PRDX6 contributes to selenocysteine metabolism and ferroptosis resistance.

Selenocysteine (Sec) metabolism is crucial for cellular function and ferroptosis prevention and has traditionally been thought to begin with the uptake of the Sec carrier selenoprotein P (SELENOP). Following uptake, Sec released from SELENOP undergoes metabolisation via selenocysteine lyase (SCLY), producing selenide, a substrate used by selenophosphate synthetase 2 (SEPHS2), which provides the essential selenium donor - selenophosphate - for the biosynthesis of the selenocysteine tRNA. Here, we report the discovery of an alternative pathway mediating Sec metabolisation that is independent of SCLY and mediated by peroxiredoxin 6 (PRDX6). Mechanistically, we demonstrate that PRDX6 can readily react with selenide and interact with SEPHS2, potentially acting as a selenium delivery system. Moreover, we demonstrate the presence and functional significance of this alternative route in cancer cells where we reveal a notable association between elevated expression of PRDX6 with a highly aggressive neuroblastoma subtype. Altogether, our study sheds light on a previously unrecognized aspect of Sec metabolism and its implications in ferroptosis, offering new avenues for therapeutic exploitation.

Identification of human TRIAC transmembrane transporters.

BACKGROUND: TRIAC (3,5,3'-triiodothyroacetic acid) is a T3-receptor agonist pharmacologically used in patients to mitigate T3 resistance. It is additionally explored to treat some symptoms of patients with inactivating mutations in the thyroid hormone (TH) transporter MCT8 (SLC16A2). MCT8 is expressed along the blood-brain-barrier, on neurons, astrocytes, and oligodendrocytes. Hence, pathogenic variants in MCT8 limit the access of TH into and their functions within the brain. TRIAC was shown to enter the brain independently of MCT8 and to modulate expression of TH-dependent genes. The aim of the study was to identify transporters that facilitate TRIAC uptake into cells. METHODS: We performed a whole-genome RNAi screen in HepG2 cells stably expressing a T3-receptor-dependent luciferase reporter gene. Validation of hits from the primary and confirmatory secondary screen involved a counter screen with siRNAs and compared the cellular response to TRIAC to the effect of T3, in order to exclude siRNAs targeting the gene expression machinery. MDCK1 cells were stably transfected with cDNA encoding C-terminally myc-tagged versions of the identified TRIAC-preferring transporters. Several individual clones were selected after immunocytochemical characterization for biochemical characterization of their 125I-TRIAC transport activities. RESULTS: We identified SLC22A9 and SLC29A2 as transporters mediating cellular uptake of TRIAC. SLC22A9 encodes the organic anion transporting polypeptide 7 (OAT7), a sodium-independent organic anion transporter expressed in the plasma membrane in brain, pituitary, liver and other organs. Competition with the SLC22A9/OAT7 substrate estrone-3-sulfate reduced 125I-TRIAC uptake. SLC29A2 encodes the equilibrative nucleoside transporter 2 (ENT2), which is ubiquitously expressed including pituitary and brain. Co-incubation with the SLC29A2/ENT2 inhibitor nitrobenzyl-6-thioinosine reduced 125I-TRIAC uptake. Moreover, ABCD1, an ATP-dependent peroxisomal pump, was identified as a 125I-TRIAC exporter in transfected MDCK1 cells. CONCLUSIONS: Knowledge of TRIAC transporter expression patterns, also during brain development, may thus in the future help to interpret observations on TRIAC effects as well as understand why TRIAC may not show a desirable effect on cells or organs not expressing appropriate transporters. The identification of ABCD1 highlights the sensitivity of our established screening assay, but it may not hold significant relevance for patients undergoing TRIAC treatment.

Development and evaluation of artificial organ models for ERCP training in patients with surgically altered anatomies.

Endoscopy training models (ETM) using artificial organs are practical, hygienic and comfortable for trainees. However, few models exist for training endoscopic retrograde cholangiopancreatography (ERCP) in patients with surgically altered anatomy. This training is necessary as the number of bariatric surgeries performed worldwide increases. ETM with human-like anatomy were developed to represent the postoperative anatomy after Billroth II (BII) reconstruction for a standard duodenoscope and the situs of a long-limbed Roux-en-Y (RY) for device-assisted enteroscopy (DAE). In three independent workshops, the models were evaluated by international ERCP experts. In RY model, a simulation for small bowel behavior in endoscopy was created. Thirty-three experts rated the ETM in ERCP expert courses. The BII model was evaluated as suitable for training (school grades 1.36), with a haptic and visual impression rating of 1.73. The RY model was rated 1.50 for training suitability and 2.06 for overall impression. Animal tissue-free ETMs for ERCP in surgically altered anatomy were successfully created. Evaluation by experienced endoscopists indicated that the models are suitable for hands-on ERCP training, including device-assisted endoscopy. It is expected that patient care will improve with appropriate training in advanced procedures.

LRP8-mediated selenocysteine uptake is a targetable vulnerability in MYCN-amplified neuroblastoma.

Ferroptosis has emerged as an attractive strategy in cancer therapy. Understanding the operational networks regulating ferroptosis may unravel vulnerabilities that could be harnessed for therapeutic benefit. Using CRISPR-activation screens in ferroptosis hypersensitive cells, we identify the selenoprotein P (SELENOP) receptor, LRP8, as a key determinant protecting MYCN-amplified neuroblastoma cells from ferroptosis. Genetic deletion of LRP8 leads to ferroptosis as a result of an insufficient supply of selenocysteine, which is required for the translation of the antiferroptotic selenoprotein GPX4. This dependency is caused by low expression of alternative selenium uptake pathways such as system Xc- . The identification of LRP8 as a specific vulnerability of MYCN-amplified neuroblastoma cells was confirmed in constitutive and inducible LRP8 knockout orthotopic xenografts. These findings disclose a yet-unaccounted mechanism of selective ferroptosis induction that might be explored as a therapeutic strategy for high-risk neuroblastoma and potentially other MYCN-amplified entities.

Endoscopic negative pressure therapy for duodenal leaks.

Background and study aim: Endoscopic negative pressure therapy (ENPT) is well established in the treatment of perforations of various etiologies in the upper and lower gastrointestinal tract. For duodenal perforations exist only case reports and series. Different indications are possible for ENPT in duodenal position: primary therapy for leaks, preemptive therapy after surgery for example, after ulcer suturing or resection with anastomoses, or as second line therapy in cases of recurrent anastomotic insufficiencies with leakage of duodenal secretion. Methods: A retrospective 4-year case series of negative pressure therapy in duodenal position indicated by different etiologies and a comprehensive review of current literature on endoscopic negative pressure duodenal therapy are presented. Results: Patients with primary duodenal leaks n= 6 and with duodenal stump insufficiencies n = 4 were included. In seven patients ENPT was the first line and sole therapy. Primary surgery for duodenal leak was performed in n = 3 patients. Mean duration of ENPT was 11.0 days, mean hospital stay was 30.0 days. Re-operation after start of ENPT was necessary in two patients with duodenal stump insufficiencies. Surgery after termination of the ENPT was not necessary in any patient. Discussion: In our case series and in the literature, ENPT has been shown to be very successful in the therapy of duodenal leaks. A challenge in ENPT for duodenal leaks is the appropriate length of the probe to safely reach the leak and keep the open pore element at the end of the probe in place despite intestinal motility.

Insights into the Mechanism of Human Deiodinase 1.

The three isoenzymes of iodothyronine deiodinases (DIO1-3) are membrane-anchored homo-dimeric selenoproteins which share the thioredoxin-fold structure. Several questions regarding their catalytic mechanisms still remain open. Here, we addressed the roles of several cysteines which are conserved among deiodinase isoenzymes and asked whether they may contribute to dimerization and reduction of the oxidized enzyme with physiological reductants. We also asked whether amino acids previously identified in DIO3 play the same role in DIO1. Human DIO1 and 2 were recombinantly expressed in insect cells with selenocysteine replaced with cysteine (DIO1U126C) or in COS7 cells as selenoprotein. Enzyme activities were studied by radioactive deiodination assays with physiological reducing agents and recombinant proteins were characterized by mass spectrometry. Mutation of Cys124 in DIO1 prevented reduction by glutathione, while 20 mM dithiothreitol still regenerated the enzyme. Protein thiol reductants, thioredoxin and glutaredoxin, did not reduce DIO1U126C. Mass spectrometry demonstrated the formation of an intracellular disulfide between the side-chains of Cys124 and Cys(Sec)126. We conclude that the proximal Cys124 forms a selenenyl-sulfide with the catalytic Sec126 during catalysis, which is the substrate of the physiological reductant glutathione. Mutagenesis studies support the idea of a proton-relay pathway from solvent to substrate that is shared between DIO1 and DIO3.

Endocrine and molecular factors of increased female reproductive performance in the Dummerstorf high-fertility mouse line FL1.

The Dummerstorf high-fertility mouse line FL1 is a worldwide unique selection experiment for increased female reproductive performance. After more than 190 generations of selection, these mice doubled the amount of offspring per litter compared to the unselected control line. FL1 females have a superior lifetime fecundity and the highest Silver fecundity index that has been described in mice, while their offspring show no signs of growth retardation. The reasons for the increased reproductive performance remained unclear. Thus, this study aims to characterize the Dummerstorf high-fertility mouse line FL1 on endocrine and molecular levels on the female side. We analyzed parameters of the hypothalamic pituitary gonadal axis on both hormonal and transcriptional levels. Gonadotropin-releasing hormone and follicle-stimulating hormone (FSH) concentrations were decreased in FL1 throughout the whole estrous cycle. Luteinizing hormone (LH) was increased in FL1 mice in estrus. Progesterone concentrations were decreased in estrus in FL1 mice and not affected in diestrus. We used a holistic gene expression approach in the ovary to obtain a global picture of how the high-fertility phenotype is achieved. We found several differentially expressed genes in the ovaries of FL1 mice that are associated with different female fertility traits. Our results indicate that ovulation rates in mice can be increased despite decreased FSH levels. Cycle-related alterations of progesterone and LH levels have the potential to improve follicular maturation, and interactions of endocrine and molecular factors lead to enhanced follicular survival, more successful folliculogenesis and therefore higher ovulation rates in female FL1 mice.

Endoscopic Management for Post-Surgical Complications after Resection of Esophageal Cancer.

BACKGROUND: Esophageal cancer (EC) is the sixth-leading cause of cancer-related deaths in the world. Esophagectomy is the most effective treatment for patients without invasion of adjacent organs or distant metastasis. Complications and relevant problems may occur in the early post-operative course or in a delayed fashion. Here, innovative endoscopic techniques for the treatment of postsurgical problems were developed during the past 20 years. METHODS: Endoscopic treatment strategies for the following postoperative complications are presented: anastomotic bleeding, anastomotic insufficiency, delayed gastric passage and anastomotic stenosis. Based on a literature review covering the last two decades, therapeutic procedures are presented and analyzed. RESULTS: Addressing the four complications mentioned, clipping, stenting, injection therapy, dilatation, and negative pressure therapy are successfully utilized as endoscopic treatment techniques today. CONCLUSION: Endoscopic treatment plays a major role in both early-postoperative and long-term aftercare. During the past 20 years, essential therapeutic measures have been established. A continuous development of these techniques in the field of endoscopy can be expected.

Genomic variants reducing expression of two endocytic receptors in 46,XY differences of sex development.

Transporter-dependent steroid hormone uptake into target cells was demonstrated in genetically engineered mice and fruit flies. We hypothesized that mutations in such transporters may cause differences in sex development (DSD) in humans. Exome sequencing was performed in 16 genetically unsolved cases of 46,XY DSD selected from an anonymized collection of 708 lines of genital fibroblasts (GF) that were taken from individuals with incomplete virilization. Selection criteria were based on available biochemical characterization of GF compatible with reduced androgen uptake. Two unrelated individuals were identified with mutations in LDL receptor-related protein 2 (LRP2), a gene previously associated with partial sex steroid insensitivity in mice. Like Lrp2-/- mice, affected individuals had non-descended testes. Western blots on GF confirmed reduced LRP2 expression, and endocytosis of sex hormone-binding globulin was reduced. In three unrelated individuals, two with undescended testes, mutations in another endocytic receptor gene, limb development membrane protein 1 like (LMBR1L), were detected. Two of these individuals had mutations affecting the same codon. In a transfected cell model, mutated LMBR1L showed reduced cell surface expression. Our findings suggest that endocytic androgen uptake in complex with sex hormone-binding globulin is relevant in human. LMBR1L may play a similar role in androgen uptake.

Evaluating the diagnostic value of zoom endoscopic surveillance compared to routine biopsy after intestinal transplantation.

BACKGROUND: After intestinal transplantation, close allograft monitoring especially during the early postoperative period is crucial since the intestine is a highly immunogenic organ. Current protocols are based on endoscopic and histologic examination with the latter one being linked to the risk of bleeding and perforation. AIMS: Evaluation of the diagnostic value of endoscopy utilizing magnification to predict acute cellular rejection compared to routine allograft biopsies. METHODS: Fourteen patients underwent the protocol with longitudinal zoom endoscopic and histological graft monitoring during the first year after transplantation. The intestinal mucosa was analyzed during endoscopy utilizing the SASAKI score while a minimum of two biopsies were taken during each examination. A new graduation of severity for acute cellular rejection based on the findings of the SASAKI score is established. RESULTS: Endoscopic findings of 385 examinations and more than 1000 intestinal allograft biopsies were analyzed. A total of 7 acute cellular rejection episodes in 6/14 patients occurred. Allograft endoscopy was able to diagnose ACR with a sensitivity of 76% and a specificity of 82%. CONCLUSIONS: Our results will be critical for refining protocols for allograft monitoring after intestinal transplantation thus paving the way towards less invasive measures.

Role of Rendezvous-Procedure in the Treatment of Complications after Laparoscopic Sleeve Gastrectomy.

INTRODUCTION: Laparoscopic sleeve gastrectomy is one of the most commonly performed bariatric procedures worldwide with good results, high patient acceptance, and low complication rates. The most relevant perioperative complication is the staple line leak. For the treatment of this complication, endoscopic negative pressure therapy has proven particularly effective. The correct time to start endoscopic negative pressure therapy has not been the subject of studies to date. METHODS: Twelve patients were included in this retrospective data analysis over three years. Endoscopic negative pressure therapy was carried out using innovative open pore suction devices. Patients were treated with simultaneous surgery and endoscopy, so called rendezvous-procedure (Group A) or solely endoscopically, or in sequence surgically and endoscopically (Group B). Therapy data of the procedures and outcome measures, including duration of therapy, therapy success, and change of treatment strategy, were collected and analysed. RESULTS: In each group, six patients were treated (mean age 52.96 years, 4 males, 8 females). Poor initial clinical situation, time span of endoscopic negative pressure therapy (Group A 31 days vs. Group B 18 days), and mean length of hospital stay (Group A 39.5 days vs. Group B 20.17 days) were higher in patients with rendezvous procedures. One patient in Group B died during the observation time. DISCUSSION: Rendezvous procedures for patients with staple line leaks after sleeve gastrectomy is indicated for serious ill patients with perigastric abscesses and in need of laparoscopic lavage. The one-stage complication management with the rendezvous procedure seems not to result in an obvious advantage in the further outcome in patients with staple line leaks after laparoscopic sleeve gastrectomy.

The Effect of tRNA[Ser]Sec Isopentenylation on Selenoprotein Expression.

Transfer RNA[Ser]Sec carries multiple post-transcriptional modifications. The A37G mutation in tRNA[Ser]Sec abrogates isopentenylation of base 37 and has a profound effect on selenoprotein expression in mice. Patients with a homozygous pathogenic p.R323Q variant in tRNA-isopentenyl-transferase (TRIT1) show a severe neurological disorder, and hence we wondered whether selenoprotein expression was impaired. Patient fibroblasts with the homozygous p.R323Q variant did not show a general decrease in selenoprotein expression. However, recombinant human TRIT1R323Q had significantly diminished activities towards several tRNA substrates in vitro. We thus engineered mice conditionally deficient in Trit1 in hepatocytes and neurons. Mass-spectrometry revealed that hypermodification of U34 to mcm5Um occurs independently of isopentenylation of A37 in tRNA[Ser]Sec. Western blotting and 75Se metabolic labeling showed only moderate effects on selenoprotein levels and 75Se incorporation. A detailed analysis of Trit1-deficient liver using ribosomal profiling demonstrated that UGA/Sec re-coding was moderately affected in Selenop, Txnrd1, and Sephs2, but not in Gpx1. 2'O-methylation of U34 in tRNA[Ser]Sec depends on FTSJ1, but does not affect UGA/Sec re-coding in selenoprotein translation. Taken together, our results show that a lack of isopentenylation of tRNA[Ser]Sec affects UGA/Sec read-through but differs from a A37G mutation.

Missense mutation in selenocysteine synthase causes cardio-respiratory failure and perinatal death in mice which can be compensated by selenium-independent GPX4.

Selenoproteins are a small family of proteins containing the trace element selenium in form of the rare amino acid selenocysteine (Sec), which is decoded by the UGA codon. In humans, a number of pathogenic variants in genes encoding distinct selenoproteins or selenoprotein biosynthesis factors have been identified. Pathogenic variants in selenocysteine synthase (SEPSECS), which catalyzes the last step in Sec-tRNA[Ser]Sec biosynthesis, were reported in children suffering from progressive cerebello-cerebral atrophy. To understand the pathomechanism associated with SEPSECS deficiency, we generated a novel mouse model recapitulating the respective human pathogenic p.Y334C variant in the murine Sepsecs gene (SepsecsY334C). Unlike in patients, pups homozygous for the p.Y334C variant died perinatally with signs of cardio-respiratory failure. Perinatal death is reminiscent of the Sedaghatian spondylometaphyseal dysplasia disorder in humans, which is caused by pathogenic variants in the gene encoding the selenoprotein and key ferroptosis regulator glutathione peroxidase 4 (GPX4). Protein expression levels of distinct selenoproteins in SepsecsY334C/Y334C mice were found to be generally reduced in brain and isolated cortical neurons, while transcriptomics analysis uncovered an upregulation of NRF2-regulated genes. Crossbreeding of SepsecsY334C/Y334C mice with mice harboring a targeted mutation of the catalytically active Sec to Cys in GPX4 rescued perinatal death of SepsecsY334C/Y334C mice, showing that the cardio-respiratory defects of SepsecsY334C/Y334C mice were caused by the lack of GPX4. Like in SepsecsY334C/Y334C mice, selenoprotein expression levels remained low and NRF2-regulated genes remained highly expressed in these compound mutant mice, indicating that selenium-independent GPX4, along with a sustained antioxidant response are sufficient to compensate for dysfunctional Sec-tRNA[Ser]Sec biosynthesis. Our findings imply that children with pathogenic variants in SEPSECS or GPX4 may even benefit from treatments that incompletely compensate for impaired GPX4 activity.

The Thyroid Hormone Transporter Mct8 Restricts Cathepsin-Mediated Thyroglobulin Processing in Male Mice through Thyroid Auto-Regulatory Mechanisms That Encompass Autophagy.

The thyroid gland is both a thyroid hormone (TH) generating as well as a TH responsive organ. It is hence crucial that cathepsin-mediated proteolytic cleavage of the precursor thyroglobulin is regulated and integrated with the subsequent export of TH into the blood circulation, which is enabled by TH transporters such as monocarboxylate transporters Mct8 and Mct10. Previously, we showed that cathepsin K-deficient mice exhibit the phenomenon of functional compensation through cathepsin L upregulation, which is independent of the canonical hypothalamus-pituitary-thyroid axis, thus, due to auto-regulation. Since these animals also feature enhanced Mct8 expression, we aimed to understand if TH transporters are part of the thyroid auto-regulatory mechanisms. Therefore, we analyzed phenotypic differences in thyroid function arising from combined cathepsin K and TH transporter deficiencies, i.e., in Ctsk-/-/Mct10-/-, Ctsk-/-/Mct8-/y, and Ctsk-/-/Mct8-/y/Mct10-/-. Despite the impaired TH export, thyroglobulin degradation was enhanced in the mice lacking Mct8, particularly in the triple-deficient genotype, due to increased cathepsin amounts and enhanced cysteine peptidase activities, leading to ongoing thyroglobulin proteolysis for TH liberation, eventually causing self-thyrotoxic thyroid states. The increased cathepsin amounts were a consequence of autophagy-mediated lysosomal biogenesis that is possibly triggered due to the stress accompanying intrathyroidal TH accumulation, in particular in the Ctsk-/-/Mct8-/y/Mct10-/- animals. Collectively, our data points to the notion that the absence of cathepsin K and Mct8 leads to excessive thyroglobulin degradation and TH liberation in a non-classical pathway of thyroid auto-regulation.

Pyogenic Liver Abscesses Caused by Acute Appendicitis: Frequency and Diagnostic and Therapeutic Recommendations.

Background: Pyogenic liver abscesses (PLA) are caused by biliary diseases or hematogenous spreading of mostly intra-abdominal infections. Liver abscesses resulted in hematogenous spreading of infections via the portal vein, such as abscesses caused by acute appendicitis. Pyogenic liver abscesses associated with appendicitis have rarely been described in the literature, especially in adults. The standard therapeutic procedures for liver abscesses are broad-spectrum antibiotic therapy and percutaneous drainage. Surgery for liver abscesses is required in cases of unsuccessful processes. Patients and Methods: A retrospective analysis of patients with liver abscesses between January 2005 and June 2013 was performed. Parameters investigated included demographics, etiologies of abscesses, treatment modalities, and germ spectrum including antibiotic profile. Five cases of PLA caused by appendicitis were reviewed in detail. Results: During the study period, 49 patients with PLA and 1,986 patients with acute appendicitis were treated in our hospital. Twenty-one patients with PLA were treated with antibiotic agents and computed tomography (CT)-guided drainage. Liver resections were necessary in 29 of the patients with PLA. In five patients with PLA, abscesses were caused by an acute appendicitis (9.4% of all PLA, 0.25% of all appendicitis operations). Diagnosis of appendicitis as cause of PLA was made during surgery for liver resections in three patients. Previous imaging was not clear in all cases of PLA caused by appendicitis. The most common pre-operative symptoms in patients with PLA caused by appendicitis were fever and right upper quadrant tenderness. Discussion: Pyogenic liver abscesses caused by acute appendicitis are rare. In the study period of eight and one-half years nearly 2,000 cases of acute appendicitis were treated and five of these patients developed liver abscesses (0.25%). Pyogenic liver abscesses should be considered in patients with unusual high infectious parameters, septic symptoms, and detection of unknown liver lesions.

The Protein Translocation Defect of MCT8L291R Is Rescued by Sodium Phenylbutyrate.

INTRODUCTION: The monocarboxylate transporter 8 (MCT8; SLC16A2) is a specific transporter for thyroid hormones. MCT8 deficiency, formerly known as the Allan-Herndon-Dudley syndrome, is a rare genetic disease that leads to neurological impairments and muscle weakness. Current experimental treatment options rely on thyromimetic agonists that do not depend on MCT8 for cellular uptake. Another approach comes from studies with the chemical chaperone sodium phenylbutyrate (NaPB), which was able to stabilize MCT8 mutants having protein folding defects in vitro. In addition, NaPB is known as a compound that assists with plasma membrane translocation. OBJECTIVE: The pathogenic MCT8L291R leads to the same severe neurological impairments found for other MCT8-deficient patients but, unexpectedly, lacks alterations in plasma 3,3',5-triiodothyronine (T3) levels. Here we tried to unravel the underlying mechanism of MCT8 deficiency and tested whether the pathogenic MCT8L291R mutant responds to NaPB treatment. Therefore, we overexpressed the mutant in Madin-Darby canine kidney cells in the human choriocarcinoma cell line JEG1 and in COS7 cells of African green monkey origin. RESULTS: In our recent study we describe that the MCT8L291R mutation most likely leads to a translocation defect. The pathogenic mutant is not located at the plasma membrane, but shows overlapping expression with a marker protein of the lysosome. Mutation of the corresponding amino acid in murine Mct8 (Mct8L223R) displays a similar effect on cell surface expression and transport function as seen before for MCT8L291R. NaPB was able to correct the translocation defect of MCT8L291R/Mct8L223R and restored protein function by increasing T3 transport activity. Furthermore, we detected enhanced mRNA levels of wild-type and mutant MCT8/Mct8 after NaPB treatment. The increase in mRNA levels could be an explanation for the positive effect on protein expression and function detected for wild-type MCT8. CONCLUSION: NaPB is not only suitable for the treatment of mutations leading to misfolding and protein degradation, but also for a mutant wrongly sorted inside a cell which is otherwise functional.

SLC20A1 Is Involved in Urinary Tract and Urorectal Development.

Previous studies in developing Xenopus and zebrafish reported that the phosphate transporter slc20a1a is expressed in pronephric kidneys. The recent identification of SLC20A1 as a monoallelic candidate gene for cloacal exstrophy further suggests its involvement in the urinary tract and urorectal development. However, little is known of the functional role of SLC20A1 in urinary tract development. Here, we investigated this using morpholino oligonucleotide knockdown of the zebrafish ortholog slc20a1a. This caused kidney cysts and malformations of the cloaca. Moreover, in morphants we demonstrated dysfunctional voiding and hindgut opening defects mimicking imperforate anus in human cloacal exstrophy. Furthermore, we performed immunohistochemistry of an unaffected 6-week-old human embryo and detected SLC20A1 in the urinary tract and the abdominal midline, structures implicated in the pathogenesis of cloacal exstrophy. Additionally, we resequenced SLC20A1 in 690 individuals with bladder exstrophy-epispadias complex (BEEC) including 84 individuals with cloacal exstrophy. We identified two additional monoallelic de novo variants. One was identified in a case-parent trio with classic bladder exstrophy, and one additional novel de novo variant was detected in an affected mother who transmitted this variant to her affected son. To study the potential cellular impact of SLC20A1 variants, we expressed them in HEK293 cells. Here, phosphate transport was not compromised, suggesting that it is not a disease mechanism. However, there was a tendency for lower levels of cleaved caspase-3, perhaps implicating apoptosis pathways in the disease. Our results suggest SLC20A1 is involved in urinary tract and urorectal development and implicate SLC20A1 as a disease-gene for BEEC.

Endoscopic negative pressure therapy with open-pore film drainage and open-pore polyurethane sponge drainage for iatrogenic perforation of the esophagus.

BACKGROUND: Management of iatrogenic esophageal perforation (IEP) is challenging. Endoscopic negative pressure therapy (ENPT) is an emerging and effective tool for the treatment of gastrointestinal and anastomotic leaks. We have used ENPT as first-line therapy for IEP since 2017. The aim of this study was to present our results with this strategy in patients with IEP. METHODS: Nine patients were treated with ENPT for IEP between August 2017 and August 2019. Their treatment characteristics, including duration of therapy, strategy used, and outcomes, were analyzed. Treatment included ENPT with open-pore film drainage (OFD) and open-pore polyurethane foam drainage (OPD). RESULTS: Early diagnosis (< 24 hours) of IEP occurred in four patients. After a mean (standard deviation) of 19.0 (13.5) days of ENPT, 6.4 (3.4) endoscopies, and 38.1 (40.3) days of hospitalization, endoscopic treatment was effective and successful in all of the patients. Additional video-assisted thoracic surgery (VATS) was done in four patients. CONCLUSIONS: ENPT is an effective new method for the management of IEP. ENPT with OFD and OPD can be combined with minimally invasive operative methods for sepsis control in IEP.

Selenium Utilization by GPX4 Is Required to Prevent Hydroperoxide-Induced Ferroptosis.

Selenoproteins are rare proteins among all kingdoms of life containing the 21st amino acid, selenocysteine. Selenocysteine resembles cysteine, differing only by the substitution of selenium for sulfur. Yet the actual advantage of selenolate- versus thiolate-based catalysis has remained enigmatic, as most of the known selenoproteins also exist as cysteine-containing homologs. Here, we demonstrate that selenolate-based catalysis of the essential mammalian selenoprotein GPX4 is unexpectedly dispensable for normal embryogenesis. Yet the survival of a specific type of interneurons emerges to exclusively depend on selenocysteine-containing GPX4, thereby preventing fatal epileptic seizures. Mechanistically, selenocysteine utilization by GPX4 confers exquisite resistance to irreversible overoxidation as cells expressing a cysteine variant are highly sensitive toward peroxide-induced ferroptosis. Remarkably, concomitant deletion of all selenoproteins in Gpx4cys/cys cells revealed that selenoproteins are dispensable for cell viability provided partial GPX4 activity is retained. Conclusively, 200 years after its discovery, a specific and indispensable role for selenium is provided.