for regulating cell cycle progression, such as cyclin-dependent kinases and cyclins, were downregulated in the studied tissues. Thus, ammonium nutrition could be considered to reduce cell proliferation; however, the cause of this phenomenon may be secondary. Reactive oxygen species (ROS), which are produced in large amounts in response to ammonium nutrition, can act as intermediates in this process. Indeed, high ROS levels resulting from H2O2 treatment or reduced ROS production in rbohc mutants, similar to ammonium-triggered ROS, correlated with altered cell cycle-related gene expression. It can be concluded that the characteristic ammonium growth suppression may be executed by enhanced ROS metabolism to inhibit cell cycle activity. This study provides a base for future research in determining the mechanism behind ammonium-induced dwarfism in plants, and strategies to mitigate such stress.

Predictors of outcome for treatment of enterovaginal fistula : Therapeutical strategies for treatment.

BACKGROUND: Enterovaginal fistulas represent a serious complication of various diseases and therapeutic procedures, often associated with complicated clinical courses and massive impairment of quality of life. As underlying conditions and procedures are multifarious, therapeutic approaches are challenging and have to be tailored individually. As the therapeutic management is complex and individualized, multiple surgical interventions might be necessary. METHODS: The aim of this study was to identify possible predictors for outcome in the treatment enterovaginal fistula patients. The study was realized as a retrospective analysis. Ninety-two patients treated with enterovaginal fistulas between 2004 and 2016 were analyzed. Patient characteristics, therapeutic data, and endoscopic findings were stratified according to etiology, closure rate and time, as well as recurrence of fistula. Main outcome measure was the overall rate of fistula closure. RESULTS: Overall therapeutic success rate was 67.4%. Postoperatively derived fistulas were most frequent (40.2%), mainly after rectal surgery (59.5%). Postoperative and non-IBD-inflammation associated fistulas had better outcome than IBD-, radiotherapy-, and tumor-related fistulas (p = 0.001). Successful fistula closure was observed more frequently after radical surgical interventions, best results observed after transabdominal surgery (p < 0.001). Fistula recurrence was also less frequently observed after radical surgical therapies (p = 0.029). A temporary stoma was associated with higher incidence of fistula closure (p = 0.013) and lower incidence of fistula recurrence (p = 0.042) in the postoperative subgroup, as well as shortened therapy period in all groups (p = 0.031). CONCLUSION: Enterovaginal fistulas are a result of various etiologies, and treatment should be adjusted accordingly. A very sustainable, rapid, and persistent therapeutic success can be expected after radical surgical approaches with temporary diverting stoma. This is especially true for postoperatively derived fistulas.

Duodenal tropism of SARS-CoV-2 and clinical findings in critically ill COVID-19 patients.

PURPOSE: Duodenal involvement in COVID-19 is poorly studied. Aim was to describe clinical and histopathological characteristics of critically ill COVID-19 patients suffering from severe duodenitis that causes a significant bleeding and/or gastrointestinal dysmotility. METHODS: In 51 critically ill patients suffering from SARS-CoV-2 pneumonia, severe upper intestinal bleeding and/or gastric feeding intolerance were indications for upper gastrointestinal endoscopy. Duodenitis was diagnosed according to macroscopic signs and mucosal biopsies. Immunohistochemistry was performed to detect viral specific protein and ACE2. In situ hybridization was applied to confirm viral replication. RESULTS: Nine of 51 critically ill patients (18%) suffering from SARS-CoV-2 pneumonia had developed upper GI bleeding complications and/or high gastric reflux. Five of them presented with minor and four (44%) with severe duodenitis. In two patients, erosions had caused severe gastrointestinal bleeding requiring PRBC transfusions. Immunohistochemical staining for SARS-CoV-2 spike protein was positive inside duodenal enterocytes in three of four patients suffering from severe duodenitis. Viral replication could be confirmed by in situ hybridization. CONCLUSION: Our data suggest that about 8% of critically ill COVID-19 patients may develop a severe duodenitis presumably associated with a direct infection of the duodenal enterocytes by SARS-CoV-2. Clinical consequences from severe bleeding and/or upper gastrointestinal dysmotility seem to be underestimated.

Endoscopic vacuum therapy for the treatment of colorectal leaks - a systematic review and meta-analysis.

BACKGROUND: During the last two decades, vacuum-assisted wound therapy has been successfully transferred to an endoscopic treatment approach of various upper and lower gastrointestinal leaks called endoscopic vacuum therapy (EVT). As mostly small case series are published in this field, the aim of our systematic review and meta-analysis was to evaluate the efficacy and safety of EVT in the treatment of colorectal leaks. METHODS: A systematic search of MEDLINE/PubMed and Cochrane databases was performed using search terms related to EVT and colorectal defects (anastomotic leakage, rectal stump insufficiency) according to the PRISMA guidelines. Randomized controlled trials (RCTs), observational studies, and case series published by December 2020 were eligible for inclusion. A meta-analysis was conducted on the success of EVT, stoma reversal rate after EVT as well as procedure-related complications. Statistical interferences were based on pooled estimates from random effects models using DerSimonian-Laird estimator. RESULTS: Only data from observational studies and case series were available. Twenty-four studies reporting on 690 patients with colorectal defects undergoing EVT were included. The mean rate of success was 81.4% (95% CI: 74.0%-87.1%). The proportion of diverted patients was 76.4% (95% CI: 64.9%-85.0%). The mean rate of ostomy reversal across the studies was 66.7% (95% CI: 58.0%-74.4%). Sixty-four patients were reported with EVT-associated complications, the weighted mean complication rate across the studies was 12.1% (95% CI: 9.7%-15.2%). CONCLUSIONS: Current medical evidence on EVT in patients with colorectal leaks lacks high quality data from RCTs. Based on the data available, EVT can be seen as a feasible treatment option with manageable risks for selected patients with colorectal leaks.

Endoscopic vacuum therapy for in- and outpatient treatment of colorectal defects.

BACKGROUND: Evidence for endoscopic vacuum therapy (EVT) for colorectal defects is still based on small patient series from various institutions, employing different treatment algorithms and methods. As EVT was invented at our institution 20 years ago, the aim was to report the efficacy and safety of EVT for colorectal defects as well as to analyze factors associated with efficacy, therapy duration, and outpatient treatment. METHODS: Cohort study with analysis of prospectively collected data of patients receiving EVT for colorectal defects at a tertiary referral center in Germany (n = 281). RESULTS: The majority of patients had malignant disease (83%) and an American Society of Anesthesiologists classification of III/IV (81%). Most frequent indications for EVT were anastomotic leakage after sigmoid or rectal resection (67%) followed by rectal stump leakage (20%). EVT was successful in 256 out of 281 patients (91%). EVT following multi-visceral resection (P = 0.037) and recent surgical revision after primary surgery (P = 0.009) were risk factors for EVT failure. EVT-associated adverse events occurred in 27 patients (10%). Median treatment duration was 25 days. Previous chemo-radiation (P = 0.006) was associated with a significant longer duration of EVT. Outpatient treatment was conducted in 49% of patients with a median hospital stay reduction of 15 days and 98% treatment success. Younger patient age (P = 0.044) was associated with the possibility of outpatient treatment. Restoration of intestinal continuity was achieved in 60% of patients where technically possible with a 12-month rate of 52%. CONCLUSIONS: In patients with colorectal defects, EVT appears to be a safe and effective, minimally invasive option for in- and outpatient treatment.

Endoscopic vacuum therapy for treatment of rectal stump leakage.

BACKGROUND: Symptomatic rectal stump leakage (RSL) is a serious complication after discontinuity resection and requires immediate surgical, interventional, or endoscopic therapy. Re-operations are associated with high morbidity and mortality in these mostly very ill patients. Endoscopic vacuum therapy (EVT) has been established for management of anastomotic leakage; however, its effectiveness for RSL treatment has not been analyzed in detail yet. METHODS: A retrospective analysis of patients treated with EVT for RSL between 2001 and 2018 analyzing factors predicting therapy success and duration was carried out. RESULTS: Fifty-six patients with RSL at a median age of 66 years were included. Of these, 18 patients (32%) had been referred for EVT from external departments or institutions. RSL was associated with a relevant clinical deterioration in all patients, and 55 patients (98%) had been classified as ASA 3 and 4, preoperatively. In 9 patients (16%), additional surgical revision was necessary with initiation of EVT. In 47 patients (84%), EVT was successful and local control of the inflammatory focus was achieved. The median duration of therapy was 20 days. Two patients (4%) suffered from minor EVT-associated bleeding that was endoscopically controlled. Preoperative radiation of the pelvis was significantly associated with EVT failure (P = 0.035), whereas patient age represented a predictive factor for therapy length (P = 0.039). In 12 patients (21%), restoration of intestinal continuity was achieved in the further course. CONCLUSIONS: We present the first specific series on EVT for RSL. EVT for RSL was shown to be an effective and safe minimal-invasive treatment option, avoiding surgical revision in the majority of patients.

Suppression of External NADPH Dehydrogenase-NDB1 in Arabidopsis thaliana Confers Improved Tolerance to Ammonium Toxicity via Efficient Glutathione/Redox Metabolism.

Environmental stresses, including ammonium (NH4⁺) nourishment, can damage key mitochondrial components through the production of surplus reactive oxygen species (ROS) in the mitochondrial electron transport chain. However, alternative electron pathways are significant for efficient reductant dissipation in mitochondria during ammonium nutrition. The aim of this study was to define the role of external NADPH-dehydrogenase (NDB1) during oxidative metabolism of NH4⁺-fed plants. Most plant species grown with NH4⁺ as the sole nitrogen source experience a condition known as “ammonium toxicity syndrome”. Surprisingly, transgenic Arabidopsis thaliana plants suppressing NDB1 were more resistant to NH4⁺ treatment. The NDB1 knock-down line was characterized by milder oxidative stress symptoms in plant tissues when supplied with NH4⁺. Mitochondrial ROS accumulation, in particular, was attenuated in the NDB1 knock-down plants during NH4⁺ treatment. Enhanced antioxidant defense, primarily concerning the glutathione pool, may prevent ROS accumulation in NH4⁺-grown NDB1-suppressing plants. We found that induction of glutathione peroxidase-like enzymes and peroxiredoxins in the NDB1-surpressing line contributed to lower ammonium-toxicity stress. The major conclusion of this study was that NDB1 suppression in plants confers tolerance to changes in redox homeostasis that occur in response to prolonged ammonium nutrition, causing cross tolerance among plants.

Nitrogen Source Dependent Changes in Central Sugar Metabolism Maintain Cell Wall Assembly in Mitochondrial Complex I-Defective frostbite1 and Secondarily Affect Programmed Cell Death.

For optimal plant growth, carbon and nitrogen availability needs to be tightly coordinated. Mitochondrial perturbations related to a defect in complex I in the Arabidopsis thalianafrostbite1 (fro1) mutant, carrying a point mutation in the 8-kD Fe-S subunit of NDUFS4 protein, alter aspects of fundamental carbon metabolism, which is manifested as stunted growth. During nitrate nutrition, fro1 plants showed a dominant sugar flux toward nitrogen assimilation and energy production, whereas cellulose integration in the cell wall was restricted. However, when cultured on NH4⁺ as the sole nitrogen source, which typically induces developmental disorders in plants (i.e., the ammonium toxicity syndrome), fro1 showed improved growth as compared to NO3- nourishing. Higher energy availability in fro1 plants was correlated with restored cell wall assembly during NH4⁺ growth. To determine the relationship between mitochondrial complex I disassembly and cell wall-related processes, aspects of cell wall integrity and sugar and reactive oxygen species signaling were analyzed in fro1 plants. The responses of fro1 plants to NH4⁺ treatment were consistent with the inhibition of a form of programmed cell death. Resistance of fro1 plants to NH4⁺ toxicity coincided with an absence of necrotic lesion in plant leaves.

Short-term ammonium supply induces cellular defence to prevent oxidative stress in Arabidopsis leaves.

Plants can assimilate nitrogen from soil pools of both ammonium and nitrate, and the relative levels of these two nitrogen sources are highly variable in soil. Long-term ammonium nutrition is known to cause damage to Arabidopsis that has been linked to mitochondrial oxidative stress. Using hydroponic cultures, we analysed the consequences of rapid shifts between nitrate and ammonium nutrition. This did not induce growth retardation, showing that Arabidopsis can compensate for the changes in redox metabolism associated with the variations in nitrogen redox status. During the first 3 h of ammonium treatment, we observed distinct transient shifts in reactive oxygen species (ROS), low-mass antioxidants, ROS-scavenging enzymes, and mitochondrial alternative electron transport pathways, indicating rapid but temporally separated changes in chloroplastic, mitochondrial and cytosolic ROS metabolism. The fast induction of antioxidant defences significantly lowered intracellular H2 O2 levels, and thus protected Arabidopsis leaves from oxidative stress. On the other hand elevated extracellular ROS production in response to ammonium supply may be involved in signalling. The response pattern displays an intricate plasticity of Arabidopsis redox metabolism to minimise stress in responses to nutrient changes.

Post-therapeutic response evaluation by a combination of endoscopy and CT scan in esophagogastric adenocarcinoma after chemotherapy: better than its reputation.

BACKGROUND: Neoadjuvant chemotherapy is an accepted standard of care for locally advanced esophagogastric cancer. As only a subgroup benefits, a response-based tailored treatment would be of interest. The aim of our study was the evaluation of the prognostic and predictive value of clinical response in esophagogastric adenocarcinomas. METHODS: Clinical response based on a combination of endoscopy and computed tomography (CT) scan was evaluated retrospectively within a prospective database in center A and then transferred to center B. A total of 686/740 (A) and 184/210 (B) patients, staged cT3/4, cN0/1 underwent neoadjuvant chemotherapy and were then re-staged by endoscopy and CT before undergoing tumor resection. Of 184 patients, 118 (B) additionally had an interim response assessment 4-6 weeks after the start of chemotherapy. RESULTS: In A, 479 patients (70%) were defined as clinical nonresponders, 207 (30%) as responders. Median survival was 38 months (nonresponders: 27 months, responders: 108 months, log-rank, p < 0.001). Clinical and histopathological response correlated significantly (p < 0.001). In multivariate analysis, clinical response was an independent prognostic factor (HR for death 1.4, 95% CI 1.0-1.8, p = 0.032). In B, 140 patients (76%) were nonresponders and 44 (24%) responded. Median survival was 33 months, (nonresponders: 27 months, responders: not reached, p = 0.003). Interim clinical response evaluation (118 patients) also had prognostic impact (p = 0.008). Interim, preoperative clinical response and histopathological response correlated strongly (p < 0.001). CONCLUSION: Preoperative clinical response was an independent prognostic factor in center A, while in center B its prognostic value could only be confirmed in univariate analysis. The accordance with histopathological response was good in both centers, and interim clinical response evaluation showed comparable results to preoperative evaluation.

Outcome, complications, and mortality of an intrathoracic anastomosis in esophageal cancer in patients without a preoperative selection with a risk score.

BACKGROUND: Esophagectomy for esophageal cancer remains a challenge with relatively high morbidity. We analyzed outcome, complications, and mortality after abdominothoracic esophagectomy with intrathoracic anastomosis. No routine preoperative risk stratification was performed. METHODS: One hundred eighty-seven consecutive patients (105 AEG I, 21 AEG II, 58 SCC, and 3 other entities) underwent standardized right abdominothoracic esophagectomy with intrathoracic anastomosis and two field lymphadenectomy between 2003 and 2009. Reconstruction was performed mostly with a gastric tube (n = 126) or a fundus rotation gastroplasty (n = 57). Seventy-four patients underwent neoadjuvant treatment (36 patients chemotherapy; 38 patients chemoradiotherapy). RESULTS: Postoperative morbidity was high (73.2 %). Ninety-two patients (49.2 %) suffered from surgical complications, 50 patients had major (26.7 %), and 42 minor (22.5 %) complications. Thirty-day mortality was 9/187 (4.8 %) while in-hospital mortality was doubled with 9.6 %. Six of 19 of the patients died without surgical complications. Preoperative treatment did not increase morbidity or mortality. Surgical complications with subsequent death were tracheobronchial fistula (2/3), ischemia of the gastric tube (3/6), anastomotic leakage (6/30), chylothorax (1/6), and intraoperative bleeding from the aorta (1/1). The median overall survival was 25.0 months. The occurrence of surgical or medical complications did not influence overall survival. In multivariate analysis, cT-category, pN-category, R-category, and re-intubation were independent prognostic factors. CONCLUSIONS: Abdominothoracic esophagectomy with intrathoracic anastomosis without preoperative patient selection is associated with a high risk for complications and subsequent death but ranges still within the upper range of published data. Strict patient selection is accepted to reduce postoperative morbidity and mortality but excludes a subgroup of patients from potentially curative resection.

Is preoperative chemotherapy followed by surgery the appropriate treatment for signet ring cell containing adenocarcinomas of the esophagogastric junction and stomach?

BACKGROUND: Recent data suggest primary resection as the preferable approach in patients with signet ring cell gastric cancer (SRC). The aim of our retrospective exploratory study was to evaluate the influence of SRC on prognosis and response in esophagogastric adenocarcinoma treated with neoadjuvant chemotherapy. METHODS: A total of 723 locally advanced esophagogastric adenocarcinomas (cT3/4 N any) documented in a prospective database from two academic centers were classified according to the WHO definition for SRC (more than 50 % SRC) and analyzed for their association with response and prognosis after neoadjuvant treatment. RESULTS: A total of 235 tumors (32.5 %) contained SRC. Median survival of SRC was 26.3 compared with 46.6 months (p < 0.001) for non-SRC. SRC were significantly associated with female gender, gastric localization, advanced ypT and R1/2 categories, and lower risk of surgical complications and anastomotic leakage (each p < 0.001). Clinical (21.1 vs. 33.7 %, p = 0.001) and histopathological response (less than 10 % residual tumor: 16.3 vs. 28.9 %, p < 0.001) were significantly less frequent in SRC. Clinical response (p = 0.003) and complete histopathological response (pCR) (3.4 %) (p = 0.003) were associated with improved prognosis in SRC. Clinical response, surgical complications, ypTN categories, but not SRC were independent prognostic factors in forward Cox regression analysis in R0 resected patients. Risk of peritoneal carcinomatosis was increased (p < 0.001), while local (p = 0.015) and distant metastases (p = 0.02) were less frequent than in non-SRC. CONCLUSIONS: Prognosis of SRC is unfavorable. Although response to neoadjuvant chemotherapy is rare in SRC, it is associated with improved outcome. Thus, chemotherapy might not generally be abandoned in SRC. A stratification based on SRC should be included in clinical trials.

Interim endoscopy results during neoadjuvant therapy for gastric cancer correlate with histopathological response and prognosis.

BACKGROUND: Neoadjuvant chemotherapy for locally advanced gastric cancer leads to major histopathological response in less than 30 % of patients. Data on interim endoscopic response assessment do not exist. This exploratory prospective study evaluates early endoscopy after 50 % of the chemotherapy as predictor for later response and prognosis. METHODS: Forty-seven consecutive patients were included (45 resected; 33 R0 resections). All patients received baseline endoscopy and CT scans, after 50 % of their chemotherapy (EGD-1, CT-1) and after completion of chemotherapy (EGD-2, CT-2). Interim endoscopic response (EGD-1) was assessed after having received 50 % (6 weeks) of the planned 12 weeks of neoadjuvant chemotherapy. Post-chemotherapy response was clinically assessed by a combination of CT scan (CT-2) and endoscopy (EGD-2). Histopathological response was determined by a standardized scoring system (Becker criteria). Endoscopic response was defined as a reduction of >75 % of the tumor mass. RESULTS: Twelve patients were responders at EGD-1 and 13 at EGD-2. Nine patients (19.1 %) were clinical responders and 7 patients (15.6 %) were histopathological responders after chemotherapy. Specificity, accuracy, and negative predictive value of the interim EGD-1 for subsequent histopathological response were 31/38 (82 %), 36/47 (76 %), and 31/33 (93 %); and for recurrence or death, 28/30 (93.3 %), 38/47 (80.9 %), and 28/35 (80.0 %). Response at EGD-1 was significantly associated with histopathological response (p = 0.010), survival (p < 0.001), and recurrence-free survival (p = 0.009). CONCLUSIONS: Interim endoscopy after 6 weeks predicts response and prognosis. Therefore, tailoring treatment according to interim endoscopic assessment could be feasible, but the findings of this study should be validated in a larger patient cohort.

A prospective study of short-term apoplastic responses to ammonium treatment.

The integration of external stimuli into plant cells has been extensively studied. Ammonium is a metabolic trigger because it affects plant nutrition status; on the contrary, it is also a stress factor inducing oxidative changes. Plants, upon quick reaction to the presence of ammonium, can avoid the development of toxicity symptoms, but their primary ammonium sensing mechanisms remain unknown. This study aimed to investigate the different signaling routes available in the extracellular space in response to supplying ammonium to plants. During short-term (30 min-24 h) ammonium treatment of Arabidopsis seedlings, no indication of oxidative stress development or cell wall modifications was observed. However, specific changes in reactive oxygen species (ROS) and redox status were observed in the apoplast, consequently leading to the activation of several ROS (RBOH, NQR), redox (MPK, OXI), and cell-wall (WAK, FER, THE, HERK) related genes. Therefore, it is expected that immediately after ammonium supply, a defense signaling route is initiated in the extracellular space. To conclude, the presence of ammonium is primarily perceived as a typical immune reaction.

Model-Informed Approaches and Innovative Clinical Trial Design for Adeno-Associated Viral Vector-Based Gene Therapy Product Development: A White Paper.

The promise of viral vector-based gene therapy (GT) as a transformative paradigm for treating severely debilitating and life-threatening diseases is slowly coming to fruition with the recent approval of several drug products. However, they have a unique mechanism of action often necessitating a tortuous clinical development plan. Expertise in such complex therapeutic modality is still fairly limited in this emerging class of adeno-associated virus (AAV) vector-based gene therapies. Because of the irreversible mode of action and incomplete understanding of genotype-phenotype relationship and disease progression in rare diseases careful considerations should be given to GT product's benefit-risk profile. In particular, special attention needs to be paid to safe dose selection, reliable dose exposure response (including clinically relevant endpoints), or creative approaches in study design targeting small patient populations during clinical development. We believe that quantitative tools encompassed within model-informed drug development (MIDD) framework fits quite well in the development of such novel therapies, as they enable us to benefit from the totality of data approach in order to support dose selection as well as optimize clinical trial designs, end point selection, and patient enrichment. In this thought leadership paper, we provide our collective experiences, identify challenges, and suggest areas of improvement in applications of modeling and innovative trial design in development of AAV-based GT products and reflect on the challenges and opportunities for incorporating MIDD tools and more in rational development of these products.

Loss of endothelial thrombomodulin predicts response to steroid therapy and survival in acute intestinal graft-versus-host disease.

Steroid-refractory graft-versus-host disease causes significant morbidity and mortality after allogeneic stem cell transplantation. The pathomechanism of steroid resistance is currently not understood, but it has been suggested that endothelial cell dysfunction plays a role. Endothelial thrombomodulin was quantified along with histological markers of epithelial damage and cytotoxic T cells in colon biopsies from 51 allografted patients, and retrospectively correlated with response to steroids and survival. Loss of endothelial thrombomodulin was the strongest predictor of response to steroids (P=0.02) and nonrelapse mortality (P=0.01) in multivariate analyses adjusting for T-cell infiltrates, histological grading, vessel density, disease status, donor type, and conditioning therapy. Our data provide evidence that at disease onset, loss of endothelial thrombomodulin expression rather than excessive T-cell infiltration associates with steroid-refractory graft-versus-host disease and mortality. Prospective histological investigations are now warranted to improve diagnosis and prognostication of this core complication of stem cell transplantation.

Respiratory Burst Oxidase Homolog D as a Modulating Component of Oxidative Response under Ammonium Toxicity.

Delayed growth, a visible phenotypic component of the so-called ammonium syndrome, occurs when ammonium is the sole inorganic nitrogen source. Previously, we have shown that modification of apoplastic reactive oxygen species (apROS) metabolism is a key factor contributing to plant growth retardation under ammonium nutrition. Here, we further analyzed the changes in apROS metabolism in transgenic plants with disruption of the D isoform of the respiratory burst oxidase homolog (RBOH) that is responsible for apROS production. Ammonium-grown Arabidopsisrbohd plants are characterized by up to 50% lower contents of apoplastic superoxide and hydrogen peroxide. apROS sensing markers such as OZF1 and AIR12 were downregulated, and the ROS-responsive signaling pathway, including MPK3, was also downregulated in rbohd plants cultivated using ammonium as the sole nitrogen source. Additionally, the expression of the cell-wall-integrity marker FER and peroxidases 33 and 34 was decreased. These modifications may contribute to phenomenon wherein ammonium inhibited the growth of transgenic plants to a greater extent than that of wild-type plants. Overall, this study indicated that due to disruption of apROS metabolism, rbohd plants cannot adjust to ammonium toxicity and are more sensitive to these conditions.

Endoscopic Treatment Options for Gastrointestinal Leaks.

Background: Spontaneous or postoperative gastrointestinal defects are still life-threatening complications with elevated morbidity and mortality. Recently, endoscopic treatment options - up and foremost endoscopic vacuum therapy (EVT) - have become increasingly popular and have shown promising results in these patients. Methods: We performed an electronic systematic search of the MEDLINE databases (PubMed, EMBASE, and Cochrane) and searched for studies evaluating endoscopic options for the treatment of esophageal and colorectal leakages and/or perforations until March 2022. Results: The closure rate of both esophageal and colorectal defects by EVT is high and even exceeds the results of surgical revision in parts. Out of all endoscopic treatment options, EVT shows most evidence and appears to have the highest therapeutic success rates. Furthermore, EVT for both indications had a low rate of serious complications without relevant in-hospital mortality. In selected patients, EVT can be applied without fecal diversion and transferred to an outpatient setting. Conclusion: Despite multiple endoscopic treatment options, EVT is increasingly becoming the new gold standard in endoscopic treatment of extraperitoneal defects of the upper and lower GI tract with localized peritonitis or mediastinitis and without close proximity to major blood vessels. However, further prospective, comparative studies are needed to strengthen the current evidence.

Production of Homozygous Carrot (Daucus carota L.) Plants by Anther Culture.

Carrot is a vegetable of increasing economic importance. New hybrid cultivars are constantly required to meet the changing market needs. The application of anther culture significantly shortens the difficult and long-lasting breeding of carrot. We examined all the stages of the process of generating androgenic plants: induction of embryos in anther cultures, regeneration and acclimatization of produced plants, their evaluation, ploidy and homozygosity, and many other factors affecting their effectiveness. Every factor has been optimized by experimentally selecting the optimal level. As a result, a full protocol of producing homozygous plants using anther cultures was developed, which is presented in this chapter.

Production of Homozygous Red Beet (Beta vulgaris L. subsp. vulgaris) Plants by Ovule Culture.

Hybrid varieties dominate the red beet market. The breeding process necessary to produce these cultivars is very difficult and time consuming. The application of in vitro gynogenesis can reduce the time needed to produce the corresponding homozygous pure lines to a few months. Our research team has developed a method to obtain red beet doubled haploid plants by gynogenesis. The best medium for gynogenesis induction is the B5 medium with the addition of 0.5 mg/L IAA, 0.2 mg/L BA, and 322 mg/L putrescine, whereas the best medium for shoot induction from embryos proved to be the MS medium supplemented with 0.1 mg/L NAA, 0.1 mg/L BA, and 0.5 mg/L putrescine. The shoots obtained were rooted on MS medium containing half the concentration of microelements and 3 mg/L NAA, 160 mg/L putrescine, and 20 g/L sucrose. Ploidy evaluation of gynogenetic plants was performed by flow cytometry and homozygosity or heterozygosity was determined by two isoenzymatic systems: PGI and AAT.