Cryo-EM structure of the transposon-associated TnpB enzyme.

The class 2 type V CRISPR effector Cas12 is thought to have evolved from the IS200/IS605 superfamily of transposon-associated TnpB proteins1. Recent studies have identified TnpB proteins as miniature RNA-guided DNA endonucleases2,3. TnpB associates with a single, long RNA (ωRNA) and cleaves double-stranded DNA targets complementary to the ωRNA guide. However, the RNA-guided DNA cleavage mechanism of TnpB and its evolutionary relationship with Cas12 enzymes remain unknown. Here we report the cryo-electron microscopy (cryo-EM) structure of Deinococcus radiodurans ISDra2 TnpB in complex with its cognate ωRNA and target DNA. In the structure, the ωRNA adopts an unexpected architecture and forms a pseudoknot, which is conserved among all guide RNAs of Cas12 enzymes. Furthermore, the structure, along with our functional analysis, reveals how the compact TnpB recognizes the ωRNA and cleaves target DNA complementary to the guide. A structural comparison of TnpB with Cas12 enzymes suggests that CRISPR-Cas12 effectors acquired an ability to recognize the protospacer-adjacent motif-distal end of the guide RNA-target DNA heteroduplex, by either asymmetric dimer formation or diverse REC2 insertions, enabling engagement in CRISPR-Cas adaptive immunity. Collectively, our findings provide mechanistic insights into TnpB function and advance our understanding of the evolution from transposon-encoded TnpB proteins to CRISPR-Cas12 effectors.

Liquid Chromatography-Mass Spectrometry-Based Qualitative Profiling of mRNA Therapeutic Reagents Using Stable Isotope-Labeled Standards Followed by the Automatic Quantitation Software Ariadne.

mRNA-based medicines are a promising modality for preventing virus-caused illnesses, including COVID-19, and treating various types of cancer and genetic diseases. To develop such medicines, methods to characterize long mRNA molecules are needed for quality control and metabolic analysis. Here, we developed an analytical platform based on isotope-dilution liquid chromatography-mass spectrometry (LC-MS) that quantitatively characterizes long, modified mRNAs by comparing them to a stable isotope-labeled reference with an identical sequence to that of the target medicine. This platform also includes database searching using the mass spectra as a query, which allowed us to confirm the primary structures of 200 to 4300 nt mRNAs including chemical modifications, with sequence coverage at 100%, to detect/identify defects in the sequences, and to define the efficiencies of the 5'-capping and integrity of the polyadenylated tail. Our findings indicated that this platform should be valuable for quantitatively characterizing mRNA vaccines and other mRNA medicines.

An alpaca single-domain antibody (VHH) phage display library constructed by CDR shuffling provided high-affinity VHHs against desired protein antigens.

Antigen-combining sites of the camelid heavy-chain antibody variable domain (VHH) are constructed by three complementarity-determining regions (CDR1, CDR2 and CDR3). We prepared cDNA using mRNA extracted from peripheral lymphocytes of alpacas that had been non-immunized or immunized with human serum albumin (HSA). The VHH gene fragments encoding the amino-terminal half-containing CDR1 as well as CDR2 and the carboxy-terminal half-containing CDR3 were amplified independently by PCR, and then full-length VHH gene fragments were generated by overlap extension PCR and cloned into the phagemid vector. This protocol, referred to as CDR shuffling, allowed us to construct an alpaca VHH phage display library possessing repertoires different from those naturally occurring in animals. We asked, first, whether this library was able to provide the functional VHH fragments against HSA, an immunized antigen, and obtained 29 anti-HSA VHH clones, 41% possessed KD values of lower than 10-8 M, 5 of which had KD values of 10-10 M. We also obtained VHH clones against non-immunized protein antigens such as cardiac troponin T and I, Ebola virus glycoprotein 1 and human immunoglobulin G by biopanning. We compared the amino acid sequences and affinities and found that 43% of VHHs had KD values of less than 10-8 M, although those having KD values of 10-10 M were unavailable. These results suggested that the CDR-shuffled VHH phage display library could potentially provide VHHs against non-immunized protein antigens with similar levels of affinities to those against immunized antigens.

Combination therapy with novel androgen receptor antagonists and statin for castration-resistant prostate cancer.

BACKGROUND: One of the growth mechanisms of castration-resistant prostate cancer (CRPC) is de novo androgen synthesis from intracellular cholesterol, and statins may be able to inhibit this mechanism. In addition, statins have been reported to suppress the expression of androgen receptors (ARs) in prostate cancer cell lines. In this study, we investigated a combination therapy of novel AR antagonists and statin, simvastatin, for CRPC. METHODS: LNCaP, 22Rv1, and PC-3 human prostate cancer cell lines were used. We developed androgen-independent LNCaP cells (LNCaP-LA). Microarray analysis was performed, followed by pathway analysis, and mRNA and protein expression was evaluated by quantitative real-time polymerase chain reaction and Western blot analysis, respectively. Cell viability was determined by MTS assay and cell counts. All evaluations were performed on cells treated with simvastatin and with or without AR antagonists (enzalutamide, apalutamide, and darolutamide). RESULTS: The combination of darolutamide and simvastatin most significantly suppressed proliferation in LNCaP-LA and 22Rv1 cells. In a 22Rv1-derived mouse xenograft model, the combination of darolutamide and simvastatin enhanced the inhibition of cell proliferation. In LNCaP-LA cells, the combination of darolutamide and simvastatin led to reduction in the mRNA expression of the androgen-stimulated genes, KLK2 and PSA; however, this reduction in expression did not occur in 22Rv1 cells. The microarray data and pathway analyses showed that the number of differentially expressed genes in the darolutamide and simvastatin-treated 22Rv1 cells was the highest in the pathway termed "role of cell cycle." Consequently, we focused our efforts on the cell cycle regulator polo-like kinase 1 (PLK1), cyclin-dependent kinase 2 (CDK2), and cell cycle division 25C (CDC25C). In 22Rv1 cells, the combination of darolutamide and simvastatin suppressed the mRNA and protein expression of these three genes. In addition, in PC-3 cells (which lack AR expression), the combination of simvastatin and darolutamide enhanced the suppression of cell proliferation and expression of these genes. CONCLUSIONS: Simvastatin alters the expression of many genes involved in the cell cycle in CRPC cells. Thus, the combination of novel AR antagonists (darolutamide) and simvastatin can potentially affect CRPC growth through both androgen-dependent and androgen-independent mechanisms.

Presepsin as a predictor of septic shock in patients with urinary tract infection.

BACKGROUND: Recently, presepsin has been reported to be a useful biomarker for early diagnosis of sepsis and evaluation of prognosis in septic patients. However, few reports have evaluated its usefulness in patients with urinary tract infections (UTI). This study aimed to evaluate whether presepsin could be a valuable marker for detecting severe sepsis, and whether it could predict the therapeutic course in patients with UTI compared with markers already used: procalcitonin (PCT) and C-reactive protein (CRP). METHODS: From April 2014 to December 2016, a total of 50 patients with urinary tract infections admitted to Gunma university hospital were enrolled in this study. Vital signs, presepsin, PCT, CRP, white blood cell (WBC) count, causative agents of urinary-tract infections, and other data were evaluated on the enrollment, third, and fifth days. The patients were divided into two groups: with (n = 11) or without (n = 39) septic shock on the enrollment day, and with (n = 7) or without (n = 43) sepsis on the fifth day, respectively. Presepsin was evaluated as a biomarker for systemic inflammatory response syndrome (SIRS) or septic shock. RESULTS: Regarding the enrollment day, there was no significant difference of presepsin between the SIRS and non-SIRS groups (p = 0.276). The median value of presepsin (pg/mL) was significantly higher in the septic shock group (p < 0.001). Multivariate logistic regression analysis showed that presepsin (≥ 500 pg/ml) was an independent risk factor for septic shock (p = 0.007). ROC curve for diagnosing septic shock indicated an area under the curve (AUC) of 0.881 for presepsin (vs. 0.690, 0.583, and 0.527 for PCT, CRP and WBC, respectively). Regarding the 5th day after admission, the median presepsin value on the enrollment day was significantly higher in the SIRS groups than in the non-SIRS groups (p = 0.006). On the other hand, PCT (≥ 2 ng/ml) on the enrollment day was an independent risk factor for SIRS. ROC curve for diagnosing sepsis on the fifth day indicated an AUC of 0.837 for PCT (vs. 0.817, 0.811, and 0.802 for presepsin, CRP, and WBC, respectively). CONCLUSIONS: This study showed that presepsin may be a good marker for diagnosing septic shock based on admission data in patients with UTI.

TDP-43 regulates site-specific 2'-O-methylation of U1 and U2 snRNAs via controlling the Cajal body localization of a subset of C/D scaRNAs.

TDP-43 regulates cellular levels of Cajal bodies (CBs) that provide platforms for the assembly and RNA modifications of small nuclear ribonucleoproteins (snRNPs) involved in pre-mRNA splicing. Alterations in these snRNPs may be linked to pathogenesis of amyotrophic lateral sclerosis. However, specific roles for TDP-43 in CBs remain unknown. Here, we demonstrate that TDP-43 regulates the CB localization of four UG-rich motif-bearing C/D-box-containing small Cajal body-specific RNAs (C/D scaRNAs; i.e. scaRNA2, 7, 9 and 28) through the direct binding to these scaRNAs. TDP-43 enhances binding of a CB-localizing protein, WD40-repeat protein 79 (WDR79), to a subpopulation of scaRNA2 and scaRNA28; the remaining population of the four C/D scaRNAs was localized to CB-like structures even with WDR79 depletion. Depletion of TDP-43, in contrast, shifted the localization of these C/D scaRNAs, mainly into the nucleolus, as well as destabilizing scaRNA2, and reduced the site-specific 2'-O-methylation of U1 and U2 snRNAs, including at 70A in U1 snRNA and, 19G, 25G, 47U and 61C in U2 snRNA. Collectively, we suggest that TDP-43 and WDR79 have separate roles in determining CB localization of subsets of C/D and H/ACA scaRNAs.

Incidence of and risk factors for deep vein thrombosis in patients undergoing osteotomies around the knee: comparative analysis of different osteotomy types.

PURPOSE: To examine the incidence of and risk factors for deep vein thrombosis (DVT) among different types of osteotomies around the knee. It was hypothesized that DVT would be detected at a substantial rate after the osteotomy, and there would be differences in the incidences among the different osteotomy procedures. METHODS: Consecutive patients who underwent knee osteotomies for varus osteoarthritis and met the inclusion criteria were included in the study. Ultrasonographic evaluation was performed to detect DVT on bilateral whole leg at 1 month before and 7 days after surgery. Statistical comparison of the demographic and clinical parameters between the patients with and without DVT as well as multivariate analysis using logistic regression was conducted to identify risk factors related to the incidence of postoperative DVT. RESULTS: The study subjects comprised 159 knees in 135 patients with medial opening wedge high tibial osteotomy (MOW-HTO), 93 knees in 78 patients with lateral closed wedge high tibial osteotomy (LCW-HTO), and 74 knees in 54 patients with double level osteotomy (DLO). In the postoperative evaluation, DVT was detected in 19 of 159 knees (11.9%) in MOW-HTO, 21 of 93 knees (22.6%) in LCW-HTO, and 5 of 74 knees (6.8%) in DLO. The incidence of DVT was significantly higher after LCW-HTO than after MOW-HTO and DLO (p < 0.01). DVT at a level above the knee was noted in one case after MOW-HTO, while DVT in the remaining cases developed at a level below the knee. No cases of symptomatic pulmonary embolism were encountered during the study period. Among the potential risk factors assessed for correlation with the incidence of DVT, LCW-HTO was identified as a significant risk factor (odds ratio: 2.54; 95% CI 1.334-4.836; p < 0.01). CONCLUSIONS: This study demonstrated that DVT occurred at a substantial rate (overall incidence of 13.8%) after osteotomy around the knee even with the use of prophylactic anticoagulant. Among the different osteotomy types, the DVT rate was significantly higher after LCW-HTO than after MOW-HTO and DLO. LEVEL OF EVIDENCE: Prospectively designed observational cohort study, Level III.

Hinge fracture in lateral closed-wedge distal femoral osteotomy in knees undergoing double-level osteotomy: assessment of postoperative change in rotational alignment using CT evaluation.

PURPOSE: The purpose of this study was to examine the radiological features of hinge fracture occurring at the distal medial femoral cortex in knees undergoing biplanar lateral closed-wedge distal femoral osteotomy (LCW-DFO) in double-level osteotomy (DLO) based on pre- and postoperative CT image analyses. It was hypothesised that medial hinge fractures in LCW-DFO would occur with a similar incidence to that in high tibial osteotomy, and its occurrence would affect the clinical/radiological outcomes and induce unintended change in alignment depending on the fracture type (direction of the fracture). METHODS: A consecutive series of 36 knees (31 patients) with primary varus osteoarthritis undergoing DLO comprised the study population. The mean age at surgery was 62.0 ± 5.9 years. Presence of hinge fracture was assessed on radiographs and CT images at 1 week. The fracture type was classified depending on the direction of the fracture line: crack propagation in line with the osteotomy (type 1) and fractures extending proximally (type 2) or distally (type 3) from the tip of the wedge. Computer-assisted assessments of bony limb alignment and bony geometry were conducted on a full-length weight-bearing radiograph and CT images using image analysis software. In addition, subjective clinical results were evaluated using the Knee Injury and Osteoarthritis Outcome Score (KOOS). Radiological and clinical follow-up results at 1 and 2 years were compared to the preoperative data, while comparative analysis was made between the subjects with and without a hinge fracture. RESULTS: Postoperative image examinations revealed type 1 and 2 medial femoral hinge fractures in 4 and 7 knees, while no type 3 fracture was identified in the study population. Consequently, the overall incidence of the hinge fracture was 30.6% (11 of the 36 knees). Four of those 11 fractures (36.4%) could not be detected on plain radiographs. CT image analysis for three-dimensional bony geometry showed greater increase in internal rotation of the distal bony segment (increased femoral antetorsion by 9.5° on average) after surgery compared to the knees without a hinge fracture (P = 0.01). Clinical evaluation using the KOOS at 2 years showed no significant difference between the groups with and without hinge fractures. CONCLUSION: In LCW-DFO, medial femoral hinge fractures occurred in 30.6% of the cases. Knees with type 1 hinge fracture exhibited significantly greater increase in femoral antetorsion as compared to those without hinge fracture. In this case series, postoperative weight-bearing protocol was delayed for knees with hinge fracture. Consequently, surgical results were not affected by the occurrence of hinge fracture for up to 2 years. LEVEL OF EVIDENCE: IV (case series).

Return to sports rate after opening wedge high tibial osteotomy in athletes.

PURPOSE: We hypothesized that patient treated with OWHTO who participate in high-impact sports would attain satisfactory outcome. The purpose of this study was to examine the clinical and radiological outcomes in a consecutive series of opening-wedge high tibial osteotomy (OWHTO) in highly active patients. METHODS: Seventy-seven consecutive patients who underwent OWHTO with varus osteoarthritic knees were included in the study. The mean age of the study population was 56.1 years. All patients were followed for a minimum of 2 years. Clinical and radiological evaluations were performed at 12 and 24 months after surgery. The clinical results were evaluated using the Knee Injury and Osteoarthritis Outcome Score (KOOS) and the International Knee Documentation Committee (IKDC) Subjective Score. In regards to radiological assessment, the following parameters were measured in full-length weightbearing radiographs both pre- and postoperatively; mechanical tibiofemoral angle (mTFA), mechanical lateral distal femoral angle (mLDFA), and weight bearing line (WBL) ratio. RESULTS: Fifty-eight patients (75.3%) returned to the same high-impact sports activities as before surgery, with a mean time to return of 8.7 ± 2.7 months (6-14 months). In the clinical assessments, the IKDC subjective score and KOOS both improved from the mean preoperative scores of 38.4 and 217.4 points to the mean postoperative scores of 74.5 and 421.6 points, respectively. The mean pre-symptomatic Tegner activity scale was 5.3 ± 0.6 and significantly decreased to 4.8 ± 1.2 at 2 years postoperative (p < 0.05). In the radiological evaluation, the postoperative mTFA, mMPTA, and WBL ratio values averaged 1.3° ± 2.2° valgus, 90.7° ± 2.9°, and 51.6% ± 8.4°, respectively, at 24 months after surgery. CONCLUSIONS: Clinical outcomes based on postoperative patient-reported outcome measures and rate of return to high-impact sports activities were favorable after OWHTO in patients with knee OA who desired to continue sporting activities with the Tegner activity score of ≥ 5 points. LEVEL OF EVIDENCE: Retrospective case series, IV.

Direct Determination of Pseudouridine in RNA by Mass Spectrometry Coupled with Stable Isotope Labeling.

Pseudouridine (Ψ) is the only "mass-silent" nucleoside produced by post-transcriptional RNA modification. We developed a mass spectrometry (MS)-based technique coupled with in vivo deuterium (D) labeling of uridines for direct determination of Ψs in cellular RNA and applied it to the comprehensive analysis of post-transcriptional modifications in human ribosomal RNAs. The method utilizes human TK6/mouse FM3A cells deficient in uridine monophosphate synthase using a CRISPR-Cas9 technique to turn off de novo uridine synthesis and fully labels uridines with D at uracil positions 5 and 6 by cultivating the cells in a medium containing uridine-5,6-D2. The pseudouridylation reaction in those cells results in the exchange of the D at the C5 of uracil with hydrogen from solvent, which produces a -1 Da mass shift, thus allowing MS-based determination of RNA Ψs. We present here the experimental details of this method and show that it allows the identification of all Ψs in human major nuclear and nucleolar RNAs, including several previously unknown Ψs. Because the method allows direct determination of Ψs at the femtomole level of RNA, it will serve as a useful tool for structure/function studies of a wide variety of noncoding RNAs.

High-peak-power 918-nm laser light source based two-photon spinning-disk microscopy for green fluorophores.

High-speed imaging of living specimen was performed using two-photon microscopy equipped with a spinning-disk scanning unit. Typically, a high-peak-power laser light source is needed to simultaneously induce two-photon excitation processes at several hundred focal points, generating the limitations of excitable fluorophores. Therefore, a high-peak-power neodymium-based 918-nm laser light source was used for intravital imaging of the most popular fluorophores, green fluorescent proteins. As a result, the proposed system obtained approximately 30 times brighter fluorescent signal than that obtained using a conventional mode-locked titanium:sapphire laser light source. Furthermore, the system visualized four-dimensional (xyz-t) calcium responses of pancreatic acinar cells agonist stimulations in the living G-CaMP7-expressing mouse with 60 million µm3 volume.

Transcription repressor-mediated control of engulfment receptor expression in Drosophila phagocytes.

We previously reported that Drosophila phagocytes enhance their phagocytic activity after apoptotic cell engulfment accompanied by the activation of the transcription repressor Tailless and an increase in the levels of engulfment receptors. We herein investigated the underlying mechanisms. We found that Tailless phosphorylation levels decreased in Drosophila phagocytes following the stimulation with apoptotic cells. Anticipating the involvement of another transcription repressor, we examined the possible involvement of Krüppel, a bibliographically identified repressor whose expression is controlled by Tailless. The level of Krüppel in phagocytes decreased after the stimulation in a Tailless-dependent manner. The RNAi knockdown of Krüppel abrogated increases in the levels of engulfment receptors and phagocytic activity in stimulated phagocytes. The binding of Krüppel to the 5'-upstream regions of genes coding for engulfment receptors was demonstrated. These results suggest the following pathway: Tailless is activated by de-phosphorylation; Krüppel expression is inhibited by Tailless; the transcription of engulfment receptors-encoding genes is augmented due to a decrease of inhibition by Krüppel; and finally phagocytic activity is enhanced.

Landscape of the complete RNA chemical modifications in the human 80S ribosome.

During ribosome biogenesis, ribosomal RNAs acquire various chemical modifications that ensure the fidelity of translation, and dysregulation of the modification processes can cause proteome changes as observed in cancer and inherited human disorders. Here, we report the complete chemical modifications of all RNAs of the human 80S ribosome as determined with quantitative mass spectrometry. We assigned 228 sites with 14 different post-transcriptional modifications, most of which are located in functional regions of the ribosome. All modifications detected are typical of eukaryotic ribosomal RNAs, and no human-specific modifications were observed, in contrast to a recently reported cryo-electron microscopy analysis. While human ribosomal RNAs appeared to have little polymorphism regarding the post-transcriptional modifications, we found that pseudouridylation at two specific sites in 28S ribosomal RNA are significantly reduced in ribosomes of patients with familial dyskeratosis congenita, a genetic disease caused by a point mutation in the pseudouridine synthase gene DKC1. The landscape of the entire epitranscriptomic ribosomal RNA modifications provides a firm basis for understanding ribosome function and dysfunction associated with human disease.

Successful treatment of degenerative medial meniscal tears in well-aligned knees with fibrin clot implantation.

PURPOSE: The purpose of this study was to examine the results of meniscal repair performed for symptomatic degenerative medial meniscal tears. METHODS: Twenty-four knees in 24 patients with symptomatic degenerative medial meniscal tears (mostly complex horizontal tears) who underwent isolated arthroscopic repair combined with autologous fibrin clot implantation were included in this study. The patients were followed up for a minimum of 2 years. The overall clinical outcome was evaluated using the Lysholm score, while the activity level was graded on the Tegner Activity Scale. The assessment of healing status at the repair site was based on clinical signs/symptoms and follow-up MRI examination results. In addition, the effects of the patient's clinical and radiological factors on healing of the repaired menisci were analyzed. RESULTS: The mean age of the study subjects was 47.0 ± 8.1 years with a mean follow-up period of 39.3 ± 11.6 months. The Lysholm score significantly improved after surgery (P < 0.01). During the follow-up period, meniscal repairs were deemed to have failed in 6 of the 24 knees (25%). In the analysis of factors influencing meniscal healing, varus deformity (% of mechanical axis < 30%) was identified in all knees in the repair failure group, and the presence of varus deformity was shown to be a significant risk factor correlated with repair failure, while other factors did not significantly influence the healing status. CONCLUSIONS: The short-term follow-up results showed that arthroscopic repair of degenerative medial meniscal tears combined with fibrin clot implantation attained clinical healing in 18 of 24 knees (75%) of patients, while 6 of the 24 knees (25%) of patients experienced clinical failure. The presence of varus deformity negatively affects the healing rate. In well-aligned knees, degenerative medial meniscal tears are successfully treated by isolated repair with fibrin clot implantation. LEVEL OF EVIDENCE: IV.

Physiologic knee joint alignment and orientation can be restored by the minimally invasive double level osteotomy for osteoarthritic knees with severe varus deformity.

PURPOSE: The purpose of this study was to examine early radiological and clinical outcomes following minimally invasive double level osteotomy (DLO) procedure performed for osteoarthritic knees with severe varus deformity. METHODS: Twenty consecutive patients who underwent DLO for varus osteoarthritic knees were included in the study. All patients could be tracked for a minimum of 1 year. Periodical radiological and clinical evaluations were performed at 6 and 12 months after surgery. In the radiological assessment, the following parameters were measured on full-length weight-bearing radiographs both pre- and postoperatively: mechanical tibiofemoral angle (mTFA), mechanical lateral distal femoral angle (mLDFA), mechanical medial proximal tibial angle (mMPTA), and joint-line convergence angle (JLCA). In addition, subjective clinical results were evaluated using the Knee Injury and Osteoarthritis Outcome Score (KOOS) and the International Knee Documentation Committee (IKDC) Subjective Score. RESULTS: The mean age of the study population was 62.5 ± 6.8 years (range 45-76 years). In the radiological evaluation, the preoperative mTFA, mLDFA, mMPTA, and JLCA values averaged 13.5° ± 3.1° varus, 91.1° ± 1.4°, 82.3° ± 2.0°, and 5.8° ± 2.3°, respectively. At 6 and 12 months, all of the radiological parameters significantly improved and corrected to the values within normal range. In the clinical assessments at the follow-up evaluations, both the KOOS and IKDC subjective scores significantly improved from the preoperative values. No significant changes were noted between the 6 and 12-month results in the radiological and clinical assessments. CONCLUSIONS: The minimally invasive DLO technique is a valuable surgical technique accomplishing restoration of physiologic knee joint alignment and orientation with significant improvement in patient-registered clinical outcomes in early postoperative evaluation. Although the follow-up period is still short, the excellent clinical and radiological outcomes shown in the present study support the efficacy of this procedure. LEVEL OF EVIDENCE: Retrospective case series, Level IV.

Prevalence and clinical characteristics of overactive bladder in systemic sclerosis.

Objectives: Overactive bladder (OAB) is a clinical diagnosis defined with the presence of urinary urgency, usually accompanied by frequency and nocturia, with or without urgency urinary incontinence. Objective was to evaluate the demographic and clinical characteristics of Japanese systemic sclerosis (SSc) patients with OAB.Methods: OAB was diagnosed by OAB symptom score (OABSS) in 104 Japanese SSc patients (93 women and 11 men). Differential diseases of OAB were conducted by urologists.Results: The prevalence of OAB in SSc patients was 27.9% (29/104). SSc patients with OAB were characterized by old age, a long history of morbidity (15.4 vs. 11.2 years, p < .01), high anti-centromere antibody positive rate (75.9 vs. 44%, p < .05), high incidence of gastroesophageal reflux disease (93.1 vs. 73.3%, p < .05), low anti-SS-A antibody positive rate (6.9 vs. 26.8%, p < .05), and low incidence of internal lung disease (17.9 vs. 45.7%, p < .05) compared to SSc patients without OAB.Conclusion: This is the first study that evaluated the prevalence and clinical features of OAB in Japanese SSc patients. Since SSc patients might be prone to develop OAB, it was thought that OAB should be noted as one of the complications of SSc patients.

An exploratory retrospective multicenter study of prognostic factors in mCRPC patients undergoing enzalutamide treatment: Focus on early PSA decline and kinetics at time of progression.

BACKGROUND: Recent studies have shown that an early prostate-specific antigen (PSA) response to androgen receptor-targeting agents in metastatic castration-resistant prostate cancer (mCRPC) is associated with a better prognosis. We analyzed the early PSA response to enzalutamide (ENZ) by measuring the PSA doubling time (PSADT) and PSA velocity (PSAV) while monitoring oncologic outcomes and survival in Japanese patients. METHODS: We analyzed a total of 241 patients with mCRPC who were treated with ENZ. The patients' median age was 75 ± 7.9 years (range, 53-93 years). There were 171 (71%) predocetaxel cases, and 70 (29%) post docetaxel cases. PSA-progression-free survival (PFS) and overall survival (OS) were assessed according to Prostate Cancer Working Group 2 criteria. This study was approved by the Institutional Review Board of Gunma University Hospital (No. 1595). RESULTS: We observed 77 good response (GR; case in which PSA remained low after treatment) cases (31.9%), 125 acquired resistance (AR; decline in PSA after treatment followed by progression) cases (51.9%), and 39 primary resistance (PR; lack of decline in PSA) cases (16.2%). Predocetaxel, PSA-PFS, and OS were significantly higher compared with post docetaxel (PSA-PFS: 47.0 vs 13.4 weeks, P < .001; OS: not yet reached vs 80.7 weeks, P < .001). Multivariate analysis of prognostic factors, including PSA response at 4 weeks, was performed using Cox regression analysis. ECOG PS (0 vs 1-2), hemoglobin (Hb; ≥ 12.2 vs < 12.2 g/dL), time to CRPC ( ≥ 12 vs < 12 m), docetaxel treatment history (no vs yes), and a PSA reduction of 50% at 4 weeks were significant predictors of OS (all, P < .05). In cases of AR (n = 125), multivariate analysis showed that PSA kinetic factors, such as PSADT and PSAV (ng/mL/m), Hb, time to CRPC, PSADT ( ≥ 2 vs < 2 m), and PSAV ( < 20 vs ≥ 20 ng/mL/m), were all predictive of OS following PSA-progression (P < .05). CONCLUSIONS: Our study has demonstrated that PSA dynamics after ENZ administration may be a useful prognostic factor for mCRPC patients.

Method for Direct Mass-Spectrometry-Based Identification of Monomethylated RNA Nucleoside Positional Isomers and Its Application to the Analysis of Leishmania rRNA.

RNA post-transcriptional modifications are common in all kingdoms of life and are predominantly affiliated with methylations at various nucleobase positions. Methylations occur frequently at specific sites on the RNA nucleobases and appear to regulate site-specific intermolecular/intramolecular interactions. Herein, we present a method that utilizes liquid chromatography-mass spectrometry (LC-MS) to identify positional monomethylated RNA nucleoside isomers. The method produces profiles of in-source fragmentation and subsequent tandem mass spectrometry (MS2) (pseudo-MS3) of RNase-digested fragments of an RNA and distinguishes between positional methylated nucleobase isomers by comparing their intranucleobase fragment ion profiles with signature profiles derived from authentic isomers. For method validation, we independently determined the positions of all known monomethylated nucleoside isomers in the Escherichia coli 16S/23S rRNAs. As proof of concept, we further applied this technology to fully characterize the base-modified nucleoside positional isomers, in rRNAs derived from Leishmania donovani, a human blood parasite afflicting millions around the globe. The method described herein will be highly beneficial for the delineation of RNA modification profiles in various cellular RNAs, and as it only requires a subpicomole amount of RNA, it could also be used for the structure-function studies of RNA populations represented in minute amounts in the cell.

Long-term quality of life and nutrition status of the aboral pouch reconstruction after total gastrectomy for gastric cancer: a prospective multicenter observational study (CCOG1505).

BACKGROUND: Several efforts have been made to alleviate harms and symptoms after gastrectomy for gastric cancer. We previously conducted a randomized controlled trial (CCOG1101) to compare quality of life (QOL) and nutritional status between Roux-en-Y (RY) and aboral pouch (AP) reconstructions for up to 1 year after total gastrectomy. However, long-term outcomes after AP reconstruction remain unclear. METHODS: A prospective multicenter observational study was conducted to compare QOL, body composition, and nutritional indicators between the RY and AP reconstructions at 5 years after surgery among patients who were enrolled in the CCOG1101 trial. QOL was assessed by the PGSAS-37 questionnaires as well as the EORTC QLQ-C30 and STO22. RESULTS: Sixty patients (31 for RY and 29 for AP) were recruited for analysis. There were no significant differences in baseline and perioperative characteristics between the two groups. No significant differences were found in the EORTC QLQ-C30 global health status and functional scales. Regarding symptom scales in the QLQ-C30 and STO22, a more favorable score for the diarrhea scale was observed in the AP group. Diarrhea was also the only item in the PGSAS-37 questionnaires in which significant benefit of AP was observed. Body weight and lean body mass continued to decrease throughout the postoperative 5 years in both groups. None of the conventional nutritional indicators using the serum samples showed significant difference between the two groups. CONCLUSIONS: Long-term observation suggested little benefit of AP reconstruction after total gastrectomy other than in alleviating diarrhea.

Endovascular Therapy with a Covered Stent Graft for Pseudoaneurysm of the Peroneal Artery Complicating High Tibial Osteotomy-A Case Report.

Pseudoaneurysm of below-the-knee arteries after a high tibial osteotomy (HTO) is rare. A 69-year-old woman with history of right HTO a half year ago had performed a left HTO for osteoarthritis. Postoperatively, she had swelling and pain of the left lower leg. Computed tomography and echocardiography revealed the pseudoaneurysm of peroneal artery (PA). After the release of the covered stent graft, the pseudoaneurysm of the PA did not disappear, it was completely excluded in the completion angiogram.