ARTS and small-molecule ARTS mimetics upregulate p53 levels by promoting the degradation of XIAP.

Mutations resulting in decreased activity of p53 tumor suppressor protein promote tumorigenesis. P53 protein levels are tightly regulated through the Ubiquitin Proteasome System (UPS). Several E3 ligases were shown to regulate p53 stability, including MDM2. Here we report that the ubiquitin E3 ligase XIAP (X-linked Inhibitors of Apoptosis) is a direct ligase for p53 and describe a novel approach for modulating the levels of p53 by targeting the XIAP pathway. Using in vivo (live-cell) and in vitro (cell-free reconstituted system) ubiquitylation assays, we show that the XIAP-antagonist ARTS regulates the levels of p53 by promoting the degradation of XIAP. XIAP directly binds and ubiquitylates p53. In apoptotic cells, ARTS inhibits the ubiquitylation of p53 by antagonizing XIAP. XIAP knockout MEFs express higher p53 protein levels compared to wild-type MEFs. Computational screen for small molecules with high affinity to the ARTS-binding site within XIAP identified a small-molecule ARTS-mimetic, B3. This compound stimulates apoptosis in a wide range of cancer cells but not normal PBMC (Peripheral Blood Mononuclear Cells). Like ARTS, the B3 compound binds to XIAP and promotes its degradation via the UPS. B3 binding to XIAP stabilizes p53 by disrupting its interaction with XIAP. These results reveal a novel mechanism by which ARTS and p53 regulate each other through an amplification loop to promote apoptosis. Finally, these data suggest that targeting the ARTS binding pocket in XIAP can be used to increase p53 levels as a new strategy for developing anti-cancer therapeutics.

Clinical outcomes and treatment necessity in patients with toxin-negative Clostridioides difficile stool samples.

PURPOSE: The clinical significance of negative toxin enzyme immunoassays (EIA) for Clostridioides difficile infections (CDIs) is unclear. Our study aimed to investigate the significance of toxin EIA-negative in the diagnosis and prognosis of CDI. METHODS: All stool specimens submitted for C. difficile toxin EIA testing were cultured to isolate C. difficile. In-house PCR for tcdA, tcdB, cdtA, and cdtB genes were performed using C. difficile isolates. Stool specimens were tested with C. difficile toxins A and B using EIA kit (RIDASCREEN Clostridium difficile toxin A/B, R-Biopharm AG, Darmstadt, Germany). Characteristics and subsequent CDI episodes of toxin EIA-negative and -positive patients were compared. RESULTS: Among 190 C. difficile PCR-positive patients, 83 (43.7%) were toxin EIA-negative. Multivariate analysis revealed independent associations toxin EIA-negative results and shorter hospital stays (OR = 0.98, 95% CI 0.96-0.99, p = 0.013) and less high-risk antibiotic exposure in the preceding month (OR = 0.38, 95% CI 0.16-0.94, p = 0.035). Toxin EIA-negative patients displayed a significantly lower white blood cell count rate (11.0 vs. 35.4%, p < 0.001). Among the 54 patients who were toxin EIA-negative and did not receive CDI treatment, three (5.6%) were diagnosed with CDI after 7-21 days without complication. CONCLUSION: Our study demonstrates that toxin EIA-negative patients had milder laboratory findings and no complications, despite not receiving treatment. Prolonged hospitalisation and exposure to high-risk antibiotics could potentially serve as markers for the development of toxin EIA-positive CDI.

MEK1/2 inhibition rescues neurodegeneration by TFEB-mediated activation of autophagic lysosomal function in a model of Alzheimer's Disease.

Alzheimer's Disease (AD) is a progressive neurodegenerative disorder, which is characterized by cognitive deficit due to synaptic loss and neuronal death. Extracellular amyloid ß plaques are one of the pathological hallmarks of AD. The autophagic lysosomal pathway is the essential mechanism to maintain cellular homeostasis by driving clearance of protein aggregates and is dysfunctional in AD. Here, we showed that inhibiting MEK/ERK signaling using a clinically available MEK1/2 inhibitor, trametinib (GSK1120212, SNR1611), induces the protection of neurons through autophagic lysosomal activation mediated by transcription factor EB (TFEB) in a model of AD. Orally administered trametinib recovered impaired neural structures, cognitive functions, and hippocampal long-term potentiation (LTP) in 5XFAD mice. Trametinib also reduced Aß deposition via induction of autophagic lysosomal activation. RNA-sequencing analysis revealed upregulation of autophagic lysosomal genes by trametinib administration. In addition, trametinib inhibited TFEB phosphorylation at Ser142 and promoted its nuclear translocation, which in turn induced autophagic lysosomal related genes, indicating that trametinib activates the autophagic lysosomal process through TFEB activation. From these observations, we concluded that MEK inhibition provides neuronal protection from the Aß burden by increasing autophagic lysosomal activity. Thus, MEK inhibition may be an effective therapeutic strategy for AD.

Clinical characteristics and manifestations in patients with hypermucoviscous Klebsiella pneumoniae bacteremia from extra-hepatobiliary tract infection.

PURPOSE: Hypermucoviscous strains of Klebsiella pneumoniae (KP) are associated with invasive liver abscess syndrome. However, little is known about the characteristics of this phenotype in non-hepatobiliary infections. In this study, we investigated the clinical characteristics of patients with hypermucoviscous Kp (hmvKp) bacteremia from non-hepatobiliary tract infection. METHODS: This retrospective cohort study was implemented at Samsung Changwon Hospital. From March 2018 to December 2019, adult patients (≥ 18 years) with KP bacteremia of the extra-hepatobiliary system were enrolled. Hypermucoviscosity was defined by the string test. Clinical characteristics and 30-day all-cause mortality between patients with hmvKp and non-hmvKp bacteremia were compared. RESULTS: Among 179 cases of non-hepatobiliary KP bacteremia, 67 (37.4%) and 112 (62.6%) isolates were classified as hmvKp and non-hmvKp, respectively. In the hmvKp group, metastatic infection (9.0 vs. 1.8%, P = 0.054) and purulent or necrotizing infection (31.3 vs. 9.8%, P < 0.001) were more frequently observed. Additionally, non-hmvKp had more frequent resistance to cefotaxime (11.9 vs. 38.4%, P < 0.001). Thirty-day all-cause mortality was similar in the hmvKp (41.8%) and non-hmvKp (39.3%) groups (P = 0.643). In multivariable analysis, septic shock (adjusted hazard ratio [aHR] = 3.05, 95% confidence interval [CI]: 1.22-7.63) and Pitt bacteremia score (aHR = 1.23 per 1 point, 95% CI 1.14-1.33) were associated with increased mortality in patients with Kp bacteremia, while urinary-tract infection (aHR = 0.38, 95% CI 0.18-0.76) was associated with decreased mortality. CONCLUSION: hmvKp was associated with less frequent drug resistance and metastatic-purulent presentation in non-hepatobiliary infection like in hepatobiliary infection. However, hmvKp was not associated with clinical outcomes.

Treatment of Soft Tissue Defects after Minimally Invasive Plate Osteosynthesis in Fractures of the Distal Tibia: Clinical Results after Reverse Sural Artery Flap.

Introduction: Distal tibial fractures make up approximately 3% to 10% of all tibial fractures or about 1% of lower extremity fractures. MIPO is an appropriate procedure and method to achieve stable metal plate fixation and osseointegration by minimizing soft tissue damage and vascular integrity at the fracture site. MIPO to the medial tibia during distal tibial fractures induces skin irritation due to the thickness of the metal plate, which causes discomfort and pain on the medial side of the distal leg, and if severe, complications such as infection and skin defect may occur. The reverse sural flap is a well-researched approach for covering defects in the lower third of the leg, ankle, and foot. Materials and Methods: Among 151 patients with distal tibia fractures who underwent minimally invasive metal plate fixation, soft tissue was injured due to postoperative complications. We treated 13 cases with necrosis and exposed metal plates by retrograde nasogastric artery flap surgery. For these patients, we collected obligatory patient records, radiological data, and wound photographs of the treatment results and complications of reconstructive surgery. Results: In all the cases, flap survival was confirmed at the final outpatient follow-up. The exposed area of the metal plate was well coated, and there was no plate failure due to complete necrosis. Three out of four women complained of aesthetic dissatisfaction because the volume of the tunnel through which the skin mirror passed and the skin plate itself were thick. In two cases, defatting was performed to reduce the thickness of the plate while removing the metal plate. Conclusions: Metal plate exposure after distal tibial fractures have been treated with minimally invasive metal plate fusion and can be successfully treated with retrograde nasogastric artery flaps, and several surgical techniques are used during flap surgery.

The epidemiology of bloodstream infection contributing to mortality: the difference between community-acquired, healthcare-associated, and hospital-acquired infections.

BACKGROUND: The epidemiology of bloodstream infection (BSI) is well-established; however, little is known about the contribution of different pathogens to mortality. To understand true burden of BSI, pathogens contributing to mortality were investigated and compared according to where the BSI was acquired. METHODS: Data from deceased patients in two teaching hospitals in the Republic of Korea were collected. BSI contributing mortality was defined as BSI within 2-weeks before death. Cases were grouped by acquisition sites: community-acquired (CA)-, healthcare-associated (HCA)-, and hospital-acquired (HA)-BSI. Drug resistance, BSI focus, and appropriateness of empirical antimicrobial therapy were also compared. RESULTS: Among 1849 deceased patients in the hospitals, 280 (15.1%) patients experienced BSI within 2-weeks before death. In all, 71, 53, and 156 patients in the CA-, HCA-, and HA-BSI groups, respectively, with 316 total isolated pathogens were analyzed. The three most common pathogens were Klebsiella pneumoniae (17.1%), Escherichia coli (16.4%), and Staphylococcus aureus (11.4%). While K. pneumoniae and E. coli were the most common pathogens in CA- and HCA-BSI, Acinetobacter baumannii and Candida species were in HA-BSI. 26.3% (41/156) of patients experienced breakthrough HCA-BSI during administration of carbapenem and/or vancomycin. The proportion of central venous catheter-related infection (0%, 3.4% and 28.3%), carbapenem resistant-Gram negative bacilli (0%, 6.9% and 21.9%), and inappropriate empirical antimicrobial therapy (21.1%, 37.7% and 51.9%; all P < 0.001) were more frequently observed in HA-BSI. CONCLUSION: The epidemiology of BSI related to mortality had unique characteristics according to the acquisition site. Given the epidemiology of HA-BSI, infection control and antibiotics stewardship programs should be emphasized.

Predictive Factors of Severe Dissection after Balloon Angioplasty for Femoropopliteal Artery Disease.

BACKGROUND: Arterial dissection is one of the mechanisms of balloon angioplasty. Although some degree of dissection is unavoidable, severe dissection that impedes blood flow decreases patency and increases the need for additional procedures. To improve the results of angioplasty, it is necessary to understand the factors related to severe dissection and make efforts to reduce its occurrence. This study aimed to elucidate the predictive and protective factors associated with severe dissection in femoropopliteal balloon angioplasty. METHODS: This was a retrospective, single-center, nonrandomized study. A total of 409 limbs were studied in 334 patients with symptomatic femoropopliteal lesions treated between 2010 and 2019. Dissections after initial balloon angioplasty were classified according to the Kobayashi dissection classification (grade A: no dissection; B: mild dissection <1/3 of the lumen; C: severe dissection, ≥1/3 of the lumen) into the nonsevere dissection group (grades A and B), and severe dissection group (grade C). We compared clinical, procedural and lesion-related characteristics between the 2 groups. Factors with statistical significance in univariate analyses were entered into a multivariate logistic regression model to identify independent predictive factors of severe dissection. RESULTS: Severe dissection occurred in 237 limbs and nonsevere dissection in 172 limbs. In univariate analyses, the predictive factors of severe dissection were TransAtlantic Inter-Society Consensus II C/D grades (P < 0.001), lesion length ≥15cm (P < 0.001), chronic total occlusion (P = 0.004), and degree of stenosis ≥70% (P < 0.001). Protective factors for severe dissection were end-stage renal disease (P = 0.008), severe calcification >50% (P < 0.001), and the use of a scoring balloon (P = 0.001). In multivariate analysis, factors associated with severe dissection were lesion length ≥15cm (OR, 2.259; 95% CI: 1.417-4-3.601), occlusion or degree of stenosis ≥70% (OR, 1.931; 95% CI: 1.255-2.971), severe calcification (OR, 0.520; 95% CI: 0.338-0.800), and the use of a scoring balloon (OR, 0.467; 95% CI: 0.263-0.830). CONCLUSIONS: Lesion length ≥15cm and occlusion or stenosis ≥70% were identified as independent predictive factors of severe dissection in femoropopliteal artery balloon angioplasty. Conversely, severe calcification and the use of a scoring balloon appeared to be protective factors against severe dissection.

UBL domain of Usp14 and other proteins stimulates proteasome activities and protein degradation in cells.

The best-known function of ubiquitin-like (UBL) domains in proteins is to enable their binding to 26S proteasomes. The proteasome-associated deubiquitinating enzyme Usp14/UBP6 contains an N-terminal UBL domain and is an important regulator of proteasomal activity. Usp14 by itself represses multiple proteasomal activities but, upon binding a ubiquitin chain, Usp14 stimulates these activities and promotes ubiquitin-conjugate degradation. Here, we demonstrate that Usp14's UBL domain alone mimics this activation of proteasomes by ubiquitin chains. Addition of this UBL domain to purified 26S proteasomes stimulated the same activities inhibited by Usp14: peptide entry and hydrolysis, protein-dependent ATP hydrolysis, deubiquitination by Rpn11, and the degradation of ubiquitinated and nonubiquitinated proteins. Thus, the binding of Usp14's UBL (apparently to Rpn1's T2 site) seems to mediate the activation of proteasomes by ubiquitinated substrates. However, the stimulation of these various activities was greater in proteasomes lacking Usp14 than in wild-type particles and thus is a general response that does not involve some displacement of Usp14. Furthermore, the UBL domains from hHR23 and hPLIC1/UBQLN1 also stimulated peptide hydrolysis, and the expression of hHR23A's UBL domain in HeLa cells stimulated overall protein degradation. Therefore, many UBL-containing proteins that bind to proteasomes may also enhance allosterically its proteolytic activity.

Structural Mutations in the Organellar Genomes of Valeriana sambucifolia f. dageletiana (Nakai. ex Maekawa) Hara Show Dynamic Gene Transfer.

Valeriana sambucifolia f. dageletiana (Nakai. ex Maekawa) Hara is a broad-leaved valerian endemic to Ulleung Island, a noted hot spot of endemism in Korea. However, despite its widespread pharmacological use, this plant remains comparatively understudied. Plant cells generally contain two types of organellar genomes (the plastome and the mitogenome) that have undergone independent evolution, which accordingly can provide valuable information for elucidating the phylogenetic relationships and evolutionary histories of terrestrial plants. Moreover, the extensive mega-data available for plant genomes, particularly those of plastomes, can enable researchers to gain an in-depth understanding of the transfer of genes between different types of genomes. In this study, we analyzed two organellar genomes (the 155,179 bp plastome and the 1,187,459 bp mitogenome) of V. sambucifolia f. dageletiana and detected extensive changes throughout the plastome sequence, including rapid structural mutations associated with inverted repeat (IR) contraction and genetic variation. We also described features characterizing the first reported mitogenome sequence obtained for a plant in the order Dipsacales and confirmed frequent gene transfer in this mitogenome. We identified eight non-plastome-originated regions (NPRs) distributed within the plastome of this endemic plant, for six of which there were no corresponding sequences in the current nucleotide sequence databases. Indeed, one of these unidentified NPRs unexpectedly showed certain similarities to sequences from bony fish. Although this is ostensibly difficult to explain, we suggest that this surprising association may conceivably reflect the occurrence of gene transfer from a bony fish to the plastome of an ancestor of V. sambucifolia f. dageletiana mediated by either fungi or bacteria.

PM2.5 Exacerbates Oxidative Stress and Inflammatory Response through the Nrf2/NF-κB Signaling Pathway in OVA-Induced Allergic Rhinitis Mouse Model.

Air pollution-related particulate matter (PM) exposure reportedly enhances allergic airway inflammation. Some studies have shown an association between PM exposure and a risk for allergic rhinitis (AR). However, the effect of PM for AR is not fully understood. An AR mouse model was developed by intranasal administration of 100 µg/mouse PM with a less than or equal to 2.5 µm in aerodynamic diameter (PM2.5) solution, and then by intraperitoneal injection of ovalbumin (OVA) with alum and intranasal challenging with 10 mg/mL OVA. The effects of PM2.5 on oxidative stress and inflammatory response via the Nrf2/NF-κB signaling pathway in mice with or without AR indicating by histological, serum, and protein analyses were examined. PM2.5 administration enhanced allergic inflammatory cell expression in the nasal mucosa through increasing the expression of inflammatory cytokine and reducing the release of Treg cytokine in OVA-induced AR mice, although PM2.5 exposure itself induced neither allergic responses nor damage to nasal and lung tissues. Notably, repeated OVA-immunization markedly impaired the nasal mucosa in the septum region. Moreover, AR with PM2.5 exposure reinforced this impairment in OVA-induced AR mice. Long-term PM2.5 exposure strengthened allergic reactions by inducing the oxidative through malondialdehyde production. The present study also provided evidence, for the first time, that activity of the Nrf2 signaling pathway is inhibited in PM2.5 exposed AR mice. Furthermore, PM2.5 exposure increased the histopathological changes of nasal and lung tissues and related the inflammatory cytokine, and clearly enhanced PM2.5 phagocytosis by alveolar macrophages via activating the NF-κB signaling pathway. These obtained results suggest that AR patients may experience exacerbation of allergic responses in areas with prolonged PM2.5 exposure.

Genetic mapping of the c1 locus by GBS-based BSA-seq revealed Pseudo-Response Regulator 2 as a candidate gene controlling pepper fruit color.

KEY MESSAGE: The Pseudo-Response Regulator 2 gene was identified in the c1 locus, representing a genetic factor regulating fruit color in pepper using GBS-based BSA-seq. The loci c1, c2, and y have been widely reported as genetic determinants of various ripe fruit colors in pepper. However, c1, which may impact reduced pigmentation in red, orange, and yellow fruits, is not well understood. Two cultivars showing peach or orange fruit in Capsicum chinense 'Habanero' were found to have c2 mutation and were hypothesized to segregate c1 locus in the F2 population. Habanero peach (HP) showed a reduced level of chlorophylls, carotenoids and total soluble solids in immature and ripe fruits. A microscopic examination of the fruit pericarps revealed smaller plastids and less stacked thylakoid grana in HP. The expression of many genes related to chlorophyll and carotenoid biosynthetic pathways were reduced in HP. To identify the genomic region of the c1 locus, bulked segregant analysis combined with genotyping-by-sequencing was employed on an F2 population derived from a cross between Habanero orange and HP. One SNP at chromosome 1 was strongly associated with the peach fruit color. Pepper Pseudo-Response Regulator 2 (PRR2) was located close to the SNP and cosegregated with the peach fruit color. A 41 bp deletion at the third exon-intron junction region of CcPRR2 in HP resulted in a premature termination codon. A nonsense mutation of CaPRR2 was found in C. annuum 'IT158782' which had white ripe fruit coupled with null mutations of capsanthin-capsorubin synthase (y) and phytoene synthase 1 (c2). These results will be useful for the genetic improvement in fruit color and nutritional quality in pepper.

Deubiquitinase Usp8 regulates α-synuclein clearance and modifies its toxicity in Lewy body disease.

In Parkinson's disease, misfolded α-synuclein accumulates, often in a ubiquitinated form, in neuronal inclusions termed Lewy bodies. An important outstanding question is whether ubiquitination in Lewy bodies is directly relevant to α-synuclein trafficking or turnover and Parkinson's pathogenesis. By comparative analysis in human postmortem brains, we found that ubiquitin immunoreactivity in Lewy bodies is largely due to K63-linked ubiquitin chains and markedly reduced in the substantia nigra compared with the neocortex. The ubiquitin staining in cells with Lewy bodies inversely correlated with the content and pathological localization of the deubiquitinase Usp8. Usp8 interacted and partly colocalized with α-synuclein in endosomal membranes and, both in cells and after purification, it deubiquitinated K63-linked chains on α-synuclein. Knockdown of Usp8 in the Drosophila eye reduced α-synuclein levels and α-synuclein-induced eye toxicity. Accordingly, in human cells, Usp8 knockdown increased the lysosomal degradation of α-synuclein. In the dopaminergic neurons of the Drosophila model, unlike knockdown of other deubiquitinases, Usp8 protected from α-synuclein-induced locomotor deficits and cell loss. These findings strongly suggest that removal of K63-linked ubiquitin chains on α-synuclein by Usp8 is a critical mechanism that reduces its lysosomal degradation in dopaminergic neurons and may contribute to α-synuclein accumulation in Lewy body disease.

The deubiquitinating enzyme Usp14 allosterically inhibits multiple proteasomal activities and ubiquitin-independent proteolysis.

The proteasome-associated deubiquitinating enzyme Usp14/Ubp6 inhibits protein degradation by catalyzing substrate deubiquitination and by poorly understood allosteric actions. However, upon binding a ubiquitin chain, Usp14 enhances proteasomal degradation by stimulating ATP and peptide degradation. These studies were undertaken to clarify these seemingly opposite regulatory roles of Usp14 and their importance. To learn how the presence of Usp14 on 26S proteasomes influences its different activities, we compared enzymatic and regulatory properties of 26S proteasomes purified from wild-type mouse embryonic fibroblast cells and those lacking Usp14. The proteasomes lacking Usp14 had higher basal peptidase activity than WT 26S, and this activity was stimulated to a greater extent by adenosine 5'-O-(thiotriphosphate) (ATPγS) than with WT particles. These differences were clear even though Usp14 is present on only a minor fraction (30-40%) of the 26S in WT mouse embryonic fibroblast cells. Addition of purified Usp14 to the WT and Usp14-defficient proteasomes reduced both their basal peptidase activity and the stimulation by ATPγS. Usp14 inhibits these processes allosterically because a catalytically inactive Usp14 mutant also inhibited them. Proteasomes lacking Usp14 also exhibited greater deubiquitinating activity by Rpn11 and greater basal ATPase activity than WT particles. ATP hydrolysis by WT proteasomes is activated if they bind a ubiquitinated protein, which is loosely folded. Surprisingly, proteasomes lacking Usp14 could be activated by such proteins even without a ubiquitin chain present. Furthermore, proteasomes lacking Usp14 are much more active in degrading non-ubiquitinated proteins (e.g. Sic1) than WT particles. Thus, without a ubiquitinated substrate present, Usp14 suppresses multiple proteasomal activities, especially basal ATP consumption and degradation of non-ubiquitinated proteins. These allosteric effects thus reduce ATP hydrolysis by inactive proteasomes and nonspecific proteolysis and enhance proteasomal specificity for ubiquitinated proteins.

Ultrasound-Guided Direct Needle Engagement for Flush-Occluded Superficial Femoral Artery.

Flush ostial occlusion of the superficial femoral artery (SFA) is challenging for endovascular treatment. Neither ipsilateral nor contralateral access is easy. The drawback of ipsilateral common femoral artery (CFA) access is that the CFA is too short for catheter manipulation and sheath engagement. Contralateral retrograde CFA access could have merit, but it has the drawback of poor pushability and torquability in the manipulation of devices. The most challenging part of flush ostial occlusion is the difficulty of wire engagement into the SFA orifice. We describe a case of successful treatment of a flush-occluded long SFA lesion using ultrasound-guided direct ipsilateral ostial access.

NUDT15 genotype distributions in the Korean population.

Thiopurines have a narrow therapeutic range because of frequent toxicity (i.e. marrow suppression), which is only partly explained by TPMT genetic polymorphisms, especially within Asian populations. Recent studies have identified NUDT15 variation as another important factor affecting thiopurine metabolism. In this study, a total of four NUDT15 coding variants (p.Arg139Cys, p.Arg139His, p.Val18Ile, and p.Val18_Val19insGlyVal) were genotyped in 920 Korean individuals using direct sequencing of NUDT15 for the first time in a Korean population. The allele frequencies were 86.7% for NUDT15*1, and 4.4, 6.9, 0.4, 1.1, and 0.50% for *2, *3, *4, *5, and *6, respectively. The NUDT15 phenotypes based on diplotypes included normal activity (n=692), intermediate activity (n=209), and low activity (n=19), occurring in 75.2, 22.7, and 2.1% of the population, respectively. This study was the first to report NUDT15 variants other than NUDT15*3 in the Korean population and more individuals who were categorized as having intermediate or low NUDT15 activity in our study than in previously reported studies in the Korean population (24.8 vs. 19.4%, P<0.05). This study is useful for future clinical studies on thiopurine pharmacogenetics and dosage adjustment in the Korean population.

Why do cellular proteins linked to K63-polyubiquitin chains not associate with proteasomes?

Although cellular proteins conjugated to K48-linked Ub chains are targeted to proteasomes, proteins conjugated to K63-ubiquitin chains are directed to lysosomes. However, pure 26S proteasomes bind and degrade K48- and K63-ubiquitinated substrates similarly. Therefore, we investigated why K63-ubiquitinated proteins are not degraded by proteasomes. We show that mammalian cells contain soluble factors that selectively bind to K63 chains and inhibit or prevent their association with proteasomes. Using ubiquitinated proteins as affinity ligands, we found that the main cellular proteins that associate selectively with K63 chains and block their binding to proteasomes are ESCRT0 (Endosomal Sorting Complex Required for Transport) and its components, STAM and Hrs. In vivo, knockdown of ESCRT0 confirmed that it is required to block binding of K63-ubiquitinated molecules to the proteasome. In addition, the Rad23 proteins, especially hHR23B, were found to bind specifically to K48-ubiquitinated proteins and to stimulate proteasome binding. The specificities of these proteins for K48- or K63-ubiquitin chains determine whether a ubiquitinated protein is targeted for proteasomal degradation or delivered instead to the endosomal-lysosomal pathway.

Pseudostenosis of the External Iliac Artery after Common Iliac Artery Occlusion: 2 Case Reports.

We report 2 cases of pseudostenosis of the external iliac artery (EIA) combined with aortoiliac occlusive disease. Both cases were treated successfully by aortobi-iliac bypass surgery instead of aortobi-femoral bypass. Pseudostenoses of the EIA disappeared in the postoperative computed tomographic angiography. We are not aware of previous reports of similar findings. Pseudostenosis should be considered if the EIA shows diffuse narrowing without wall irregularity in combination with severe stenosis or occlusion of the common iliac artery.

Enhanced ubiquitin-dependent degradation by Nedd4 protects against α-synuclein accumulation and toxicity in animal models of Parkinson's disease.

Parkinson's disease is a neurodegenerative disorder, characterized by accumulation and misfolding of α-synuclein. Although the level of α-synuclein in neurons is fundamentally linked to the onset of neurodegeneration, multiple pathways have been implicated in its degradation, and it remains unclear which are the critical ubiquitination enzymes that protect against α-synuclein accumulation in vivo. The ubiquitin ligase Nedd4 targets α-synuclein to the endosomal-lysosomal pathway in cultured cells. Here we asked whether Nedd4-mediated degradation protects against α-synuclein-induced toxicity in the Drosophila and rodent models of Parkinson's disease. We show that overexpression of Nedd4 can rescue the degenerative phenotype from ectopic expression of α-synuclein in the Drosophila eye. Overexpressed Nedd4 in the Drosophila brain prevented the α-synuclein-induced locomotor defect whereas reduction in endogenous Nedd4 by RNAi led to worsening motor function and increased loss of dopaminergic neurons. Accordingly, AAV-mediated expression of wild-type but not the catalytically inactive Nedd4 decreased the α-synuclein-induced dopaminergic cell loss in the rat substantia nigra and reduced α-synuclein accumulation. Collectively, our data in two evolutionarily distant model organisms strongly suggest that Nedd4 is a modifier of α-synuclein pathobiology and thus a potential target for neuroprotective therapies.

Visualization of the segment IV hepatic artery using 128-section MDCT angiography.

AIM: To visualize the segment IV hepatic artery and to evaluate the variations in anatomy using multidetector computed tomography (MDCT) angiography. MATERIALS AND METHODS: Six hundred and seventeen patients (381 men and 236 women; mean age 62.7 ± 8.1 years; age range 22-92 years) who underwent MDCT angiography performed using a 128-section MDCT system were included in the study. The segment IV hepatic arteries of 453 patients with adequate image quality were displayed using volume rendering (VR), maximum intensity projection (MIP), and multiplanar reconstruction (MPR), and were analysed regarding the origination and variation of the arteries by two radiologists and an anatomist retrospectively. RESULTS: Segment IV arteries were categorized into five different types according to their points of origin: left hepatic artery (LHA, 51.66%), right hepatic artery (RHA, 30.68%), proper hepatic artery (PHA, 5.3%), dual (12.14%), and triple (0.22%). Segment IV arteries arising from normal LHA, RHA, and PHA were found in 73.73% of patients, and those arising from variant LHA or RHA were found in 26.27%. The patterns RN2, LA2, LA3, LA4, PN2, PV1, DA1, DA2, DV3, and DV4 were first reported in the present study. CONCLUSIONS: MDCT angiography can evaluate normal as well as anatomical variants of segment IV arteries. Predicting arterial patterns of segment IV of the liver is important in planning and performing all radiological and surgical procedures in the liver, especially in hemi-liver graft procedures.

Ubiquitin ligase Nedd4 promotes alpha-synuclein degradation by the endosomal-lysosomal pathway.

α-Synuclein is an abundant brain protein that binds to lipid membranes and is involved in the recycling of presynaptic vesicles. In Parkinson disease, α-synuclein accumulates in intraneuronal inclusions often containing ubiquitin chains. Here we show that the ubiquitin ligase Nedd4, which functions in the endosomal-lysosomal pathway, robustly ubiquitinates α-synuclein, unlike ligases previously implicated in its degradation. Purified Nedd4 recognizes the carboxyl terminus of α-synuclein (residues 120-133) and attaches K63-linked ubiquitin chains. In human cells, Nedd4 overexpression enhances α-synuclein ubiquitination and clearance by a lysosomal process requiring components of the endosomal-sorting complex required for transport. Conversely, Nedd4 down-regulation increases α-synuclein content. In yeast, disruption of the Nedd4 ortholog Rsp5p decreases α-synuclein degradation and enhances inclusion formation and α-synuclein toxicity. In human brains, Nedd4 is present in pigmented neurons and is expressed especially strongly in neurons containing Lewy bodies. Thus, ubiquitination by Nedd4 targets α-synuclein to the endosomal-lysosomal pathway and, by reducing α-synuclein content, may help protect against the pathogenesis of Parkinson disease and other α-synucleinopathies.