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Supplementary Figure S5. Related to Figure 4. A, Real-time scratch assay of MDA-MB-231 cells that are engineered to overexpressed TβRI, SMAD2 or SMAD3 using the IncuCyte system. Western blot analysis of TβRI, SMAD2 or SMAD3 expression levels in MDA-MB-231 cells (above). Representative scratch wound results at the end time point of the experiment are shown (middle). The region of the original scratch is colored in purple and the area of cell migration into the scratch is colored in yellow. Relative wound density (closure) is plotted at indicate times (below). ****, P < 0.0001, two-way analysis of variance (ANOVA). Same blot was used for TβRI and GAPDH (loading control). UCHL1 and SMAD2/3 results were derived from another blot using the same corresponding cell lysates. B, Real-time scratch assay of MDA-MB-436 shUCHL1 cells that are engineered to overexpressed TβRI, SMAD2 or SMAD3 using the IncuCyte system. Western blot of TβRI, SMAD2 or SMAD3 expression levels in MDA-MB-436 shUCHL1 cells (above). Representative scratch wound results at the end time point of the experiment are shown (middle). Relative wound density (closure) is plotted at indicate times (below). **, P < 0.01, ****, P < 0.0001, two-way analysis of variance (ANOVA). Same blot was used for TβRI and GAPDH (loading control). UCHL1 and SMAD2/3 levels in corresponding cell lysates were determined using another blot. C, Real-time scratch assay of MDA-MB-231 cells that overexpress with UCHL1 WT or UCHL1 C90A using the IncuCyte system. Representative scratch wound results at the end time point of the experiment are shown (above). Relative wound density (closure) is plotted at indicate times (below). ***, P < 0.001, ****, P < 0.0001, two-way analysis of variance (ANOVA). D, In vivo zebrafish extravasation assay of MDA-MB-231 cells that overexpress UCHL1 WT or UCHL1 C90A. Representative images of zebrafish tail fin with zoom in of invasive cells (above). Analysis of invasive cell numbers of control, UCHL1 WT and UCHL1 C90A groups in zebrafish extravasation experiment (below). **, P < 0.01, ****, P < 0.0001, two-way analysis of variance (ANOVA).

Supplementary methods, supplementary table legends, supplementary references.

Supplementary video

Supplementary Table S1-S6.

Purpose:Therapies directed to specific molecular targets are still unmet for patients with triple-negative breast cancer (TNBC). Deubiquitinases (DUB) are emerging drug targets. The identification of highly active DUBs in TNBC may lead to novel therapies.Experimental Design:Using DUB activity probes, we profiled global DUB activities in 52 breast cancer cell lines and 52 patients' tumor tissues. To validate our findings in vivo, we employed both zebrafish and murine breast cancer xenograft models. Cellular and molecular mechanisms were elucidated using in vivo and in vitro biochemical methods. A specific inhibitor was synthesized, and its biochemical and biological functions were assessed in a range of assays. Finally, we used patient sera samples to investigate clinical correlations.Results:Two DUB activity profiling approaches identified UCHL1 as being highly active in TNBC cell lines and aggressive tumors. Functionally, UCHL1 promoted metastasis in zebrafish and murine breast cancer xenograft models. Mechanistically, UCHL1 facilitates TGFβ signaling–induced metastasis by protecting TGFβ type I receptor and SMAD2 from ubiquitination. We found that these responses are potently suppressed by the specific UCHL1 inhibitor, 6RK73. Furthermore, UCHL1 levels were significantly increased in sera of patients with TNBC, and highly enriched in sera exosomes as well as TNBC cell–conditioned media. UCHL1-enriched exosomes stimulated breast cancer migration and extravasation, suggesting that UCHL1 may act in a paracrine manner to promote tumor progression.Conclusions:Our DUB activity profiling identified UCHL1 as a candidate oncoprotein that promotes TGFβ-induced breast cancer metastasis and may provide a potential target for TNBC treatment.

Excitotoxicity, a neuronal death process in neurological disorders such as stroke, is initiated by the overstimulation of ionotropic glutamate receptors. Although dysregulation of proteolytic signaling networks is critical for excitotoxicity, the identity of affected proteins and mechanisms by which they induce neuronal cell death remain unclear. To address this, we used quantitative N-terminomics to identify proteins modified by proteolysis in neurons undergoing excitotoxic cell death. We found that most proteolytically processed proteins in excitotoxic neurons are likely substrates of calpains, including key synaptic regulatory proteins such as CRMP2, doublecortin-like kinase I, Src tyrosine kinase and calmodulin-dependent protein kinase IIβ (CaMKIIβ). Critically, calpain-catalyzed proteolytic processing of these proteins generates stable truncated fragments with altered activities that potentially contribute to neuronal death by perturbing synaptic organization and function. Blocking calpain-mediated proteolysis of one of these proteins, Src, protected against neuronal loss in a rat model of neurotoxicity. Extrapolation of our N-terminomic results led to the discovery that CaMKIIα, an isoform of CaMKIIβ, undergoes differential processing in mouse brains under physiological conditions and during ischemic stroke. In summary, by identifying the neuronal proteins undergoing proteolysis during excitotoxicity, our findings offer new insights into excitotoxic neuronal death mechanisms and reveal potential neuroprotective targets for neurological disorders.

BACKGROUND: To explore the clinical value of three-dimensional image reconstruction technology (3DIT) on preoperative surgical planning and perioperative outcomes in laparoscopic pyeloplasty (LP). METHODS: Data of 25 patients with ureteropelvic junction obstruction (UPJO) admitted to our hospital from January 2018 to January 2019 was analyzed retrospectively. All patients underwent preoperative enhanced computed tomography (CT) scanning. In the 12 cases in the 3DIT group, preoperative planning involved the use of virtual operation and morphometry based on reconstruction of the CT data into three-dimensional (3D) images. Surgery in the other 13 cases was performed with traditional CT examination. Demographic, surgical outcome, and postoperative parameters were compared between these two groups. RESULTS: Reconstructed 3D images clearly showed the spatial structural relationships between the UPJO and surrounding blood vessels. In all 25 cases surgery was completed with no conversion to open surgery. Preoperative 3DIT analyses resulted in significant improvements to mean operation time (107.76 vs. 141.58 min, P=0.024), mean time of dissociating ureteropelvic junction (UPJ) (11.26 vs. 19.40 min, P=0.020), and mean estimated blood loss volume (23.84 vs. 49.16 mL, P=0.028). There were no statistically significant differences in perioperative complications, postoperative hospital stays or postoperative drainage time. CONCLUSIONS: 3DIT based on enhanced CT scans is of clinical value in the treatment of UPJO, as it can provide accurate anatomical information and reliable guidance for preoperative operation planning, and it facilitates image-guided LP.

Phomopsis vexans, which causes Phomopsis blight of eggplant, has been reported worldwide. To study the biocontrol of this disease, 162 leaf and fruit samples of eggplant Phomopsis blight were collected from Hunan, Hubei, Jiangxi, Sichuan, Zhejiang, Fujian, Guangdong and Anhui Provinces from 2017 to 2019. Eighty-seven pathogenic fungus isolates were identified as P. vexans. The following studies were conducted: screening of sporulation medium, spore morphology analysis, mycovirus detection and identification of novel mycoviruses in these isolates. The results showed that eggplant tissue medium was the most suitable medium for rapid sporulation, and all isolates had mycoviruses consisting of mainly mixed infections. The genome of these mycoviruses varied from 1-15 kb. Five novel mycoviruses infecting P. vexans were obtained, including “Phomopsis vexans fusarivirus 1” (PvFV1), “Phomopsis vexans ourmia-like virus 1” (PvOLV1), “Phomopsis vexans endornavirus 2” (PvEV2), “Phomopsis vexans partitivirus 1” (PvPV1) and “Phomopsis vexans victorivirus L1” (PvVVL1). Thus, PvVVL1 displays a unique genome structure, and this is the first report of a victorivirus consisting of two segments and of a deltapartitivirus infecting the fungus host.

Background Previous classification of renal pelvicalyceal anatomical structure may be difficult to intuitively understand and unpractical for endourological surgery. We aim to put forward a modified Takazawa anatomical classification of renal pelvicalyceal system based on three-dimensional (3D) virtual reconstruction models for endourological surgery. Methods We retrospectively collected data on 225 patients (320 kidneys) in total between Apr. 2017 and Dec. 2020, spatial anatomical structure of renal pelvis and calyces were modeled and corresponding morphological parameters were measured after 3D virtual reconstruction of computed tomography urography (CTU). The modified Takazawa renal pelvicalyceal anatomical classification was advanced based on the renal pelvicalyceal morphological parameters [bifurcated branches of renal pelvis, cross sectional area of renal pelvis and ureteropelvic junction (UPJ), infundibuloureteral angle (IUA), lower pole infundibular calyceal length (IL)] by 3D virtual reconstruction models, and comparison of renal pelvicalyceal system morphological parameters were performed to evaluate the differences in various classification types of renal pelvis and calyces. Results Anatomical structure of renal pelvis and calyces were divided into two main types (Type A and Type B) according to renal pelvic branch patterns. A single pelvis without bifurcated branch was regarded as Type A (62%) and subclassified into three subtypes: Type A1 (22%), Type A2 (27%) and Type A3 (13%), the slimline pelvis was classified as Type A1, the typical pelvis as Type A2 and the broad pelvis as Type A3. A divided pelvis with bifurcated branches was seen as Type B (38%) and subclassified into two subtypes: Type B1 (15%) with the wide and flat lower calyx branch, Type B2 (23%) with the narrow and steep lower calyx branch. Conclusions Previous studies have reported that the visualization and classification of renal pelvicalyceal anatomical structure by endocast, autopsy, ultrasonography and excretory urography, the modified Takazawa classification system based on 3D virtual reconstruction models enables to standardized different anatomical morphology of renal pelvicalyceal system and provide intuitive and concise information on anatomy, thus leading to the improvement in treatment modality.

A novel mycovirus belonging to the proposed family "Fusariviridae" was discovered in Alternaria Solani by sequencing a double-stranded RNA extracted from this phytopathogenic fungus. The virus was tentatively named “Alternaria solani fusarivirus 1” (AsFV1). AsFV1 has a single-stranded positive-sense (+ssRNA) genome of 6,845 nucleotides containing three open reading frames (ORFs) and a poly(A) tail. The largest ORF, ORF1 encodes a large polypeptide of 1,556 amino acids (aa) with conserved RNA-dependent RNA polymerase and helicase domains. The ORF2 and ORF3 have overlapping regions, encoding a putative protein of 522 amino acids (aa) and a putative protein of 105 amino acids (aa) respectively, for which function is unknown now. Multiple sequence alignments and phylogenetic analysis revealed AsFV1 belonging to Fusariviridae. This is the first report of the full-length nucleotide sequence of a fusarivirus infected with Alternaria solani.

Background To describe our technique for using an intraureteral injection of indocyanine green (ICG) and visualization under near-infrared fluorescence (NIRF) to facilitate challenging upper urinary tract reconstructions (UUTRs) and to present the comparative outcomes. Methods We collected 36 patients who underwent laparoscopic UUTRs between April 2019 and March 2020, and we divided the patients into two groups based on the use of ICG (ICG group and non-ICG group). Demographic characteristics, perioperative outcomes, and functional outcomes were compared between the two groups. Results There were 18 cases in the ICG group and 18 cases in the non-ICG group, respectively. There were no differences in the baseline characteristics between the two groups. The intraoperative time to identification of the ureter (TIU; 20.9±11.7 vs. 30.0±14.6 min, P=0.03) and length of postoperative hospital stay (LPHS; 11.1±3.0 vs. 16.6±10.0 days, P=0.03) were significantly shorter in the ICG group. There was also a trend for lesser time for locating the stricture (43.0±27.9 vs. 55.4±18.6 min, P=0.14) and lower estimated blood loss (EBL) in the ICG group patients (88.3±75.4 vs. 91.7±46.2 mL, P=0.22). During the mean 3.8-month follow-up for the ICG group and the 6.2-month for the non-ICG group, there was a trend for more severe complications in the non-ICG group. Conclusions Visualizing intraureteral ICG under NIRF is useful in challenging UUTRs, allows for rapid ureteral identification and accurate real-time delineation of the ureteral stricture margins, and provides encouraging follow-up outcomes compared with those in the non-ICG group.

A novel polymycovirus isolating from plant-pathogenic fungus Colletotrichum gloeosporioides was identified. The virus were composed nine RNA segments, ranging from 699 bp to 2,444 bp, and except that RNA5 contains two open reading frames (ORF5-1 and ORF5-2), other RNA segement contain only one ORF. In addition,the proteins encoded by ORFs 1-8 were homologous to the protein encoded by ORFs 1-8 in Colletotrichum camelliae filamentous virus 1(CcFV-1) by BLASTp. The amino acid sequence between the RNA-dependent RNA polymerase (RdRp) encoded by ORF1 and the viral methyltransferase encoded by ORF3 shared 87.6% and 83.3% identity with CcFV-1. The proline-alanine-serine-rich protein (PASrp) encoded by ORF4 shows 86.6% sequence homology to that of CcFV-1. The sequence homology of proteins encoded by ORFs2, 5-1, 6, 7 and 8 with the corresponding proteins of CcFV-1 was 86.6%, 82.5%, 89.0%, 45.7% and 95.5%, respectively. However, RNA9 is a defective RNA of RNA2, missing the 1556 bp (nt 519 to nt 2074) intermediate fragment of RNA2. Phylogenetic analysis based on RdRp protein indicated that the virus, which is the phylogenetically closest relative of CcFV-1, clustered with members of the family Polymycoviridae, so we tentatively named it "Colletotrichum gloeosporioides polymycovirus virus 1" (CgPmV1). To our knowledge, this is the first report of a defective polymycovirus in C. gloeosporioides.

The transforming growth factor-β (TGF-β) is a multifunctional cytokine critical for embryogenesis and tissue homeostasis. Alterations in TGF-β signaling pathway are observed in several types of malignant tumors and often related with cancer progression and metastasis. TGF-β signaling is transduced across the plasma membrane after ligand-receptor binding and consequent phosphorylation of the intracellular effectors SMAD2/3 by TGF-β receptors. Phosphorylated SMAD2/3 accumulates in the nucleus after complex formation with SMAD4 to act as transcription factors and regulate the expression of genes critically associated with cell proliferation and differentiation. Traditional methodologies used to assess TGF-β signaling pathway lack accuracy and/or show poor scalability, limiting in vitro experiments and almost excluding their use in vivo. Here, we describe a fast method to quantitate TGF-β signaling pathway activity in vitro and in vivo by using adenoviral reporters. Its implementation in vitro allows quantitating cell response to TGF-β at concentrations as low as pictograms/mL. Additionally, the use of an in vivo imaging system (IVIS) enables quantitating and monitoring TGF-β signaling pathway activity over time during cancer progression, eliminating the requirement of animal euthanasia at multiple time points for this purpose. Importantly, this protocol has been consistently used in different models and effectively led to the visualization and measurement of TGF-β activity levels. Improving the sensitivity, specificity, and scalability of methods focused on characterizing this and other molecular pathways will result in a better understanding of their biology in physiological and pathological processes.

Excitotoxicity is a neuronal death process initiated by over-stimulation of ionotropic glutamate receptors. Although dysregulation of proteolytic signaling networks is critical for excitotoxicity, the identity of affected proteins and mechanisms by which they induce neuronal cell death remain unclear. To address this, we used quantitative N-terminomics to identify proteins modified by proteolysis in neurons undergoing excitotoxic cell death. We found that most proteolytically processed proteins in excitotoxic neurons are likely substrates of calpains, including key synaptic regulatory proteins such as CRMP2, doublecortin-like kinase I, Src tyrosine kinase and calmodulin-dependent protein kinase IIβ (CaMKIIβ). Critically, calpain-catalyzed proteolytic processing of these proteins generates stable truncated fragments with altered activities that potentially contribute to neuronal death by perturbation of synaptic organization and function. Blocking calpain-mediated proteolysis of one of these proteins, Src protected against neuronal loss in a rat model of neurotoxicity. Extrapolation of our N-terminomic results led to the discovery that CaMKIIα, an isoform of CaMKIIβ undergoes differential processing in mouse brains under physiological conditions and during ischemic stroke. In summary, our findings inform excitotoxic neuronal death mechanism and suggest potential therapeutic strategies for neuroprotection.In BriefAmeen, et al. used a proteomic method called N-terminomics to identify proteolytic events occurring in neurons during excitotoxicity. They found that most proteolytic processing is mediated by calpains, resulting in the generation of stable truncated fragments with the potential to induce synaptic dysfunction and loss, eventually leading to neuronal death. They further showed that some of these proteolytic processed proteins, such as the protein kinases Src and CaMKII, are potential targets for neuroprotection.HighlightsIdentification of over 300 neuronal proteins cleaved by calpains to form stable truncated fragments during excitotoxicity.The calpain cleavage sites of these proteins unveil for the first time the preferred cleavage sequences of calpains in neurons.These pathological proteolytic events potentially induce synaptic dysfunction and loss, which likely contribute to excitotoxic neuronal death.Some of the neuronal proteins proteolyzed by calpains are potential targets of neuroprotection.Graphical abstract: Pathological proteolytic events in neurons during excitotoxicity unveiled by N-terminomic analyses(A) N-terminomic and global proteomic analyses identified neo-N-terminal sites and neuronal proteins undergoing significant abundance changes during excitotoxicity. (B) Informatic analysis of the proteomic results predicted (i) the preferred sequences of proteolytic processing of neuronal proteins catalyzed by calpains during excitotoxicity and (ii) perturbation of synaptic organization and functions as the major consequence of calpain-mediated proteolytic events. (C) Validation of these predictions and further experimentations unveiled: (i) calpain-mediated cleavage of proteins associated with synaptic damage in excitotoxic neurons, (ii) a new mechanism of dysregulation of CaMKIIα and CaMKIIβ, which are key protein kinases governing synaptic dysfunctions and excitotoxic neuronal death and (iii) potential therapeutic targets such as the protein kinases Src and CaMKII for neuroprotectionOne Sentence SummaryProteolytic events in neurons during excitotoxicity inform neuronal death mechanism and potential therapeutic strategies for neuroprotection.

To identify novel cancer therapies, the tumor microenvironment (TME) has received a lot of attention in recent years in particular with the advent of clinical successes achieved by targeting immune checkpoint inhibitors (ICIs). The TME consists of multiple cell types that are embedded in the extracellular matrix (ECM), including immune cells, endothelial cells and cancer associated fibroblasts (CAFs), which communicate with cancer cells and each other during tumor progression. CAFs are a dominant and heterogeneous cell type within the TME with a pivotal role in controlling cancer cell invasion and metastasis, immune evasion, angiogenesis and chemotherapy resistance. CAFs mediate their effects in part by remodeling the ECM and by secreting soluble factors and extracellular vesicles. Exosomes are a subtype of extracellular vesicles (EVs), which contain various biomolecules such as nucleic acids, lipids, and proteins. The biomolecules in exosomes can be transmitted from one to another cell, and thereby affect the behavior of the receiving cell. As exosomes are also present in circulation, their contents can also be explored as biomarkers for the diagnosis and prognosis of cancer patients. In this review, we concentrate on the role of CAFs-derived exosomes in the communication between CAFs and cancer cells and other cells of the TME. First, we introduce the multiple roles of CAFs in tumorigenesis. Thereafter, we discuss the ways CAFs communicate with cancer cells and interplay with other cells of the TME, and focus in particular on the role of exosomes. Then, we elaborate on the mechanisms by which CAFs-derived exosomes contribute to cancer progression, as well as and the clinical impact of exosomes. We conclude by discussing aspects of exosomes that deserve further investigation, including emerging insights into making treatment with immune checkpoint inhibitor blockade more efficient.

Hyperactivation of oncogenic pathways downstream of RAS and PI3K/AKT in normal cells induces a senescence-like phenotype that acts as a tumor-suppressive mechanism that must be overcome during transformation. We previously demonstrated that AKT-induced senescence (AIS) is associated with profound transcriptional and metabolic changes. Here, we demonstrate that human fibroblasts undergoing AIS display increased Cystathionine-β-synthase (CBS) expression and consequent activation of the transsulfuration pathway controlling hydrogen sulfide (H2S) and glutathione (GSH) metabolism. Activated transsulfuration pathway during AIS maintenance enhances the antioxidant capacity, protecting senescent cells from ROS-induced cell death via GSH and H2S. Importantly, CBS depletion allows cells that have undergone AIS to escape senescence and re-enter the cell cycle, indicating the importance of CBS activity in maintaining AIS. Mechanistically, we show this restoration of proliferation is mediated through suppressing mitochondrial respiration and reactive oxygen species (ROS) production and increasing GSH metabolism. These findings implicate a potential tumor-suppressive role for CBS in cells with inappropriately activated PI3K/AKT signaling. Consistent with this concept, in human gastric cancer cells with activated PI3K/AKT signaling, we demonstrate that CBS expression is suppressed due to promoter hypermethylation. CBS loss cooperates with activated PI3K/AKT signaling in promoting anchorage-independent growth of gastric epithelial cells, while CBS restoration suppresses the growth of gastric tumors in vivo. Taken together, we find that CBS is a novel regulator of AIS and a potential tumor suppressor in PI3K/AKT-driven gastric cancers, providing a new exploitable metabolic vulnerability in these cancers.

Background To analyze the safety and clinical significance of performing ureteroscopy after ureteral reconstruction with autologous onlay/graft. To describe the ureteroscopic appearances of the appendiceal onlay flap and lingual mucosa graft. Methods Beginning in August 2018, we conducted a prospective cohort study of autologous onlay/graft techniques to repair ureteral strictures. The perioperative data of 42 patients who had undergone surgery more than 6 months prior were collected prospectively. During the postoperative follow-up, ureteroscopy was performed in 27 patients (64.3%) after surgery (group A), and ureteroscopy was not performed in the other 15 patients (35.7%) (group B). We carried out a comparative study of these two groups of patients. Analyses were conducted mainly on complications related to ureteroscopy and the success rate of ureteral reconstruction surgery. Results There were no significant differences in patient demographic data or the length of ureteral reconstruction between the two groups (P>0.05). For the ureteroscopy group, the median time from repair surgery to ureteroscopy was 3 (range, 2-7) months, there was no poor healing of the anastomosis, and the ureteral lumen of all patients was unobstructed. Some expected observations can be found in the ureteral lumen, such as mucosa edema, stones, follicles and granulation tissue. Among the 27 patients, one patient (3.7%) developed bleeding intraoperatively and 7 patients (25.9%) were found to have low-grade (Clavien-Dindo I and II) postoperative complications, including 5 cases of fever and 2 cases of bleeding. The mean follow-up times of patients in group A and group B were 16.7±6.4 and 19.0±10.1 months, respectively. The objective success (imaging showed hydronephrosis ease) rate of the two groups was 100%. The subjective success (symptom relief) rates of group A and group B were 96.3% and 100%, respectively. Conclusions Patients after autologous onlay flap/graft ureteroplasty do not need to undergo routine ureteroscopy unless there is aggravation of hydronephrosis or other indications for ureteroscopy, such as stones.

In this study, a novel mycovirus designed Colletotrichum gloeosporioides ourmia-like virus 1 (CgOLV1) was isolated from a filamentous phytopathogenic fungus, Colletotrichum gloeosporioides. The virus has a genome of 2,516 nucleotides and contains a large open reading frame (ORF) encoding an RNA-dependent RNA polymerase (RdRp). Phylogenetic analysis indicated that CgOLV1 is located in the ourmia-like mycovirus clade, whose members are related to plant ourmiaviruses. To the best of our knowledge, this is the first report of an ourmia-like mycovirus in C. gloeosporioides.

Background The selection of treatment for bilateral ureteral strictures caused by radiotherapy in patients with gynecological tumors often brings great challenges to urologists. This study was designed to analyze the characteristics of radiation-induced ureteral strictures and summarize the surgical experience of bilateral ileal ureter substitution. Methods We retrospectively collected the medical records of 18 patients between June 2010 and June 2019 who had a radiation-induced bilateral ureteral strictures. Time interval from radiotherapy to the discovery of ureteral stricture was categorized into short-term (less than 12 months) and long-term (over 12 months) groups. All patients received reverse "7" bilateral ileal ureteral substitution. Surgical success was defined as no restenosis, relief of symptoms, and improved/stabilized hydronephrosis. Results The patients had been suffered from ureteral stricture for a median of 12 [2-106] months. The mean length of the left and right ureteral stricture was 9.6±2.6 and 8.8±3.2 cm, respectively. The mean length of the ileal graft was 29.1±7.4 cm. The mean operative time was 308.4±70.2 min, and the mean estimated blood loss was 254.7±166.2 mL. The postoperative hospital stays in the short-term group was significantly shorter than that in the long-term group (14.0 vs. 20.6 days, P=0.049). During a median follow-up time of 24.1 (4.5-71.9) months, the success rate was 94.4%. Eight minor complications and two major complications occurred in 7 patients. Conclusions Bilateral ileal ureter substitution can be an effective strategy for radiation-induced ureteral stricture in patients with gynecological tumors.

Epithelial-mesenchymal transition (EMT) describes an evolutionary conserved morphogenic process defined by loss of epithelial characteristics and acquisition of mesenchymal phenotype, and altered patterns of intercellular communication, leading to functional changes in cell migration and invasion. In this regard, we have previously reported that oncogenic H-Ras induced EMT in Madin-Darby Canine Kidney (MDCK) cells (21D1 cells) trigger changes in the protein distribution pattern in cells, exosomes, and soluble protein factors (secretome) which modulate the tumor microenvironment. Here, we report that shed microvesicles (also termed microparticles/ectosomes) secreted from MDCK cells following oncogenic H-Ras-induced EMT (21D1-sMVs) are biochemically distinct from exosomes and parental MDCK-sMVs. The protein spectra of RNA-binding proteins and mitochondrial proteins in 21D1-sMVs differ profoundly compared to those of exosomes, likewise proteins associated with suppression of anoikis. We show that 21D1-sMVs promote cell migration, confer anchorage-independent growth, and induce EMT in parental MDCK cells. An unexpected and novel finding was the selective sorting of tissue transglutaminase-2 (TGM2) into 21D1-sMVs; there was no evidence of TGM2 in MDCK-sMVs. Prior treatment of 21D1-sMVs with neutralizing anti-TGM2 or anti-FN1 antibodies attenuates the invasive capability of fibroblasts. These finding suggest that microvesicle-associated TGM2 may play an important contributory role in the EMT process and warrants further investigation.

Epithelial-mesenchymal transition (EMT) describes an evolutionary conserved morphogenic process defined by loss of epithelial characteristics and acquisition of mesenchymal phenotype, and altered patterns of intercellular communication, leading to functional changes in cell migration and invasion. In this regard, we have previously reported that oncogenic H-Ras induced EMT in MDCK cells (21D1 cells) trigger changes in the protein distribution pattern in cells, exosomes, and soluble protein factors (secretome) which modulate the tumour microenvironment. Here, we report that shed microvesicles (also termed microparticles/ ectosomes) secreted from MDCK cells following oncogenic H-Ras-induced EMT (21D1-sMVs) are biochemically distinct from exosomes and parental MDCK-sMVs. The protein spectra of RNA-binding proteins and mitochondrial proteins in 21D1-sMVs differ profoundly to those of exosomes, likewise proteins associated with suppression of anoikis. We show that 21D1-sMVs promote cell migration, confer anchorage-independent growth, and induce EMT in parental MDCK cells. An unexpected and novel finding was the selective sorting of tissue transglutaminase-2 (TGM2) into 21D1-sMVs; there was no evidence of TGM2 in MDCK-sMVs. Prior treatment of 21D1-sMVs with neutralizing anti-TGM2 or anti-FN1 antibodies attenuates the invasive capability of fibroblasts. These finding suggest that microvesicle-associated TGM2 may play an important contributory role in the EMT process and warrants further investigation. (199/ 200 words). This article is protected by copyright. All rights reserved.

Background This study is an initial comparative analysis of perioperative and intermediate-term functional outcomes between patients who underwent robot-assisted laparoscopic (RALS) or conventional laparoscopic surgery (LS). Materials and methods A total of 25 patients who underwent ileal ureter replacement (10 RALS and 15 LS) were followed by functional cine magnetic resonance urography (MRU) combined with a modified Whitaker test. Also, the characteristics, perioperative data and functional outcomes of the patients were compared. Results The estimated blood loss, postoperative hospital stay and time to oral intake were significantly lower in the RALS group. At the median 14-month follow-up, all the patients showed improved renal function and were symptom-free, with no signs of leakage or stenosis observed by cine MRU combined with a modified Whitaker test. Conclusions RALS with an extracorporeal bowel resection is feasible and appears to be safe, with quick postoperative recovery and encouraging outcomes.

Objectives: The aim of the study was to present our modified flap pyeloplasty techniques for recurrent ureteropelvic junction obstruction (UPJO) with a long proximal ureteral stricture and compare outcomes of laparoscopic and robotic procedures. Materials and Methods: Between March 2018 and January 2020, 21 patients underwent modified laparoscopic or robotic flap pyeloplasty for recurrent UPJO with a long proximal ureteral stricture. Our surgical modifications included the “wishbone” anastomosis and “ureteral plate” technique. Demographic, perioperative, and follow-up data were recorded and compared retrospectively between the groups. Success was defined as subjective pain alleviation and hydronephrosis improvement. Results: Thirteen modified laparoscopic flap pyeloplasty (mLFP) and 8 modified robotic flap pyeloplasty (mRFP) were performed successfully without conversion. mRFP tended to have shorter overall operative time (142.4 vs. 179.1 min, p = 0.122) and anastomosis time (43.1 vs. 61.0 min, p = 0.093) than mLFP. No difference was found in estimated blood loss (p = 0.723) and pararenal draining time (p = 0.175) between the groups. The mean postoperative hospital stay of mRFP was significantly shorter than that of mLFP (5.0 vs. 8.2 days, p = 0.015). No major complications occurred. During the mean follow-up of 17.9 months, the overall success rate was 90.5%, and there was no significant difference between 2 groups. Conclusions: The modified flap pyeloplasty could be considered a practical and effective treatment option with a high success rate for recurrent UPJO with a long proximal ureteral stricture, and the robotic procedures showed advantages of higher efficiency and faster recovery.

Objective: To share the technique of posteriorly augmented anastomotic ureteroplasty with lingual mucosal onlay grafts for long proximal ureteral strictures as well as our initial experience with t...

Here, we report the molecular characterization of a novel partitivirus from Phomopsis vexans strain PvHZ002, a plant-pathogenic fungus infecting eggplant. The virus was designated "Phomopsis vexans partitivirus 1" (PvPV1). PvPV1 contains two dsRNA segments, dsRNA1 and dsRNA2, which are 1,662 bp and 1,628 bp long, respectively. Each segment contains a single open reading frame, putatively encoding RNA-dependent RNA polymerase (dsRNA 1) and capsid protein (dsRNA 2). A homology search and phylogenetic analysis showed that PvPV1 clustered with viruses of the genus Deltapartitivirus of the family Partitiviridae.

To present our experience of laparoscopic and robotic ureteroplasty using onlay flap or graft for the management of long proximal or middle ureteral strictures and summarize our treatment strategies for these challenging scenarios. From March 2018 to January 2020, 53 patients with long proximal/middle ureteral strictures (2–6 cm) who underwent laparoscopic or robotic onlay flap/graft ureteroplasty were retrospectively enrolled. Different reconstruction techniques were chosen based on our management strategy: pelvic flap (PF) was the first choice for proximal stricture if pelvic tissue was sufficient for repair, while appendiceal flap (AF) was preferred over oral mucosa graft for both proximal and middle strictures. A total of 28 PFs, 9 AFs and 16 lingual mucosa grafts (LMGs) onlay ureteroplasty were performed successfully, with 33 laparoscopic procedures and 20 robotic procedures being undertaken. No intraoperative complications or conversion occurred. The median stricture length was 4 cm (range 2–6 cm). Compared with laparoscopic procedures, robotic procedures showed significantly shorter operative time (P = 0.008), shorter postoperative hospital stay (P = 0.011) but higher hospital cost (P

Metastasis is the leading cause of death for cancer patients. During cancer progression, the initial detachment of cells from the primary tumor and the later colonization of a secondary organ are characterized as limiting steps for metastasis. Epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) are opposite dynamic multistep processes that enable these critical events in metastasis by altering the phenotype of cancer cells and improving their ability to migrate, invade and seed at distant organs. Among the molecular pathways that promote tumorigenesis in late-stage cancers, transforming growth factor-beta (TGF-beta) is described as an EMT master inducer by controlling different genes and proteins related to cytoskeleton assembly, cell-cell attachment and extracellular matrix remodeling. Still, despite the successful outcomes of different TGF-beta pharmacological inhibitors in cell culture (in vitro) and animal models (in vivo), results in cancer clinical trials are poor or inconsistent at least, highlighting the existence of crucial components in human cancers that have not been properly explored. Here we review most recent findings to provide perspectives bridging the gap between on-target anti-TGF-beta therapiesin vitroand in pre-clinical models and the poor clinical outcomes in treating cancer patients. Specifically, we focus on (i) the dual roles of TGF-beta signaling in cancer metastasis; (ii) dynamic signaling; (iii) functional differences of TGF-beta free in solution vs. in exosomes; (iv) the regulatory effects of tumor microenvironment (TME) - particularly by cancer-associated fibroblasts - on TGF-beta signaling pathway. Clearly identifying and establishing those missing links may provide strategies to revitalize and clinically improve the efficacy of TGF-beta targeted therapies.

Here, a novel mycovirus, Botryosphaeria dothidea mitovirus 1 (BdMV1), was isolated from a phytopathogenic fungus, Botryosphaeria dothidea, and its molecular characteristics were determined. BdMV1 has a genome of 2,667 nt that contains a single large open reading frame (ORF) using the fungal mitochondrial genetic code. The ORF encodes an RNA-dependent RNA polymerase (RdRp) of 727 amino acids with a molecular mass of 81.64 kDa. BLASTp analysis revealed that the RdRp domain of BdMV1 has 39.59% and 39.18% sequence identity to Plasmopara viticola associated mitovirus 43 and Setosphaeria turcica mitovirus 1, respectively. Phylogenetic analysis further suggested that BdMV1 is a new member of the genus Mitovirus within the family Mitoviridae. To the best of our knowledge, this is the first report of a mitovirus in B. dothidea.

TGF beta-SMAD3 signaling is a major driving force for cancer metastasis, while BMP-SMAD1/5 signaling can counteract this response. Analysis of gene expression profiles revealed that an increased TGF beta-SMAD3 and a reduced BMP-SMAD1/5 targeted gene expression signature correlated with shortened distant metastasis free survival and overall survival of patients. At molecular levels, we discovered that TGF beta abolished BMP-induced SMAD1/5 activation in the highly-invasive breast cancer MDA-MB-231 cells, but to a less extent in the non-invasive cancer and normal breast cells. This suggests an inverse correlation between BMP signaling and invasiveness of tumor cells and TGF beta signaling acts in a double whammy fashion in driving cancer invasion and metastasis. Sustained ERK activation by TGF beta was specifically observed in MDA-MB-231 cells, and MEK inhibitor (MEKi) treatment restored BMP-SMAD1/5 signaling while not affecting SMAD2/3 activation. FK506 potently activated BMP, but not TGF beta signaling in breast cancer cells. MEKi or FK506 alone inhibited MDA-MB-231 extravasation in a zebrafish xenograft cancer model. Importantly, when administrated at suboptimal concentrations MEKi and FK506 strongly synergized in promoting BMP-SMAD1/5 signaling and inhibiting cancer cell extravasation. Furthermore, this combination of suboptimal concentrations treatment in a mouse tumor model resulted in real-time reduction of BMP-SMAD1/5 signaling in live tumors, and consequently potently inhibited tumor self-seeding, liver and bone metastasis, but not lung and brain metastasis. Mechanistically, it is the first time to identify BMP-SMAD1/5 signaling as an underlying molecular driver for organ-specific metastasis. Combining of MEKi and FK506, or their analogues, may be explored for clinical development of breast cancer.

Hyperactivation of SRC-family protein kinases (SFKs) contributes to the initiation and progression of human colorectal cancer (CRC). Since oncogenic mutations of SFK genes are rare in human CRC, we investigated if SFK hyperactivation is linked to dysregulation of their upstream inhibitors, C-terminal SRC kinase (CSK) and its homolog CSK-homologous kinase (CHK/MATK). We demonstrate that expression of CHK/MATK but not CSK was significantly downregulated in CRC cell lines and primary tumours compared to normal colonic tissue. Investigation of the mechanism by which CHK/MATK expression is down-regulated in CRC cells uncovered hypermethylation of the CHK/MATK promoter in CRC cell lines and primary tumours. Promoter methylation of CHK/MATK was also observed in several other tumour types. Consistent with epigenetic silencing of CHK/MATK, genetic deletion or pharmacological inhibition of DNA methyltransferases increased CHK/MATK mRNA expression in CHK/MATK-methylated colon cancer cell lines. SFKs were hyperactivated in CHK/MATK-methylated CRC cells despite expressing enzymatically active CSK, suggesting loss of CHK/MATK contributes to SFK hyperactivation. Re-expression of CHK/MATK in CRC cell lines led to reduction in SFK activity via a non-catalytic mechanism, a reduction in anchorage-independent growth, cell proliferation and migration in vitro, and a reduction in tumour growth and metastasis in a zebrafish embryo xenotransplantation model in vivo, collectively identifying CHK/MATK as a novel putative tumour suppressor gene in CRC. Furthermore, our discovery that CHK/MATK hypermethylation occurs in the majority of tumours warrants its further investigation as a diagnostic marker of CRC.

Comment on “USP26 regulates TGF‐β signaling by deubiquitinating and stabilizing SMAD7” by Kit Leng Lui et al. [Image: see text]

Purpose: Therapies directed to specific molecular targets are still unmet for patients with triple-negative breast cancer (TNBC). Deubiquitinases (DUB) are emerging drug targets. The identification of highly active DUBs in TNBC may lead to novel therapies. Experimental Design: Using DUB activity probes, we profiled global DUB activities in 52 breast cancer cell lines and 52 patients' tumor tissues. To validate our findings in vivo, we employed both zebrafish and murine breast cancer xenograft models. Cellular and molecular mechanisms were elucidated using in vivo and in vitro biochemical methods. A specific inhibitor was synthesized, and its biochemical and biological functions were assessed in a range of assays. Finally, we used patient sera samples to investigate clinical correlations. Results: Two DUB activity profiling approaches identified UCHL1 as being highly active in TNBC cell lines and aggressive tumors. Functionally, UCHL1 promoted metastasis in zebrafish and murine breast cancer xenograft models. Mechanistically, UCHL1 facilitates TGFβ signaling–induced metastasis by protecting TGFβ type I receptor and SMAD2 from ubiquitination. We found that these responses are potently suppressed by the specific UCHL1 inhibitor, 6RK73. Furthermore, UCHL1 levels were significantly increased in sera of patients with TNBC, and highly enriched in sera exosomes as well as TNBC cell–conditioned media. UCHL1-enriched exosomes stimulated breast cancer migration and extravasation, suggesting that UCHL1 may act in a paracrine manner to promote tumor progression. Conclusions: Our DUB activity profiling identified UCHL1 as a candidate oncoprotein that promotes TGFβ-induced breast cancer metastasis and may provide a potential target for TNBC treatment.