An engineered ligand-responsive Csy4 endoribonuclease controls transgene expression from Sendai virus vectors.

BACKGROUND: Viral vectors are attractive gene delivery vehicles because of their broad tropism, high transduction efficiency, and durable expression. With no risk of integration into the host genome, the vectors developed from RNA viruses such as Sendai virus (SeV) are especially promising. However, RNA-based vectors have limited applicability because they lack a convenient method to control transgene expression by an external inducer. RESULTS: We engineered a Csy4 switch in Sendai virus-based vectors by combining Csy4 endoribonuclease with mutant FKBP12 (DD: destabilizing domain) that becomes stabilized when a small chemical Shield1 is supplied. In this Shield1-responsive Csy4 (SrC) switch, Shield1 increases Csy4 fused with DD (DD-Csy4), which then cleaves and downregulates the transgene mRNA containing the Csy4 recognition sequence (Csy4RS). Moreover, when Csy4RS is inserted in the viral L gene, the SrC switch suppresses replication and transcription of the SeV vector in infected cells in a Shield1-dependent manner, thus enabling complete elimination of the vector from the cells. By temporally controlling BRN4 expression, a BRN4-expressing SeV vector equipped with the SrC switch achieves efficient, stepwise differentiation of embryonic stem cells into neural stem cells, and then into astrocytes. CONCLUSION: SeV-based vectors with the SrC switch should find wide applications in stem cell research, regenerative medicine, and gene therapy, especially when precise control of reprogramming factor expression is desirable.

Holmium laser enucleation of the prostate as a secondary surgery following prostatic urethral lift.

Introduction: Prostatic urethral lift is a treatment option for benign prostatic hyperplasia, yet information on surgeries following this procedure is scarce. Case presentation: A 71-year-old man with persistent urinary retention following prostatic urethral lift underwent a secondary holmium laser enucleation of the prostate. The morcellation procedure, complicated by the presence of implants from the previous surgery, broke the morcellator blade. The fragmented blade was successfully retrieved without any organ damage. Postoperatively, the patient encountered no complications and showed improvement in his urinary symptoms. Conclusion: This case highlights the potential risk of device breakage when a secondary surgery, specifically the morcellation process, is performed following prostatic urethral lift. Care must be taken to prevent interaction between the implants and the morcellator. Our case demonstrates the efficacy of holmium laser enucleation of the prostate as a salvage surgical intervention for patients in whom prostatic urethral lift has failed.

Analysis of distribution, collection, and confirmation of capacity dependency of small extracellular vesicles toward a therapy for liver cirrhosis.

BACKGROUND: The progression of liver fibrosis leads to portal hypertension and liver dysfunction. However, no antifibrotic agents have been approved for cirrhosis to date, making them an unmet medical need. Small extracellular vesicles (sEVs) of mesenchymal stem cells (MSCs) are among these candidate agents. In this study, we investigated the effects of sEVs of MSCs, analyzed their distribution in the liver post-administration, whether their effect was dose-dependent, and whether it was possible to collect a large number of sEVs. METHODS: sEVs expressing tdTomato were generated, and their uptake into constituent liver cells was observed in vitro, as well as their sites of uptake and cells in the liver using a mouse model of liver cirrhosis. The efficiency of sEV collection using tangential flow filtration (TFF) and changes in the therapeutic effects of sEVs in a volume-dependent manner were examined. RESULTS: The sEVs of MSCs accumulated mostly in macrophages in damaged areas of the liver. In addition, the therapeutic effect of sEVs was not necessarily dose-dependent, and it reached a plateau when the dosage exceeded a certain level. Furthermore, although ultracentrifugation was commonly used to collect sEVs for research purposes, we verified that TFF could be used for efficient sEV collection and that their effectiveness is not reduced. CONCLUSION: In this study, we identified some unknown aspects regarding the dynamics, collection, and capacity dependence of sEVs. Our results provide important fundamentals for the development of therapies using sEVs and hold potential implications for the therapeutic applications of sEV-based therapies for liver cirrhosis.

Leiomyoma of the Prostate Treated via Holmium Laser Enucleation: A Case Report and Literature Review.

Leiomyoma is a rare tumor that arises from mesenchymal cells, with few reported cases of treatment using holmium laser enucleation of the prostate. A 74-year-old man with dysuria had a mass near the bladder neck in magnetic resonance imaging; this entity was suspected to be a leiomyoma. The patient underwent holmium laser enucleation of the prostate and one lobe was removed. However, the mass was firm and morcellation was difficult to break into small pieces. Therefore, it was fragmented via trans-urethral resection and removed with a curette. The postoperative course was favorable, with a positive clinical outcome. This case highlights the efficacy of holmium laser enucleation of the prostate in the management of prostatic leiomyoma and emphasizes its importance as a viable treatment option.

Surface modulation of extracellular vesicles with cell-penetrating peptide-conjugated lipids for improvement of intracellular delivery to endothelial cells.

Exosomes (diameter 30-200 nm) are a subtype of extracellular vesicles secreted by cells containing DNA, microRNA (miRNA), and proteins. Exosomes are expected to be valuable as a means of delivering drugs or functional miRNAs in treatment of diseases. However, the delivery of exosomes is not sufficiently effective, even though exosomes have intrinsic delivery functions. Cell-penetrating peptides (CPPs) are short peptide families that facilitate cellular intake of molecules and vesicles. We previously reported that the modification of cells, and liposomes with CPP-conjugated-lipids, CPPs conjugated with poly (ethylene glycol)-conjugated phospholipids (PEG-lipid), that induce adhesion by CPPs, can be useful for cell-based assays and harvesting liposomes. In this study, we aimed to modulate the exosome surface using Tat peptide (YGRKKRRQRRR)-PEG-lipids to improve intracellular delivery to endothelial cells. We isolated and characterized exosomes from the medium of HEK 293 T cell cultures. Tat conjugated PEG-lipids with different spacer molecular weights and lipid types were incorporated into exosomes using fluorescein isothiocyanate labeling to optimize the number of Tat-PEG-lipids immobilized on the exosome surface. The exosomes modified with Tat-PEG-lipids were incubated with human umbilical vein endothelial cells (HUVECs) to study the interaction. Tat conjugated with 5 kDa PEG and C16 lipids incorporated on the exosome surface were highly detected inside HUVECs by flow cytometry. Fluorescence was negligible in HUVECs for control groups. Thus, Tat-PEG-lipids can be modified on the exosome surface, improving the intracellular delivery of exosomes.

Simplified approach to the medial internal iliac region using a uretero-hypogastric nerve fascia development procedure for extended pelvic lymph node dissection during robot-assisted radical prostatectomy for high-risk prostate cancer.

INTRODUCTION: Although several clinical guidelines for prostate cancer (PC) recommend extended pelvic lymph node dissection (ePLND) during radical prostatectomy for high-risk cases, there are several issues to consider, including certain technical aspects. A simplified approach to the medial internal iliac region and paravesical arteries has not been established. The uretero-hypogastric nerve fascia (UHF) envelopes the ureter, hypogastric nerve, and pelvic autonomic nerves. To preserve the UHF, it is possible to approach the medial side of the internal iliac vessels without injuring any important tissue. We analyzed technical feasibility and lymph node (LN) yields. PATIENTS AND METHODS: After obtaining institutional review board approval, 265 high-risk PC patients with ePLND were identified. A da Vinci S or Xi robotic surgical system (Intuitive Surgical, Sunnyvale, CA, USA) was used. We divided the patients into conventional (non-UHF) method and modified (UHF) groups. The numbers of LNs removed, procedure-related complications, and surgical outcomes were analyzed. RESULTS: The median number of LNs removed was 19.0 in the non-UHF group and 22.0 in the UHF group (p = 0.004). Significantly more LNs were removed from the internal iliac region in the UHF group (p = 0.042). There was no difference in overall operative, console, or LN dissection time, or the severe complication rate (Clavien-Dindo grade ≥ III), between the non-UHF and UHF groups. CONCLUSIONS: Our simplified approach using the UHF development technique is technically feasible, has no major complications, and allows for the removal of significantly more LNs compared with the conventional method.

Rapid and Highly Sensitive Method for Evaluating Surface Coatings against an Enveloped RNA Virus.

The COVID-19 pandemic has increased public health vigilance worldwide. The coronavirus (SARS-CoV-2) can spread via aerosols, and droplet-borne viruses remain viable on nonliving surfaces for long duration. Hence, effective antiviral coatings are highly useful in eliminating viral persistence on nonliving surfaces. Although innovative antiviral coatings have been designed, conventional procedures for antiviral assays are generally laborious, time-consuming, and have a high limit of detection. In the present study, we report a rapid and highly sensitive method for evaluating antiviral coatings by measuring the luciferase activity derived from recombinant Sendai virus (SeV). The physicochemical characteristics of SeV, which has a single-stranded RNA genome encapsulated within a lipid envelope, allow us to exploit it as an indicator of the physicochemical potential of coating materials against enveloped RNA viruses in general. We demonstrate that SeV-based assay systems allow for the rapid and quantitative evaluation of the surface coatings composed of iodine solubilized in polyvinyl acetate. Additionally, we have investigated the effect of mucins, the dominant protein component of saliva, on the antiviral activity of surface coatings. The presence of mucins in the SeV suspension considerably rescues luciferase activity at the viral-surface interface, presumably due to mucin-mediated viral protection. Our findings provide insights into a procedure capable of the rapid evaluation and optimization of surface coatings, and suggest an important role of the mucin in the valid evaluation of antiviral agents.

Tendon-Specific Dicer Deficient Mice Exhibit Hypoplastic Tendon Through the Downregulation of Tendon-Related Genes and MicroRNAs.

Tendon is a fibrous connective tissue, that is, transmitting the forces that permit body movement. However, tendon/ligament biology is still not fully understood and especially, the role of miRNAs in tendon/ligament is sparse and uncharacterized in in vivo models. The objectives of this study were to address the function of DICER using mice with tendon/ligament-specific deletion of Dicer (Dicer conditional knockout; cKO), and to identify key miRNAs in tendon/ligament. Dicer cKO mice exhibited hypoplastic tendons through structurally abnormal collagen fibrils with downregulation of tendon-related genes. The fragility of tendon did not significantly affect the tensile strength of tendon in Dicer cKO mice, but they showed larger dorsiflexion angle in gait compared with Control mice. We identified two miRNAs, miR-135a and miR-1247, which were highly expressed in the Achilles tendon of Control mice and were downregulated in the Achilles tendon of Dicer cKO mice compared with Control mice. miR-135a mimic increased the expression of tendon-related genes in injured Achilles tendon-derived fibroblasts. In this study, Dicer cKO mice exhibited immature tendons in which collagen fibrils have small diameter with the downregulation of tendon-related genes such as transcriptional factor, extracellular matrix, and miRNAs. Thus, DICER plays an important role in tendon maturation, and miR-135a may have the potential to become key miRNA for tendon maturation and healing.

Live-cell imaging of microRNA expression with post-transcriptional feedback control.

MicroRNAs (miRNAs) are small noncoding RNAs that regulate complex gene expression networks in eukaryotic cells. Because of their unique expression patterns, miRNAs are potential molecular markers for specific cell states. Although a system capable of imaging miRNA in living cells is needed to visually detect miRNA expression, very few fluorescence signal-on sensors that respond to expression of target miRNA (miR-ON sensors) are available. Here we report an miR-ON sensor containing a bidirectional promoter-driven Csy4 endoribonuclease and green fluorescent protein, ZsGreen1, for live-cell imaging of miRNAs with post-transcriptional feedback control. Csy4-assisted miR-ON (Csy4-miR-ON) sensors generate negligible background but respond sensitively to target miRNAs, allowing high-contrast fluorescence detection of miRNAs in various human cells. We show that Csy4-miR-ON sensors enabled imaging of various miRNAs, including miR-21, miR-302a, and miR-133, in vitro as well as in vivo. This robust tool can be used to evaluate miRNA expression in diverse biological and medical applications.

A large adrenocortical adenoma surrounded with a renal arteriovenous malformation.

INTRODUCTION: Large adrenal adenomas are clinically rare. We report a case of a large adrenal adenoma with a renal arteriovenous malformation, mimicking a malignant adrenal tumor in preoperative imaging. CASE PRESENTATION: A 66-year-old woman presented to a local hospital with abdominal pain. A right adrenal tumor was detected, 66 mm in diameter and surrounded by thick and tortuous vessels. Based on the imaging findings, pheochromocytoma was suspected. However, clinical symptoms and endocrine abnormalities were absent, and radionuclide accumulation in scintigraphy was negative. Laparoscopic right adrenalectomy was performed. Intraoperatively, a notable growth of vessels forming a nidus surrounding the tumor was observed. Pathologically, this was diagnosed as an adrenocortical adenoma in conjunction with a renal arteriovenous malformation. CONCLUSION: We report a case of a large adrenal tumor surrounded with an arteriovenous malformation. To the best of our knowledge, this is the first reported case of this combination.

Optimizing first trocar access for robot-assisted radical prostatectomy: Optical trocar access through the upper abdominal quadrant using the Kii Fios First Entry trocar.

OBJECTIVES: A pre-equipped metal trocar is required to use as a camera trocar due to a specification change in the da Vinci X/Xi system (Intuitive Surgical). We observed slight slippage of a trocar placed by the open method. With optical trocar access (OTA), the initial trocar is viewed directly with a laparoscope during placement. Reports regarding OTA for robotic surgery are limited, particularly for robot-assisted radical prostatectomy (RARP). We modified the OTA procedure such that it was appropriate for RARP. PATIENTS AND METHODS: A total of 158 patients were enrolled in this study. The first trocar placement time (FTPT) was compared between the open and OTA groups. In the OTA group, the trocar was mainly placed through the upper abdominal quadrant. We also analyzed the differences between the conventional and modified OTA procedures using the Kii Fios First Entry trocar (Applied Medical). We examined the factors affecting the FTPT using linear regression models. A P value <.05 was considered significant. RESULTS: The FTPT was significantly shorter in the OTA group than the open group (P < .0001). The modified method was associated with a shorter FTPT (P = .0001). None of the patient characteristics affected the FTPT in either group. No major complications were observed. CONCLUSIONS: OTA was applied successfully during RARP. Use of the Kii Fios First Entry trocar with upper abdominal quadrant placement was suitable for RARP.

Association of short sleep duration and long media use with caries in school children.

BACKGROUND: Individual lifestyle factors are associated with dental caries in children. The aim of this study was to investigate comprehensively the association between dental caries in elementary school children and lifestyle factors of the children and their parents. METHODS: Children from five elementary schools in Takaoka city, Toyama Prefecture, Japan (1,699: 848 boys, 851 girls, age range 6-12 years) participated in a survey conducted in March 2016. Questions on socioeconomic status (SES) and lifestyle factors in the children and their parents were included. Children who received treatment for three or more dental caries were defined as having many caries. Logistic regression analysis was performed to evaluate whether these factors were associated with the number of caries. RESULTS: The percentage of children with many caries (≥3) was 38.4%. In univariate analysis, being in 5th or 6th grade, frequent snacking, short sleep duration, long hours of media use, paternal smoking, and parental skipping of breakfast were significantly associated with many caries. Lack of affluence was marginally significant. In multivariate analysis, the association of short hours of sleep and long hours of media use remained significant. The adjusted odds ratio (OR) for media use of 1-2 h and more than 2 h were 1.79 (95% confidence interval [CI]: 1.30-2.46) and 2.24 (95% CI: 1.61-3.11) respectively. The adjusted OR for less than 8 h of sleep was 1.49 (95% CI: 1.07-2.06). CONCLUSIONS: Long duration of media use and short duration of sleep were associated with dental caries. Establishing a healthy lifestyle is necessary for preventing dental caries in children.

A Sendai Virus-Based Cytoplasmic RNA Vector as a Novel Platform for Long-Term Expression of MicroRNAs.

Cytoplasmic RNA virus-derived vectors have emerged as attractive vehicles for microRNA (miRNA) delivery as they possess no potential risk of chromosomal insertion. However, their relatively short-term expression limits their use in biological applications that require long-term miRNA manipulation, such as somatic cell reprogramming. Here, we show that a cytoplasmic RNA virus vector based on a replication-defective and persistent Sendai virus (SeVdp) serves as an effective platform for long-term production of miRNAs capable of inducing sequence-specific target suppression. The SeVdp vector was able to simultaneously deliver embryonic stem cell-enriched miRNAs, as well as multiple transcription factors, into fibroblasts, resulting in effective reprogramming into induced pluripotent stem cells. Furthermore, we report that the murine miR-367 hairpin produced elevated levels of mature miRNA when it was incorporated into the SeVdp vector and served as an effective backbone for production of artificial miRNAs. These SeVdp vector-derived artificial miRNAs efficiently inhibited expression of target genes. Our findings provide novel insights into a powerful tool for long-term and targeted gene silencing in areas such as regenerative medicine, gene therapy, and cell therapy.

Cefiderocol (S-649266), A new siderophore cephalosporin exhibiting potent activities against Pseudomonas aeruginosa and other gram-negative pathogens including multi-drug resistant bacteria: Structure activity relationship.

The structure-activity relationship (SAR) for a novel series of catechol conjugated siderophore cephalosporins is described with their in vitro activities against multi-drug resistant Gram-negative pathogens including Pseudomonas aeruginosa, Acinetobacter baumannii, Stenotrophomonas maltophilia and Enterobacteriaceae. Cefiderocol (3) was one of the best molecules which displayed well-balanced and potent activities against multi-drug resistant Gram-negative pathogens including carbapenem resistant bacteria among the prepared compounds with the modified C-7 side chain and the modified C-3 side chain. Cefiderocol (3) is a highly promising parenteral cephalosporin for the treatment of multi-drug resistant Gram-negative infection.

Endoscopic Treatment of Intrauterine Device Migration into the Bladder with Stone Formation.

Background: An intrauterine device is commonly used for contraception globally. Although intrauterine device placement is an effective and safe method of contraception, migration into the bladder with stone formation is a rare and serious complication. The management approaches for an intrauterine device embedded in the bladder include endoscopic procedures and open surgical removal. In this study, we report the case of a patient with recurrent urinary tract infection associated with intrauterine device migration and urolithiasis, who successfully underwent endoscopic treatment combined with laser fragmentation. Case Presentation: A 22-year-old woman presented to our hospital with a 1-month history of lower abdominal pain, hematuria, and pain on urination. Transvaginal ultrasound showed a hyperechoic lesion in the bladder. A plain abdominal radiograph showed the presence of a T-shaped intrauterine device with calculus formation in the pelvis. CT revealed a vesical stone fixed to the top of the bladder wall, and there was no vesicovaginal fistula formation. She had undergone intrauterine device insertion several years previously. Cystoscopy confirmed the diagnosis. She underwent endoscopic lithotripsy, and the intrauterine device was extracted from the bladder wall. Repair of the bladder wall and disappearance of symptoms were confirmed. Conclusion: Endoscopic treatment combined with laser fragmentation of stones surrounding a migrated intrauterine device should be considered as a minimally invasive approach, which can be performed safely.

Sensitive and long-term monitoring of intracellular microRNAs using a non-integrating cytoplasmic RNA vector.

MicroRNAs (miRNAs) are small noncoding RNAs that modulate gene expression at the post-transcriptional level. Different types of cells express unique sets of miRNAs that can be exploited as potential molecular markers to identify specific cell types. Among the variety of miRNA detection methods, a fluorescence-based imaging system that utilises a fluorescent-reporter gene regulated by a target miRNA offers a major advantage for long-term tracking of the miRNA in living cells. In this study, we developed a novel fluorescence-based miRNA-monitoring system using a non-integrating cytoplasmic RNA vector based on a replication-defective and persistent Sendai virus (SeVdp). Because SeVdp vectors robustly and stably express transgenes, this system enabled sensitive monitoring of miRNAs by fluorescence microscopy. By applying this system for cellular reprogramming, we found that miR-124, but not miR-9, was significantly upregulated during direct neuronal conversion. Additionally, we were able to isolate integration-free human induced pluripotent stem cells by long-term tracking of let-7 expression. Notably, this system was easily expandable to allow detection of multiple miRNAs separately and simultaneously. Our findings provide insight into a powerful tool for evaluating miRNA expression during the cellular reprogramming process and for isolating reprogrammed cells potentially useful for medical applications.

Simple and effective generation of transgene-free induced pluripotent stem cells using an auto-erasable Sendai virus vector responding to microRNA-302.

Transgene-free induced pluripotent stem cells (iPSCs) are valuable for both basic research and potential clinical applications. We previously reported that a replication-defective and persistent Sendai virus (SeVdp) vector harboring four reprogramming factors (SeVdp-iPS) can efficiently induce generation of transgene-free iPSCs. This vector can express all four factors stably and simultaneously without chromosomal integration and can be eliminated completely from reprogrammed cells by suppressing vector-derived RNA-dependent RNA polymerase. Here, we describe an improved SeVdp-iPS vector (SeVdp(KOSM)302L) that is automatically erased in response to microRNA-302 (miR-302), uniquely expressed in pluripotent stem cells (PSCs). Gene expression and genome replication of the SeVdp-302L vector, which contains miRNA-302a target sequences at the 3' untranslated region of L mRNA, are strongly suppressed in PSCs. Consequently, SeVdp(KOSM)302L induces expression of reprogramming factors in somatic cells, while it is automatically erased from cells successfully reprogrammed to express miR-302. As this vector can reprogram somatic cells into transgene-free iPSCs without the aid of exogenous short interfering RNA (siRNA), the results we present here demonstrate that this vector may become an invaluable tool for the generation of human iPSCs for future clinical applications.

Transplantation of allogeneic adipose-derived mesenchymal stem cells in a cerebral palsy patient (Retracted).

This article was retracted on 25/08/2017.

Novel Strategy to Control Transgene Expression Mediated by a Sendai Virus-Based Vector Using a Nonstructural C Protein and Endogenous MicroRNAs.

Tissue-specific control of gene expression is an invaluable tool for studying various biological processes and medical applications. Efficient regulatory systems have been utilized to control transgene expression in various types of DNA viral or integrating viral vectors. However, existing regulatory systems are difficult to transfer into negative-strand RNA virus vector platforms because of significant differences in their transcriptional machineries. In this study, we developed a novel strategy for regulating transgene expression mediated by a cytoplasmic RNA vector based on a replication-defective and persistent Sendai virus (SeVdp). Because of the capacity of Sendai virus (SeV) nonstructural C proteins to specifically inhibit viral RNA synthesis, overexpression of C protein significantly reduced transgene expression mediated by SeVdp vectors. We found that SeV C overexpression concomitantly reduced SeVdp mRNA levels and genomic RNA synthesis. To control C expression, target sequences for an endogenous microRNA were incorporated into the 3' untranslated region of the C genes. Incorporation of target sequences for miR-21 into the SeVdp vector restored transgene expression in HeLa cells by decreasing C expression. Furthermore, the SeVdp vector containing target sequences for let-7a enabled cell-specific control of transgene expression in human fibroblasts and induced pluripotent stem cells. Our findings demonstrate that SeV C can be used as an effective regulator for controlling transgene expression. This strategy will contribute to efficient and less toxic SeVdp-mediated gene transfer in various biological applications.