The MEKK1-MKK1/2-MPK4 cascade phosphorylates and stabilizes STOP1 to confer aluminum resistance in Arabidopsis.

Aluminum (Al) toxicity can seriously restrict crop production on acidic soils, which comprise 40% of the world's potentially arable land. The zinc finger transcription factor STOP1 has a conserved and essential function in mediating plant Al resistance. Al stress induces STOP1 accumulation via post-transcriptional regulatory mechanisms. However, the upstream signaling pathway involved in Al-triggered STOP1 accumulation remains unclear. Here, we report that the MEKK1-MKK1/2-MPK4 cascade positively regulates STOP1 phosphorylation and stability. Mutations of MEKK1, MKK1/2, or MPK4 lead to decreased STOP1 stability and Al resistance. Al stress induces the kinase activity of MPK4, which interacts with and phosphorylates STOP1. The phosphorylation of STOP1 reduces its interaction with the F-box protein RAE1 that mediates STOP1 degradation, thereby leading to enhanced STOP1 stability and Al resistance. Taken together, our results suggest that the MEKK1-MKK1/2-MPK4 cascade is important for Al signaling and confers Al resistance through phosphorylation-mediated enhancement of STOP1 accumulation in Arabidopsis.

EXECUTER2 modulates the EXECUTER1 signalosome through its singlet oxygen-dependent oxidation.

Oxidative post-translational modifications of specific chloroplast proteins contribute to the initiation of retrograde signaling. The Arabidopsis thaliana EXECUTER1 (EX1) protein, a chloroplast-localized singlet oxygen (1O2) sensor, undergoes tryptophan (Trp) 643 oxidation by 1O2, a chloroplast-derived and light-dependent reactive oxygen species. The indole side chain of Trp is vulnerable to 1O2, leading to the generation of oxidized Trp variants and priming EX1 for degradation by a membrane-bound FtsH protease. The perception of 1O2 via Trp643 oxidation and subsequent EX1 proteolysis facilitate chloroplast-to-nucleus retrograde signaling. In this study, we discovered that the EX1-like protein EX2 also undergoes 1O2-dependent Trp530 oxidation and FtsH-dependent turnover, which attenuates 1O2 signaling by decelerating EX1-Trp643 oxidation and subsequent EX1 degradation. Consistent with this finding, the loss of EX2 function reinforces EX1-dependent retrograde signaling by accelerating EX1-Trp643 oxidation and subsequent EX1 proteolysis, whereas overexpression of EX2 produces molecular phenotypes opposite to those observed in the loss-of- function mutants of EX2. Intriguingly, phylogenetic analysis suggests that EX2 may have emerged evolutionarily to attenuate the sensitivity of EX1 toward 1O2. Collectively, these results suggest that EX2 functions as a negative regulator of the EX1 signalosome through its own 1O2-dependent oxidation, providing a new mechanistic insight into the regulation of EX1-mediated 1O2 signaling.

Extra-axial tentorial medulloblastoma: a rare presentation of a common posterior fossa tumour.

Medulloblastoma is a common paediatric posterior fossa tumour typically presenting as midline intra-axial mass involving the cerebellar vermis and/or roof of fourth ventricle with typical radiological features. These can be extra-axial in extremely rare instances with less than 50 cases reported so far in literature. We present a case of 18-year-old boy presenting with ataxia and headache. MRI showed dural mass (involving the left tentorium cerebellum) with typical imaging features of extra-axial lesion. The patient underwent near total excision of the tumour. Histopathology along with immunohistochemistry revealed the mass to be medulloblastoma. We present this case to highlight rarity of this location for medulloblastoma and the importance of considering this in the differential diagnosis of atypical posterior fossa extra-axial lesions. This can help in performing other relevant preoperative workup similar on the lines of medulloblastoma and planning of relevant management.

Degradation of STOP1 mediated by the F-box proteins RAH1 and RAE1 balances aluminum resistance and plant growth in Arabidopsis thaliana.

The C2H2-type zinc finger transcription factor sensitive to proton rhizotoxicity 1 (STOP1) is crucial for aluminum (Al) resistance in Arabidopsis. The F-box protein Regulation of AtALMT1 Expression 1 (RAE1) was recently reported to regulate the stability of STOP1. There is a unique homolog of RAE1, RAH1 (RAE1 homolog 1), in Arabidopsis, but the biological function of RAH1 is still not known. In this study, we characterize the role of RAH1 and/or RAE1 in the regulation of Al resistance and plant growth. We demonstrate that RAH1 can directly interact with STOP1 and promote its ubiquitination and degradation. RAH1 is preferentially expressed in root caps and various vascular tissues, and its expression is induced by Al and controlled by STOP1. Mutation of RAH1 in rae1 but not the wild-type (WT) background increases the level of STOP1 protein, leading to increased expression of STOP1-regulated genes and enhanced Al resistance. Interestingly, the rah1rae1 double mutant shows reduced plant growth compared with the WT and single mutants under normal conditions, and introduction of stop1 mutation into the double mutant background can rescue its reduced plant growth phenotype. Our results thus reveal that RAH1 plays an unequally redundant role with RAE1 in the modulation of STOP1 stability and plant growth, and dynamic regulation of the STOP1 level is critical for the balance of Al resistance and normal plant growth.

Golgi-localised manganese transporter PML3 regulates Arabidopsis growth through modulating Golgi glycosylation and cell wall biosynthesis.

Golgi is a critical compartment for both the reutilisation of the essential micronutrient manganese (Mn) and its detoxification. However, whether Mn plays a role in the Golgi remains to be demonstrated in plants. We characterised the function of PML3, a member of the Unknown Protein Family UPF0016, in Mn transport and the regulation of plant growth, Golgi glycosylation and cell wall biosynthesis in Arabidopsis. We also investigated the relationship of PML3 with NRAMP2, a trans-Golgi network localised Mn transporter. PML3-GFP is preferentially localised in the cis-Golgi. PML3 can transport Mn to rescue the hypersensitivity of yeast mutant Δpmr1 to excess Mn. Two mutant alleles of PML3 displayed reduced plant growth and impaired seed development under Mn-deficient conditions. The pml3 mutants also showed impaired Golgi glycosylation and cell wall biosynthesis under Mn deficiency. Double mutations of PML3 and NRAMP2 showed improved plant growth compared with that of single mutants under Mn deficiency, implying that PML3 and NRAMP2 play opposite roles in the regulation of Golgi Mn levels. Our results suggest that PML3 mediates Mn uptake into the Golgi compartments, which is required for proper protein glycosylation and cell wall biosynthesis under Mn-deficient conditions.

Endovascular Embolization in Problematic Hemodialysis Arteriovenous Fistulas: A Nonsurgical Technique.

INTRODUCTION: In the past, surgical techniques were considered gold standard practice for obliterating the accessory veins, reducing the flow across the high flowing arteriovenous fistulas (AVFs), or for closing the problematic hemodialysis AVFs. However, recently endovascular embolization has emerged as a safe and cost-effective alternative to these surgical techniques. In this study, technical and clinical success, and safety of endovascular embolization have been evaluated for accessory vein obliteration, flow reduction, and fistula closure in problematic AVFs using various embolizing agents. METHODS: This is a retrospective study of patients with problematic hemodialysis AVFs, who underwent endovascular embolization for accessory vein obliteration, flow reduction, and AVF closure at our center from February 2017 to January 2019 with various embolic agents like vascular plugs (VP), thrombin, coils, and glue. Follow-up was done at 1 week, 3 months, 6 months, and annually thereafter. RESULTS: In this study 30 patients with problematic hemodialysis AVFs [Left brachiocephalic fistula (BCF) (n = 22), right BCF (n = 4), and left radiocephalic fistula (RCF) (n = 4)], underwent endovascular embolization for accessory vein obliteration (n = 6), flow reduction (n = 3), and AVF closure (n = 21). Of the 6 patients undergoing embolization for obliteration of accessory collateral, 4 patients had nonmaturing AVFs and 2 patients had symptoms of venous hypertension (VH). Post embolization, all 4 AVFs matured over a month and symptoms of VH completely resolved within a week. Three patients who underwent embolization for flow reduction had patent AVF (on doppler) post procedure and they achieved adequate flow during dialysis with complete resolution of symptoms of VH. Out of 21 patients, who underwent endovascular closure, complete AVF thrombosis was seen in 18 patients only with the use of VP, while 4 patients required additional procedure to achieve complete thrombosis of AVF. CONCLUSION: Endovascular embolization in problematic hemodialysis AVF is a safe and cost-effective alternative to open surgical methods and vascular plug could be embolic agent of choice for AVF closure.

Primitive neuroectodermal tumour (PNET) of the renal capsule mimicking solid adrenal tumour.

Primitive neuroectodermal tumour (PNET) of renal capsule is a rare entity. We report a case of a 17-year-old girl, who presented with symptoms of epigastric and right hypochondrium pain since 1 year. She was afebrile and physical examination revealed a soft, non-tender, firm, bimanually palpable and ballotable mass along right flank. Ultrasound abdomen showed a large heteroechoic mass in right suprarenal region with indistinct planes with upper pole of right kidney. On CT, a large right suprarenal mass was noted with origin likely from right adrenal gland. Surgery was done and intraoperatively, the large mass in right suprarenal region showed involvement of the upper pole of the right kidney. The right adrenal gland was small in size, compressed and displaced by the lesion. Histopathology revealed the mass to be PNET of kidney. We report the relevant imaging findings of the case with review of literature of this entity.

Pilot study comparing dominant intraprostatic lesion volume using Ga-68 prostate-specific membrane antigen PET-computed tomography and multiparametric MRI.

PURPOSE: The standard imaging used for delineation of dominant intraprostatic lesion (DIL) is multiparametric MRI (mpMRI). The use of biologic imaging such as Ga-68 prostate-specific membrane antigen (PSMA) PET-computed tomography (PET-CT) for this purpose is being explored in view of increased sensitivity of this modality and the associated ease of delineation. MATERIALS AND METHODS: The primary objective of the study was to compare the autogenerated volumes of the DIL in Ga-68 PSMA PET-CT with the standard volume delineated in mpMRI. Twenty patients with biopsy-proven untreated prostatic adenocarcinoma were included. Multiple percentages of the maximum standardized uptake value (%SUVmax) were used to autogenerate DIL volumes in Ga-68 PSMA PET-CT and these volumes were numerically matched with the consensus DIL volume in mpMRI. PSMA tumor volume (PSMA-TV) and total lesion PSMA (TL-PSMA) were also calculated for each lesion. RESULTS: Median volume of DIL in mpMRI was 4 cm (interquartile range, IQR = 2.5-7.6 cm). The IQR for interobserver variability was 0.5-2.5 cm. Median SUVmax of the DIL was 14.1 (IQR = 10.2-22.3). Median %SUVmax corresponding to mpMRI volume was 41% of SUVmax (IQR = 34-55%). There was a strong negative correlation between MRI volume and %SUVmax (r = -0.829, P < 0.001). There was a significant correlation between TL-PSMA and prostate-specific antigen (r = 0.609, P = 0.004). CONCLUSIONS: The median DIL volume was 4 cm and median %SUVmax corresponding to MR volume of DIL was 41%. A strong inverse relationship is found between mpMRI-defined DIL volume and the %SUVmax which generates similar volume in Ga-68 PSMA PET-CT. TL-PSMA could be a quantitative biomarker for tumor load and prognosis.

Glue Embolization of Adrenal Artery Pseudoaneurysm: Case Series with Review of Literature.

Mortality rates for pseudoaneurysm (PSA) rupture are high and immediate intervention in the form of embolization can be life saving for the patient. Adrenal artery PSAs are rare with scarce references in literature. These arteries are small in caliber and require modification of the cannulation techniques for endovascular access. In situations, where the distal artery cannot be cannulated or the ostium cannot be negotiated, and percutaneous direct needle puncture (PDNP) techniques can be used. We discuss two patients with adrenal artery PSA that presented to us and their successful embolization with N-butyl cyanoacrylate through endovascular and PDNP techniques along with relevant review of the literature.

Outcome of portosystemic shunt surgery on pre-existing cholangiopathy in children with extrahepatic portal vein obstruction.

BACKGROUND: This study was performed to assess the effect of portosystemic shunt surgery (PSS) on portal cavernoma cholangiopathy (PCC) in children with extrahepatic portal vein obstruction (EHPVO). METHODS: Children with EHPVO and PCC (unfit for Meso-Rex shunt) underwent magnetic resonance cholangiogram (MRC) and magnetic resonance portovenogram (MRPV) before non-selective PSS. PCC was graded by modified Llop classification. Those with patent shunt were re-evaluated at least 6 months after surgery with MRC, MRPV and compared with pre-shunt images. RESULTS: Twenty-five children underwent PSS (central end to side splenorenal shunt with splenectomy [n = 24], mesocaval shunt [n = 1]). Pre-surgery MRC showed PCC grade I in 11, grade II in 1 and grade III in 13. MRPV showed superior mesenteric vein (SMV) block in 20. Re-assessment for PCC 18 (6 to 54) months after surgery showed grade I in 6 and grade III in 19. Thus, PCC was progressive in 6 and static in 19. Density of peribiliary collaterals decreased in 5 (SMV patent, static PCC), increased in 3 and remained unchanged in 17. Splenomegaly-related problems, gastroesophageal varices and other intra-abdominal (esophageal, perisplenic and perigastric) collaterals ameliorated in all. CONCLUSION: Non-selective PSS decompresses esophago-gastro-splenic venous circuit effectively but fails to ameliorate cholangiopathy and peribiliary collaterals. Persistence of cholangiopathy is attributable to SMV block.

Incidence of internal mammary node in locally advanced breast cancer and its correlation with metastatic disease: a retrospective observational study.

OBJECTIVE: To determine incidence of internal mammary nodes (IMN) at baseline CT of locally advanced breast cancer (LABC) and ascertain prognostic implication. METHODS AND MATERIALS: Retrospective review of all LABC patients from 1 January 2012 through 31 December 2014 was performed after approval from institutional review board. CTs of 182 patients enrolled were reviewed by two radiologists independently, and IMNs were documented based on size, location and relation with location of breast primary. 3-year follow-up was analysed and incidence of metastases was calculated as overall incidence, incidence in patients with and without discernible IMN at baseline imaging. Results are presented as numbers and percentages. Differences in metastases of two groups were compared using χ2 test. 95% CI was calculated and p < 0.05 was considered significant. RESULTS: 77 of 182 had identifiable IMN (42.3% incidence). Majority of identifiable nodes were on ipsilateral side of primary (incidence 90.90%) with higher incidence in patients with upper-outer quadrant tumours (55.9%). Majority were seen in second intercostal space (44.4%). 36 (19.7%) developed distant metastases within 3 years of therapy. Of these, 21 (27.3%) had IMN as compared with 15 (14.3 %) without IMN on baseline imaging. Patients with identifiable IMN on baseline CT had significantly higher incidence of distant metastases (p = 0.0321). CONCLUSION: Significant number LABC patients have identifiable IMN on baseline imaging with patients showing IMN on baseline CT showing significantly higher rate of metastatic disease following therapy. ADVANCES IN KNOWLEDGE: Many LABC patients have identifiable IMNs on baseline imaging which show higher incidence of subsequent metastatic disease.

Oxidative post-translational modification of EXECUTER1 is required for singlet oxygen sensing in plastids.

Environmental information perceived by chloroplasts can be translated into retrograde signals that alter the expression of nuclear genes. Singlet oxygen (1O2) generated by photosystem II (PSII) can cause photo-oxidative damage of PSII but has also been implicated in retrograde signaling. We previously reported that a nuclear-encoded chloroplast FtsH2 metalloprotease coordinates 1O2-triggered retrograde signaling by promoting the degradation of the EXECUTER1 (EX1) protein, a putative 1O2 sensor. Here, we show that a 1O2-mediated oxidative post-translational modification of EX1 is essential for initiating 1O2-derived signaling. Specifically, the Trp643 residue in DUF3506 domain of EX1 is prone to oxidation by 1O2. Both the substitution of Trp643 with 1O2-insensitive amino acids and the deletion of the DUF3506 domain abolish the EX1-mediated 1O2 signaling. We thus provide mechanistic insight into how EX1 senses 1O2 via Trp643 located in the DUF3506 domain.

F-box protein RAE1 regulates the stability of the aluminum-resistance transcription factor STOP1 in Arabidopsis.

Aluminum (Al) toxicity is a major factor limiting crop production on acid soils, which represent over 30% of the world's arable land. Some plants have evolved mechanisms to detoxify Al. Arabidopsis, for example, secretes malate via the AtALMT1 transporter to chelate and detoxify Al. The C2H2-type transcription factor STOP1 plays a crucial role in Al resistance by inducing the expression of a set of genes, including AtALMT1 Here, we identify and characterize an F-box protein-encoding gene regulation of Atalmt1 expression 1 (RAE1) that regulates the level of STOP1. Mutation and overexpression of RAE1 increases or decreases the expression of AtALMT1 and other STOP1-regulated genes, respectively. RAE1 interacts with and promotes the degradation of STOP1 via the ubiquitin-26S proteasome pathway, while Al stress promotes the accumulation of STOP1. We find that STOP1 up-regulates RAE1 expression by directly binding to the RAE1 promoter, thus forming a negative feedback loop between STOP1 and RAE1. Our results demonstrate that RAE1 influences Al resistance through the ubiquitination and degradation of STOP1.

Enhanced activity of Withania somnifera family-1 glycosyltransferase (UGT73A16) via mutagenesis.

This work used an approach of enzyme engineering towards the improved production of baicalin as well as alteration of acceptor and donor substrate preferences in UGT73A16. The 3D model of Withania somnifera family-1 glycosyltransferase (UGT73A16) was constructed based on the known crystal structures of plant UGTs. Structural and functional properties of UGT73A16 were investigated using docking and mutagenesis. The docking studies were performed to understand the key residues involved in substrate recognition. In the molecular model of UGT73A16, substrates binding pockets are located between N- and C-terminal domains. Modeled UGT73A16 was docked with UDP-glucose, UDP-glucuronic acid (UDPGA), kaempferol, isorhamnetin, 3-hydroxy flavones, naringenin, genistein and baicalein. The protein-ligand interactions showed that His 16, Asp 246, Lys 255, Ala 337, Gln 339, Val 340, Asn 358 and Glu 362 amino acid residues may be important for catalytic activity. The kinetic parameters indicated that mutants A337C and Q339A exhibited 2-3 fold and 6-7 fold more catalytic efficiency, respectively than wild type, and shifted the sugar donor specificity from UDP-glucose to UDPGA. The mutant Q379H displayed large loss of activity with UDP-glucose and UDPGA strongly suggested that last amino acid residue of PSPG box is important for glucuronosylation and glucosylation and highly specific to sugar binding sites. The information obtained from docking and mutational studies could be beneficial in future to engineer this biocatalyst for development of better ones.

Singlet oxygen- and EXECUTER1-mediated signaling is initiated in grana margins and depends on the protease FtsH2.

Formation of singlet oxygen ((1)O2) has been implicated with damaging photosystem II (PSII) that needs to undergo continuous repair to maintain photosynthetic electron transport. In addition to its damaging effect, (1)O2 has also been shown to act as a signal that triggers stress acclimation and an enhanced stress resistance. A signaling role of (1)O2 was first documented in the fluorescent (flu) mutant of Arabidopsis It strictly depends on the chloroplast protein EXECUTER1 (EX1) and happens under nonphotoinhibitory light conditions. Under severe light stress, signaling is initiated independently of EX1 by (1)O2 that is thought to be generated at the acceptor side of active PSII within the core of grana stacks. The results of the present study suggest a second source of (1)O2 formation in grana margins close to the site of chlorophyll synthesis where EX1 is localized and the disassembly of damaged and reassembly of active PSII take place. The initiation of (1)O2 signaling in grana margins depends on EX1 and the ATP-dependent zinc metalloprotease FtsH. As FtsH cleaves also the D1 protein during the disassembly of damaged PSII, EX1- and (1)O2-mediated signaling seems to be not only spatially but also functionally associated with the repair of PSII.