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Supplementary Figure 1 from Degradation of Tob1 Mediated by SCFSkp2-Dependent Ubiquitination

The cyclin-dependent kinase inhibitor p27Kip1 is degraded in late G1 phase by the ubiquitin-proteasome pathway, allowing cells to enter S phase. Due to accelerated degradation of p27Kip1, various human cancers express low levels of p27Kip1 associated with poor prognosis. S-phase kinase–associated protein 2, the F-box protein component of an SCF ubiquitin ligase complex, is implicated in degradation of p27Kip1 during S-G2 phases. Recently, Kip1 ubiquitination–promoting complex has been reported as another ubiquitin ligase that targets cytoplasmic p27Kip1 exported from the nucleus in G0-G1 phases. Here, we identified a RING-H2–type ubiquitin ligase, Pirh2, as a p27Kip1-interacting protein. Endogenous Pirh2 physically interacted with endogenous p27Kip1 in mammalian cells. Pirh2 directly ubiquitinated p27Kip1 in an intact RING finger domain-dependent manner in vivo, as well as in vitro. Ablation of endogenous Pirh2 by small interfering RNA increased the steady-state level of p27Kip1 and decelerated p27Kip1 turnover. Depletion of Pirh2 induced accumulation of p27Kip1 in both the nucleus and cytoplasm. Pirh2 expression was induced from late G1-S phase, whereas p27Kip1 was decreased in synchronization with accumulation of Pirh2. Furthermore, reduction of Pirh2 resulted in an impairment of p27Kip1 degradation and an inhibition of cell cycle progression at G1-S transition in a p53-independent manner. Overall, the results indicate that Pirh2 acts as a negative regulator of p27Kip1 function by promoting ubiquitin-dependent proteasomal degradation. [Cancer Res 2007;67(22):10789–95]

Supplementary Figures 1-6 from Up-regulation of GPR48 Induced by Down-regulation of p27Kip1 Enhances Carcinoma Cell Invasiveness and Metastasis

Supplementary Figure 1 from Pirh2 Promotes Ubiquitin-Dependent Degradation of the Cyclin-Dependent Kinase Inhibitor p27Kip1

Tob1, a member of the Tob/BTG family, is involved in the control of G1-S progression by suppressing cyclin D1 expression and acts as a tumor suppressor gene. Tob1 was reported to have a quick turnover through the ubiquitin-proteasome pathway, but proteins involved in this process are still unknown. We showed that Skp2, a substrate-targeting subunit of the SCF (Skp1/Cul1/F-box protein) ubiquitin ligase complex, was involved in ubiquitin-dependent degradation of Tob1. Skp2 interacted with Tob1 and facilitated ubiquitination of Tob1 in intact cells as well as in vitro. Skp2 mutants without the F-box or leucine rich repeat were not able to bind to Tob1 and did not enhance ubiquitination of Tob1. Tob1 was stabilized in both Skp2−/− mouse fibroblasts and Skp2 knockdown HeLa cells. Moreover, cyclin D1 expression was suppressed in Skp2 knockdown HeLa cells. These data suggest that Tob1 is a novel target for degradation by the SCF-Skp2 ubiquitin ligase. (Cancer Res 2006; 66(17): 8477-83)

A reduced expression level of the cyclin-dependent kinase inhibitor p27Kip1 is associated with increased tumor malignancy and poor prognosis in individuals with various types of cancer. To investigate the basis for this relation, we applied microarray analysis to screen for genes differentially expressed between p27+/− and parental (p27+/+) HCT116 human colon carcinoma cells. Expression of the gene for G protein–coupled receptor 48 (GPR48) was increased in the p27+/− cells. Forced expression of GPR48 increased both in vitro invasive activity and lung metastasis potency of HCT116 cells. In contrast, depletion of endogenous GPR48 by RNA interference reduced the invasive potential of HeLa and Lewis lung carcinoma cells not only in vitro but also in vivo. Moreover, GPR48 expression was significantly associated with lymph node metastasis and inversely correlated with p27 expression in human colon carcinomas. GPR48 may thus play an important role in invasiveness and metastasis of carcinoma and might therefore represent a potential prognostic marker or therapeutic target. (Cancer Res 2006; 66(24): 11623-31)

Supplementary Figure 2 from Degradation of Tob1 Mediated by SCFSkp2-Dependent Ubiquitination

We propose a generalization of the rolling guidance filter (RGF) to a similarity-based clustering (SBC) algorithm which can handle general vector data. The proposed RGF-based SBC algorithm makes the similarities between data clearer than the original similarity values computed from the original data. On the basis of the similarity values, we assign cluster labels to data by an SBC algorithm. Experimental results show that the proposed algorithm achieves better clustering result than the result by the naive application of the SBC algorithm to the original similarity values. Additionally, we study the convergence of a unimodal vector dataset to its mean vector.

Terrestrial plants respond to and resist gravitational force. The response is termed "gravity resistance", and centrifugal hypergravity conditions are efficient for investigating its nature and mechanism. A functional screening of Arabidopsis T-DNA insertion lines for the suppression rate of elongation growth of hypocotyls under hypergravity conditions was performed in this study to identify the genes required for gravity resistance. As a result, we identified

Urinary retention and hematuria owing to radiation-induced mucositis are occasional late adverse events in patients with prostate cancer. Moreover, radiation-induced secondary malignancies are late adverse events, although they are extremely rare. Herein, we describe a case of radiation-induced secondary malignancy of the prostate that was initially difficult to distinguish from radiation mucositis. A 74-year-old man with prostate cancer underwent brachytherapy and external beam radiotherapy 9 years ago. Twenty-eight months after irradiation, he presented with urinary retention and hematuria owing to radiation mucositis and underwent transurethral resection of the prostate. At 89 months after irradiation, the patient again showed urinary retention and hematuria. The cause of urinary retention and hematuria could not be identified on cystoscopy. Despite receiving medications, the patient’s symptoms did not improve. Therefore, transurethral fulguration was performed, and prostate biopsy revealed spindle cell sarcoma. A diagnosis of radiation-induced undifferentiated pleomorphic/spindle cell sarcoma was made, and the patient underwent total cystectomy and construction of the ileal conduit. Two weeks after the surgery, computed tomography revealed peritoneal dissemination. The patient died 5 weeks after the surgery. The case findings indicate that clinicians should consider the possibility of radiation-induced secondary malignancy; moreover, thorough pathological examination of the prostate with CT and MRI is important to distinguish RISM from radiation mucositis even if no tumors are found on cystoscopy.

Background Inflammatory bowel disease, such as Crohn's disease and ulcerative colitis, is characterized by chronic intestinal inflammation leading to intestinal mucosal damage. Inflammatory bowel disease causes dysregulation of mucosal T cell responses, especially the responses of CD4+ T cells. Previously, we demonstrated that indoleamine-2,3-dioxygenase plays an immunosuppressive role in 2,4,6-trinitrobenzene sulfate (TNBS)-induced colitis. Although indoleamine-2,3-dioxygenase exerts immunosuppressive effects by altering the local concentration of tryptophan (Trp) and immunomodulatory Trp metabolites, the specific changes in immune regulation during colitis caused by Trp metabolites and its related enzymes remain unclear. Aim To investigate role of kynurenine 3-monooxygenase (KMO) in TNBS-induced colitis and involvement of Trp metabolites in maintenance of intestinal homeostasis. Methods Colitis was induced in eight-week-old male KMO+/+ or KMO-/- mice of C57BL/6N background using TNBS. Three days later, the colon was used for hematoxylin-eosin staining for histological grading, immunohistochemical or immunofluorescence staining for KMO, cytokines, and immune cells. Inflammatory and anti-inflammatory cytokines were measured using quantitative RT-PCR, and kynurenine (Kyn) pathway metabolites were measured by high-performance liquid chromatography. The cell proportions of colonic lamina propria and mesenteric lymph nodes were analyzed by flow cytometry. Results KMO expression levels in the colonic mononuclear phagocytes, including dendritic cells and macrophages increased upon TNBS induction. Notably, KMO deficiency reduced TNBS-induced colitis, resulting in an increased frequency of Foxp3+ regulatory T cells and increased mRNA and protein levels of anti-inflammatory cytokines, including transforming growth factor-β and interleukin-10. Conclusion Absence of KMO reduced TNBS-induced colitis via generation of Foxp3+ regulatory T cells by producing Kyn. Thus, Kyn may play a therapeutic role in colon protection during colitis.

In our previous report, intravitreal injection using 0.25% povidone-iodine to irrigate the conjunctival sac together with pre- and post-injection topical antibiotics achieved an incidence of post-injection endophthalmitis significantly lower than other reports. In this study, we examined whether similarly low incidence is achieved without using any topical antibiotics. Prospective cohort study. We evaluated intravitreal injections of anti-vascular endothelial growth factor agents conducted by vitreoretinal specialists at the outpatient injection room of a single university hospital. This study had two protocols. First stage: We performed more than 3000 injections with pre-injection but without post-injection topical antibiotics. Final stage: After confirming no case of endophthalmitis in the first stage, we performed more than 12,500 injections without either pre- or post-injection topical antibiotics. In both protocols, we used 0.25% povidone-iodine to sterilize the conjunctival sac both before and after injection. First stage was performed between April 2015 and January 2016. No case of suspected or proven infectious endophthalmitis occurred in 6039 injections [95% confidence interval (CI) 0–0.000497%]. Final stage was performed between February 2016 and November 2017. No case of suspected or proven infectious endophthalmitis occurred in 12,523 injections (95% CI 0–0.00024%). This result was comparable to our previous study using both pre- and post-injection topical antibiotics (0/15,144 injections, 95% CI 0–0.000198%). Using conjunctival sac irrigation with 0.25% povidone-iodine before and after intravitreal injection, the incidence of endophthalmitis remains low even when the use of pre- or post-injection topical antibiotics is discontinued.

Background Renal artery stenting is performed for renal artery injuries to preserve renal function and prevent renovascular hypertension. However, its indications are controversial and its long-term prognosis remains unknown. Here, we evaluate the characteristics and long-term outcomes of renal artery stenting for blunt renal artery injuries at our institution. Methods We retrospectively reviewed patients with blunt renal artery injuries who had been treated with stenting over a 12-year period at our institution. Five patients (three men and two women) were included. Results Trauma resulted from falls in three patients and motor vehicle accidents in two. All patients had experienced multiple injuries (median injury severity score, 24 [range, 16-48]; median revised trauma score, 5.9672 [4.0936-7.8408]; and median probability of survival, 0.689 [0.533-0.980]). All renal artery injuries involved stenosis because of traumatic arterial dissection or intimal tear; no cases of total occlusion were observed. No complications due to the intervention itself were observed. Although two patients developed reversible acute renal failure, none required long-term hemodialysis. One patient with renovascular hypertension was treated with antihypertensive agents for a month and subsequently became normotensive without further medication. All patients underwent postoperative computed tomography, which revealed no stent occlusion or renal atrophy. Renal scintigraphy for three patients demonstrated preserved differential renal function. All five patients survived. Conclusions Renal artery stenting for hemodynamically stable blunt renal artery injuries with stenosis is suggested to be safe and helps in avoiding long-term hemodialysis and renovascular hypertension.

In prostate stereotactic body radiation therapy (SBRT), hydrogel spacers are increasingly used. This study aimed to perform a dosimetry comparison of treatment plans using CyberKnife (CK), commonly used for prostate SBRT, Helical TomoTherapy (HT), and TrueBeam (TB) in patients with hydrogel spacer implantations. The data of 20 patients who received hydrogel spacer implantation for prostate SBRT were retrospectively analyzed. The prescription dose was 36.25 Gy in five fractions to 95% of the planning target volume (PTV; D95). The conformity index (CI), gradient index (GI), homogeneity index (HI), and dose‐volume histogram (DVH) were analyzed for the three modalities, using the same PTV margins. The monitor unit (MU) and the beam‐on‐time (BOT) values were subsequently compared. The CI of TB (0.93 ± 0.02) was significantly superior to those of CK (0.82 ± 0.03, p

Fluorescence labeling of the target molecules using a small molecule-based probe is superior than a method using genetically expressed green fluorescence protein (GFP) in terms of convenience in its preparation and functionalization. Fluorophore-nitrilotriacetic acid (NTA) conjugates with several ester protecting groups were synthesized and evaluated for their cell membrane permeability by fluorescence microscopy analysis. One of the derivatives, acetoxymethyl (AM)-protected NTA conjugate is hydrolyzed, resulting in intracellular accumulation, thus providing localized fluorescence intensity in cells. This modification is expected as an effective method for converting a non-cell membrane permeable NTA-BODIPY conjugates to a cell membrane permeable derivatives.

Shiga toxin (Stx) is a main virulence factor of Enterohemorrhagic Escherichia coli (EHEC) that causes diarrhea and hemorrhagic colitis and occasionally fatal systemic complications. Stx induces rapid apoptotic cell death in some cells, such as human myelogenous leukemia THP-1 cells expressing CD77, a receptor for Stx internalization, and the induction of apoptotic cell death is thought to be crucial for the fatal systemic complications. Therefore, in order to suppress the fatal toxicity, it is important to understand the mechanism how cells can escape from apoptotic cell death in the presence of Stx. In this study, we isolated resistant clones to Stx-induced apoptosis from highly sensitive THP-1 cells by continuous exposure with lethal dose of Stx. All of the ten resistant clones lost the expression of CD77 as a consequence of the reduction in CD77 synthase mRNA expression. These results suggest that downregulation of CD77 or CD77 synthase expression could be a novel approach to suppress the fatal toxicity of Stx in EHEC infected patient.

Chronic myelogenous leukemia (CML) is characterized by the oncogenic fusion protein, BCR-ABL protein kinase, against which clinically useful inhibitors have been developed. An alternative approach to treat CML is to degrade the BCR-ABL protein. Recently, potent degraders against BCR-ABL have been developed by conjugating dasatinib to ligands for E3 ubiquitin ligases. Since the degraders contain the dasatinib moiety, they also inhibit BCR-ABL kinase activity, which complicates our understanding of the impact of BCR-ABL degradation by degraders in CML growth inhibition. To address this issue, we chose DAS-IAP, as a potent BCR-ABL degrader, and developed a structurally related inactive degrader, DAS-meIAP, which inhibits kinase activity but does not degrade the BCR-ABL protein. DAS-IAP showed slightly weaker activity than DAS-meIAP in inhibiting cell growth when CML cells were treated for 48 h. However, DAS-IAP showed sustained growth inhibition even when the drug was removed after short-term treatment, whereas CML cell growth rapidly resumed following removal of DAS-meIAP and dasatinib. Consistently, suppression of BCR-ABL levels and downstream kinase signaling were maintained after DAS-IAP removal, whereas kinase signaling rapidly recovered following removal of DAS-meIAP and dasatinib. These results indicate that BCR-ABL degrader shows more sustained inhibition of CML cell growth than ABL kinase inhibitor.

Aberrant expression of proteins often underlies many diseases, including cancer. A recently developed approach in drug development is small molecule-mediated, selective degradation of dysregulated proteins. We have devised a protein-knockdown system that utilizes chimeric molecules termed specific and nongenetic IAP-dependent protein erasers (SNIPERs) to induce ubiquitylation and proteasomal degradation of various target proteins. SNIPER(ER)-87 consists of an inhibitor of apoptosis protein (IAP) ligand LCL161 derivative that is conjugated to the estrogen receptor α (ERα) ligand 4-hydroxytamoxifen by a PEG linker, and we have previously reported that this SNIPER efficiently degrades the ERα protein. Here, we report that derivatization of the IAP ligand module yields SNIPER(ER)s with superior protein-knockdown activity. These improved SNIPER(ER)s exhibited higher binding affinities to IAPs and induced more potent degradation of ERα than does SNIPER(ER)-87. Further, they induced simultaneous degradation of cellular inhibitor of apoptosis protein 1 (cIAP1) and delayed degradation of X-linked IAP (XIAP). Notably, these reengineered SNIPER(ER)s efficiently induced apoptosis in MCF-7 human breast cancer cells that require IAPs for continued cellular survival. We found that one of these molecules, SNIPER(ER)-110, inhibits the growth of MCF-7 tumor xenografts in mice more potently than the previously characterized SNIPER(ER)-87. Mechanistic analysis revealed that our novel SNIPER(ER)s preferentially recruit XIAP, rather than cIAP1, to degrade ERα. Our results suggest that derivatized IAP ligands could facilitate further development of SNIPERs with potent protein-knockdown and cytocidal activities against cancer cells requiring IAPs for survival.

Objective This study aimed to evaluate the usefulness of a new wet laboratory (wet lab) system using porcine eyes with eyelids. Design Teaching device trial. Participants Porcine eyes with orbital tissues and eyelids. Methods Twenty porcine eyes with orbital tissues and eyelids were enucleated from pigs butchered at age 6 months. These eyes were positioned in the eye sockets of a model head and stabilized with a pin. Eye draping, dressing with tape, and speculum placement were conducted. The vertical and horizontal widths of the palpebra under the speculum setting were compared with those of 55 patients who underwent cataract surgery. The rotation and torsion of the porcine eye in the new wet lab system were also compared with those of a conventional wet lab system. For comparison with actual cataract surgery, 5 ophthalmologists, including residents, were asked to respond to a questionnaire survey. Results The horizontal widths of the palpebra under the speculum setting were 27.5 ± 3.1 mm in porcine eyes and 28.6 ± 5.1 mm in human eyes, and the vertical widths were 16.9 ± 1.3 mm and 16.1 ± 1.5 mm (p = 0.53, 0.05). The amounts of rotation and torsion were significantly greater with the new wet lab system. Ophthalmologists evaluated the new wet lab system as being more realistic than the conventional system, in terms of both natural eye movement and restriction of the surgical field by the eyelid and the speculum. Conclusions Wet lab training using porcine eyes with eyelids is more practical than older systems as it reproduces an ocular surgical field very similar to that of humans.

In recent years, the induction of target-protein degradation via the ubiquitin-proteasome system (UPS) mediated by small molecules has attracted attention, and this approach has applications in pharmaceutical development. However, this technique requires a ligand for the target protein that can be incorporated into tailor-made molecules, and there are many proteins for which such ligands have not been found. In this study, we developed a protein-knockdown method that recognizes a His-tag fused to a protein of interest. This strategy theoretically allows comprehensive targeting of proteins of interest by a particular molecule recognizing the tag. As expected, our hybrid molecule 10 [SNIPER(CH6)] efficiently degraded His-tagged CRABP-II and Smad2 in cells. This system provides an easy method to determine the susceptibility of proteins of interest to UPS-mediated degradation. Furthermore, we hope that this method will become an efficient tool to analyze the function of the UPS.

We previously developed a hybrid small molecule SNIPER (Specific and Nongenetic IAP-dependent Protein ERaser) against transforming acidic coiled-coil-3 (TACC3), SNIPER(TACC3), that induces proteasomal degradation of TACC3 protein. In this study, we found that SNIPER(TACC3) induces cytoplasmic vacuolization derived from endoplasmic reticulum (ER) and paraptosis-like cell death selectively in cancer cells. Mechanistic analysis suggests that accumulation of ubiquitylated protein aggregates that requires X-linked inhibitor of apoptosis protein (XIAP) induces ER stress, which results in ER-stress responses involving X-box binding protein-1 (XBP-1) and ER-derived vacuolization in cancer cells. Importantly, inhibition of proteasome enhanced the SNIPER(TACC3)-induced vacuolization, and the combination treatment of SNIPER(TACC3) and bortezomib exhibited a synergistic anticancer activity in several cancer cell lines. The induction of paraptosis-like cell death in cancer cells by SNIPER(TACC3) could be applied to treat cancer cells resistant to undergo apoptosis by overexpression of XIAP.

Purpose To verify that ocular surface irrigation with 0.025% povidone-iodine (PI) or 0.0025% polyvinyl alcohol-iodine (PAI) during cataract surgery minimizes bacterial contamination of the anterior chamber. Methods The study was a prospective, interventional case series. First, the bactericidal effect of PI or PAI against Staphylococcus aureus was evaluated in vitro. Next, in 400 eyes undergoing cataract surgery, the ocular surface was irrigated every 20 seconds during surgery with balanced salt solution (BSS; 200 eyes) or BSS containing 0.025% PI (100 eyes) or 0.0025% PAI (100 eyes). At the completion of surgery, anterior chamber fluid was cultured bacteriologically. Visual acuity (VA) and corneal endothelial cell density were measured before and 7 days after surgery. Results A marked bactericidal effect was observed when S. aureus was directly exposed for 15 seconds to 0.01% PI or 0.001% PAI diluted in BSS. When the two solutions were stored at room temperature, bactericidal effect did not attenuate after 60 min. The bacterial detection rate at the completion of surgery was significantly reduced in 0.025% PI (0%, 0/100 eyes) or 0.0025% PAI group (0%, 0/100 eyes) compared to BSS group (5%, 10/200 eyes) (p = 0.0340). No differences in postoperative visual acuity and postoperative corneal endothelial cell density were observed between three groups. Conclusion In cataract surgery, irrigation every 20 seconds of the operative field with 0.025% PI or 0.0025% PAI, both of which contain 0.0025% available iodine concentration, achieved a very low bacterial contamination rate in the anterior chamber.

Many diseases, especially cancers, result from aberrant or overexpression of pathogenic proteins. Specific inhibitors against these proteins have shown remarkable therapeutic effects, but these are limited mainly to enzymes. An alternative approach that may have utility in drug development relies on selective degradation of pathogenic proteins via small chimeric molecules linking an E3 ubiquitin ligase to the targeted protein for proteasomal degradation. To this end, we recently developed a protein knockdown system based on hybrid small molecule SNIPERs (Specific and Nongenetic IAP-dependent Protein Erasers) that recruit inhibitor of the apoptosis protein (IAP) ubiquitin ligases to specifically degrade targeted proteins. Here, we extend our previous study to show a proof of concept of the SNIPER technology in vivo. By incorporating a high affinity IAP ligand, we developed a novel SNIPER against estrogen receptor α (ERα), SNIPER(ER)-87, that has a potent protein knockdown activity. The SNIPER(ER) reduced ERα levels in tumor xenografts and suppressed the growth of ERα-positive breast tumors in mice. Mechanistically, it preferentially recruits X-linked IAP (XIAP) rather than cellular IAP1, to degrade ERα via the ubiquitin-proteasome pathway. With this IAP ligand, potent SNIPERs against other pathogenic proteins, BCR-ABL, bromodomain-containing protein 4 (BRD4), and phosphodiesterase-4 (PDE4) could also be developed. These results indicate that forced ubiquitylation by SNIPERs is a useful method to achieve efficient protein knockdown with potential therapeutic activities and could also be applied to study the role of ubiquitylation in many cellular processes.

Chromosomal translocation occurs in some cancer cells, resulting in the expression of aberrant oncogenic fusion proteins that include BCR-ABL in chronic myelogenous leukemia (CML). Inhibitors of ABL tyrosine kinase, such as imatinib and dasatinib, exhibit remarkable therapeutic effects, although emergence of drug resistance hampers the therapy during long-term treatment. An alternative approach to treat CML is to downregulate expression of the BCR-ABL protein. Recently, we have devised a protein knockdown system by hybrid molecules named Specific and Nongenetic inhibitor of apoptosis protein [IAP]-dependent Protein Erasers (SNIPER). This system is designed to induce IAP-mediated ubiquitylation and proteasomal degradation of target proteins. In this review, we describe the development of SNIPER against BCR-ABL, and discuss the features and prospect for treatment of CML.

A patient with acquired optic disc pit (ODP) maculopathy underwent vitrectomy with anterior capsule transplantation to the ODP and gas tamponade. Structural changes were evaluated by enhanced depth imaging optical coherence (OCT) tomography. During vitrectomy, the eye was confirmed to have preexisting posterior vitreous detachment. Postoperative OCT showed complete closure of the optic pit resulting in rapid absorption of subretinal fluid. The authors' observations suggest that the anterior capsule is a useful material for achieving optic pit closure. To the authors' knowledge, this is the first report describing application of the anterior capsule to the treatment of ODP maculopathy. [ Ophthalmic Surg Lasers Imaging Retina. 2019;50:649–652.]

Purpose To investigate the safety and effectiveness of intravitreal injection (IVI) of 1.25% povidone iodine (PI) followed by vitrectomy using 0.025% PI irrigation for treating endophthalmitis. Methods Prospective case series study. Nine eyes of 8 patients with postoperative or endogenous endophthalmitis treated at the Nihon University Hospital between April 2015 and October 2017 were studied. First, IVI of 0.1 mL/1.25%PI was conducted, followed by anterior chamber irrigation and vitrectomy using 0.025%PI irrigation solution. Corneal epithelial damage, anterior chamber inflammation, and vitreous inflammation were assessed and fundus examinations were performed, using a slit-lamp microscope and indirect ophthalmoscopy. A specular microscope, Goldmann perimetry, and electroretinography (ERG) were also used. Results In all but case 7, endophthalmitis resolved rapidly and good visual acuity was maintained. No adverse events were noted. Furthermore, the perioperative ERG showed improvements in the oscillatory potentials amplitudes on ERG and flicker ERG, as well as in the implicit time of the a-wave, suggesting functional recovery in the retinal outer and inner layers after therapy. Conclusions IVI of PI followed by vitrectomy was thought to be a safe and effective treatment for endophthalmitis. Translational relevance We succeeded in proving the clinical safety of IVI of PI followed by vitrectomy with PI irrigation as well as previous experimental reports. PI is available in world widely, therefore this method will be optimal treatment for endophthalmitis.

Selective degradation of pathogenic proteins by small molecules in cells is a novel approach for development of therapeutic agents against various diseases, including cancer. We and others have developed a protein knockdown technology with a series of hybrid small compounds, called SNIPERs (Specific and Nongenetic IAP-dependent Protein ERasers); and peptidic chimeric molecules, called PROTACs (proteolysis-targeting chimeric molecules), which induce selective degradation of target proteins via the ubiquitin-proteasome pathway. These compounds include two different ligands connected by a linker; one is a ligand for a ubiquitin ligase and the other is a ligand for the target protein, which are expected to crosslink these proteins in cells. Theoretically, any cytosolic protein can be targeted for degradation by this technology. To date, several SNIPERs and PROTACs against various oncogenic proteins have been developed, which specifically induce polyubiquitylation and proteasomal degradation of the oncogenic proteins, resulting in cell death, growth arrest, or impaired migration of cancer cells. Thus, this protein knockdown technology has a great potential for cancer therapy.

Purpose: To investigate the usefulness of 0.16 mg/0.05 mL intravitreal bevacizumab (IVB) injection 1 day before vitrectomy for proliferative diabetic retinopathy. Methods: Sixty-two patients with proliferative diabetic retinopathy (66 eyes) with an indication for primary vitrectomy were randomized to IVB group (34 eyes) or sham control group (32 eyes). Intravitreal bevacizumab group received intravitreal injection of 0.16 mg/0.05 mL bevacizumab, and sham control group received sham injection 1 day before vitrectomy. Vitreous fluid was sampled before vitrectomy was started. Results: Frequency of reoperation due to recurrent vitreous hemorrhage within 4 weeks after surgery was significantly lower (P = 0.033) in IVB group (3.1%, 1/32) than in sham control group (20.6%, 7/34). The number of intraoperative endodiathermy spots (0.63 ± 1.0 vs. 1.3 ± 1.4, P = 0.025) and frequency of postoperative recurrent vitreous hemorrhage (3.1%, 1/32 vs. 23.5%, 8/34, P = 0.017) were significantly lower in IVB group than in sham control group. Vitreous vascular endothelial growth factor concentrations were 1315.3 ± 1153.4 pg/mL in sham control group and 25.0 ± 13.6 pg/mL in IVB group (P Conclusion: Intravitreal injection of 0.16 mg/0.05 mL bevacizumab 1 day before vitrectomy blocked vascular endothelial growth factor production in vitreous and significantly reduced the incidence of reoperation due to early postoperative recurrent vitreous hemorrhage.

To investigate the bactericidal effect of 0.025% povidone-iodine in Balanced Salt Solution PLUS (0.025% PI-BSS PLUS) and its use in vitrectomy for postoperative endophthalmitis.First, an experimental laboratory model using Staphylococcus aureus was used to evaluate the bactericidal effect of PI-BSS PLUS. Next, in a case series of 4 eyes with postoperative endophthalmitis, vitrectomy using 0.025% PI-BSS PLUS as irrigation solution was conducted, followed by postoperative intravitreal and intravenous antibiotics.In in vitro study, PI at concentrations of 0.01% and above in BSS PLUS exhibited marked bactericidal effect after 15 seconds of exposure. Bactericidal effect of 0.025% PI-BSS PLUS was maintained at room temperature storage for 15 minutes but was attenuated after 30 minutes. Among 4 eyes that underwent vitrectomy using 0.025% PI-BSS PLUS, coagulase-negative Staphylococcus sp. was isolated in 1 eye at the beginning but not at completion of surgery. In all four eyes, endophthalmitis was resolved with no adverse events. Ocular toxicity was not observed.The 0.025% PI-BSS PLUS is bactericidal and nontoxic when used as irrigation solution in vitrectomy. In 4 cases of postoperative endophthalmitis, vitrectomy using 0.025% PI-BSS PLUS followed by postoperative antibiotics resolved endophthalmitis.

Background Japanese (JPN) guidelines for the management of acute pancreatitis were published in 2006. The severity assessment criteria for acute pancreatitis were later revised by the Japanese Ministry of Health, Labour and Welfare (MHLW) in 2008, leading to their publication as the JPN Guidelines 2010. Following the 2012 revision of the Atlanta Classifications of Acute Pancreatitis, in which the classifications of regional complications of pancreatitis were revised, the development of a minimally invasive method for local complications of pancreatitis spread, and emerging evidence was gathered and revised into the JPN Guidelines. Methods A comprehensive evaluation was carried out on the evidence for epidemiology, diagnosis, severity, treatment, post-endoscopic retrograde cholangiopancreatography (ERCP) pancreatitis and clinical indicators, based on the concepts of the GRADE system (Grading of Recommendations Assessment, Development and Evaluation). With the graded recommendations, where the evidence was unclear, Meta-Analysis team for JPN Guidelines 2015 conducted an additional new meta-analysis, the results of which were included in the guidelines. Results Thirty-nine questions were prepared in 17 subject areas, for which 43 recommendations were made. The 17 subject areas were: Diagnosis, Diagnostic imaging, Etiology, Severity assessment, Transfer indication, Fluid therapy, Nasogastric tube, Pain control, Antibiotics prophylaxis, Protease inhibitor, Nutritional support, Intensive care, management of Biliary Pancreatitis, management of Abdominal Compartment Syndrome, Interventions for the local complications, Post-ERCP pancreatitis and Clinical Indicator (Pancreatitis Bundles 2015). Meta-analysis was conducted in the following four subject areas based on randomized controlled trials: (1) prophylactic antibiotics use; (2) prophylactic pancreatic stent placement for the prevention of post-ERCP pancreatitis; (3) prophylactic non-steroidal anti-inflammatory drugs (NSAIDs) for the prevention of post-ERCP pancreatitis; and (4) peritoneal lavage. Using the results of the meta-analysis, recommendations were graded to create useful information. In addition, a mobile application was developed, which made it possible to diagnose, assess severity and check pancreatitis bundles. Conclusions The JPN Guidelines 2015 were prepared using the most up-to-date methods, and including the latest recommended medical treatments, and we are confident that this will make them easy for many clinicians to use, and will provide a useful tool in the decision-making process for the treatment of patients, and optimal medical support. The free mobile application and calculator for the JPN Guidelines 2015 is available via http://www.jshbps.jp/en/guideline/jpn-guideline2015.html

Highlights • Shiga toxin (Stx) rapidly reduces the level of short-lived anti-apoptotic proteins. • Stx induces activation of caspase 9 and apoptosis. • Proteasome inhibitors prevent the reduction of anti-apoptotic proteins. • Proteasome inhibitors suppress Stx-induced apoptosis. • Bortezomib prolongs the survival of mice challenged with a lethal dose of Stx., Shiga toxin (Stx) causes fatal systemic complications. Stx induces apoptosis, but the mechanism of which is unclear. We report that Stx induced rapid reduction of short-lived anti-apoptotic proteins followed by activation of caspase 9 and the progression of apoptosis. Proteasome inhibitors prevented the reduction of anti-apoptotic proteins, and inhibited caspase activation and apoptosis, suggesting that the reduction of anti-apoptotic proteins is a prerequisite for Stx-induced apoptosis. A clinically approved proteasome inhibitor, bortezomib, prolonged the survival of mice challenged by Stx. These results imply that proteasome inhibition may be a novel approach to prevent the fatal effects of Stx.

Protein degradation technology based on hybrid small molecules is an emerging drug modality that has significant potential in drug discovery and as a unique method of post-translational protein knockdown in the field of chemical biology. Here, we report the first example of a novel and potent protein degradation inducer that binds to an allosteric site of the oncogenic BCR-ABL protein. BCR-ABL allosteric ligands were incorporated into the SNIPER (Specific and Nongenetic inhibitor of apoptosis protein [IAP]-dependent Protein Erasers) platform, and a series of in vitro biological assays of binding affinity, target protein modulation, signal transduction, and growth inhibition were carried out. One of the designed compounds, 6 (SNIPER(ABL)-062), showed desirable binding affinities against ABL1, cIAP1/2, and XIAP and consequently caused potent BCR-ABL degradation.

We designed and synthesized hybrid molecules for a protein knockdown method based on the recognition of a His-tag fused to a protein of interest (POI). The synthesized target protein degradation inducers contained three functional moieties: a His-tag ligand (nickel nitrilotriacetic acid [Ni-NTA]), an E3 ligand (bestatin [BS] or MV1), and a carrier peptide (Tat or nonaarginine [R9]). The designed hybrid molecules, BS-Tat-Ni-NTA, MV1-Tat-Ni-NTA, BS-R9-Ni-NTA, and MV1-R9-Ni-NTA, efficiently degraded His-tagged cellular retinoic acid binding protein 2 via the ubiquitin-proteasome system (UPS). This system will become a useful tool for research into selective protein degradation inducers that act via the UPS.

Targeted protein degradation using small molecules is a novel strategy for drug development. We have developed hybrid molecules named specific and nongenetic inhibitor of apoptosis protein [IAP]-dependent protein erasers (SNIPERs) that recruit IAP ubiquitin ligases to degrade target proteins. Here, we show novel SNIPERs capable of inducing proteasomal degradation of the androgen receptor (AR). Through derivatization of the SNIPER(AR) molecule at the AR ligand and IAP ligand and linker, we developed 42a (SNIPER(AR)-51), which shows effective protein knockdown activity against AR. Consistent with the degradation of the AR protein, 42a inhibits AR-mediated gene expression and proliferation of androgen-dependent prostate cancer cells. In addition, 42a efficiently induces caspase activation and apoptosis in prostate cancer cells, which was not observed in the cells treated with AR antagonists. These results suggest that SNIPER(AR)s could be leads for an anticancer drug against prostate cancers that exhibit AR-dependent proliferation.

Chromosomal translocation occurs in some cancer cells, which results in the expression of aberrant oncogenic fusion proteins that include BCR-ABL in chronic myelogenous leukemia (CML). Inhibitors of ABL tyrosine kinase, such as imatinib and dasatinib, exhibit remarkable therapeutic effects, although emergence of drug resistance hampers the therapy during long-term treatment. An alternative approach to treat CML is to downregulate the BCR-ABL protein. We have devised a protein knockdown system by hybrid molecules named Specific and Non-genetic inhibitor of apoptosis protein [IAP]-dependent Protein Erasers (SNIPER), which is designed to induce IAP-mediated ubiquitylation and proteasomal degradation of target proteins, and a couple of SNIPER(ABL) against BCR-ABL protein have been developed recently. In this study, we tested various combinations of ABL inhibitors and IAP ligands, and the linker was optimized for protein knockdown activity of SNIPER(ABL). The resulting SNIPER(ABL)-39, in which dasatinib is conjugated to an IAP ligand LCL161 derivative by polyethylene glycol (PEG) × 3 linker, shows a potent activity to degrade the BCR-ABL protein. Mechanistic analysis suggested that both cellular inhibitor of apoptosis protein 1 (cIAP1) and X-linked inhibitor of apoptosis protein (XIAP) play a role in the degradation of BCR-ABL protein. Consistent with the degradation of BCR-ABL protein, the SNIPER(ABL)-39 inhibited the phosphorylation of signal transducer and activator of transcription 5 (STAT5) and Crk like proto-oncogene (CrkL), and suppressed the growth of BCR-ABL-positive CML cells. These results suggest that SNIPER(ABL)-39 could be a candidate for a degradation-based novel anti-cancer drug against BCR-ABL-positive CML.