Nail insertion points in semi-extended nailing of tibial fractures and their influence on alignment: A retrospective cohort study comparing two nail insertion techniques.

INTRODUCTION: Semi-extended tibial nailing techniques include the extra-articular technique (EAT) and the patellar eversion technique (PET). These approaches differ regarding the exposure of the patellar retinaculum and the size of the surgical field. This study compared the postoperative alignment and intramedullary nailing entry points between the EAT and PET for tibial fractures. PATIENTS AND METHODS: A total of 54 patients (aged ≥18 years) who had undergone intramedullary nailing by the EAT (n = 29) or PET (n = 25) for a tibial shaft fracture were evaluated. The intramedullary nailing entry point and postoperative alignment were measured, and the 1-year postoperative follow-up results were compared. RESULTS: For the EAT and PET, the intramedullary nailing entry point was located at a mean distance of 4.04 mm medial to the optimal entry point and 0.27 mm lateral to the optimal entry point, respectively. The mean angular deformation observed in anteroposterior radiographs following surgery using the EAT and PET were 2.49° and 0.32° valgus, respectively. CONCLUSION: The intramedullary nailing entry point affected postoperative alignment. Intramedullary nailing may result in malalignment while performing the EAT due to the interference of the patella at the time of nailing.

G-quadruplex-forming nucleic acids interact with splicing factor 3B subunit 2 and suppress innate immune gene expression.

G-quadruplex (G4), a non-canonical higher-order structure formed by guanine-rich nucleic acid sequences, affects various genetic events in cis, including replication, transcription and translation. Whereas up-regulation of innate immune/interferon-stimulated genes (ISGs) is implicated in cancer progression, G4-forming oligonucleotides that mimic telomeric repeat-containing RNA suppress ISG induction in three-dimensional (3D) culture of cancer cells. However, it is unclear how G4 suppresses ISG expression in trans. In this study, we found that G4 binding to splicing factor 3B subunit 2 (SF3B2) down-regulated STAT1 phosphorylation and ISG expression in 3D-cultured cancer cells. Liquid chromatography-tandem mass spectrometry analysis identified SF3B2 as a G4-binding protein. Either G4-forming oligonucleotides or SF3B2 knockdown suppressed ISG induction, whereas Phen-DC3, a G4-stabilizing compound, reversed the inhibitory effect of G4-forming oligonucleotides on ISG induction. Phen-DC3 inhibited SF3B2 binding to G4 in vitro. SF3B2-mediated ISG induction appeared to occur independently of RNA splicing because SF3B2 knockdown did not affect pre-mRNA splicing under the experimental conditions, and pharmacological inhibition of splicing by pladienolide B did not repress ISG induction. These observations suggest that G4 disrupts the ability of SF3B2 to induce ISGs in cancer. We propose a new mode for gene regulation, which employs G4 as an inhibitory trans-element.

Generation of Insulin-Producing Cells from Canine Adipose Tissue-Derived Mesenchymal Stem Cells.

The potential of mesenchymal stem cells (MSCs) to differentiate into nonmesodermal cells such as pancreatic beta cells has been reported. New cell-based therapy using MSCs for diabetes mellitus is anticipated as an alternative treatment option to insulin injection or islet transplantation in both human and veterinary medicine. Several protocols were reported for differentiation of MSCs into insulin-producing cells (IPCs), but no studies have reported IPCs generated from canine MSCs. The purpose of this study was to generate IPCs from canine adipose tissue-derived MSCs (AT-MSCs) in vitro and to investigate the effects of IPC transplantation on diabetic mice in vivo. Culturing AT-MSCs with the differentiation protocol under a two-dimensional culture system did not produce IPCs. However, spheroid-like small clusters consisting of canine AT-MSCs and human recombinant peptide µ-pieces developed under a three-dimensional (3D) culture system were successfully differentiated into IPCs. The generated IPCs under 3D culture condition were stained with dithizone and anti-insulin antibody. Canine IPCs also showed gene expression typical for pancreatic beta cells and increased insulin secretion in response to glucose stimulation. The blood glucose levels in streptozotocin-induced diabetic mice were decreased after injection with the supernatant of canine IPCs, but the hyperglycemic states of diabetic mice were not improved after transplanting IPCs subcutaneously or intramesenterically. The histological examination showed that the transplanted small clusters of IPCs were successfully engrafted to the mice and included cells positive for insulin by immunofluorescence. Several factors, such as the transplanted cell number, the origin of AT-MSCs, and the differentiation protocol, were considered potential reasons for the inability to improve the hyperglycemic state after IPC transplantation. These findings suggest that canine AT-MSCs can be differentiated into IPCs under a 3D culture system and IPC transplantation may be a new treatment option for dogs with diabetes mellitus.

From the wings to the center stage of chromosomes.

Telomere-binding protein TRF2 protects the linear chromosome ends, telomeres, from being recognized as damaged DNA. TRF2 also regulates gene expression outside telomeres, but the detailed mechanism has not been fully understood. Mukherjee and colleagues have employed ChIP-Seq and biochemical analyses to identify G-quadruplexes at gene promoters across the genome as nontelomeric TRF2-binding sites. TRF2 occupancy on such target sites leads to epigenetic gene repression, implicating TRF2-G-quadruplex interaction as a sophisticated regulator of gene expression.

Revisiting Telomere Shortening in Cancer.

Telomeres, the protective structures of chromosome ends are gradually shortened by each cell division, eventually leading to senescence or apoptosis. Cancer cells maintain the telomere length for unlimited growth by telomerase reactivation or a recombination-based mechanism. Recent genome-wide analyses have unveiled genetic and epigenetic alterations of the telomere maintenance machinery in cancer. While telomerase inhibition reveals that longer telomeres are more advantageous for cell survival, cancer cells often have paradoxically shorter telomeres compared with those found in the normal tissues. In this review, we summarize the latest knowledge about telomere length alterations in cancer and revisit its rationality. Finally, we discuss the potential utility of telomere length as a prognostic biomarker.

MERIT40-dependent recruitment of tankyrase to damaged DNA and its implication for cell sensitivity to DNA-damaging anticancer drugs.

Tankyrase, a member of the poly(ADP-ribose) polymerase (PARP) family, regulates various intracellular responses, such as telomere maintenance, Wnt/ß-catenin signaling and cell cycle progression through its interactions with multiple target proteins. Tankyrase contains a long stretch of 24 ankyrin repeats that are further divided into five subdomains, called ANK repeat clusters (ARCs). Each ARC works as an independent ligand-binding unit, which implicates tankyrase as a platform for multiple protein-protein interactions. Furthermore, tankyrase distributes to various intracellular loci, suggesting potential distinct but yet unidentified physiological functions. To explore the novel functions of tankyrase, we performed liquid chromatography-mass spectrometry analysis and identified the BRE-BRCC36-MERIT40 complex, a regulator of homologous recombination, as tankyrase-binding proteins. Among the complex components, MERIT40 was directly associated with tankyrase via a tankyrase-binding consensus motif, as previously reported. In X-ray-irradiated non-small cell lung cancer cells, tankyrase localized to DNA double-stranded break sites in a MERIT40-dependent manner. MERIT40 knockdown increased the cell sensitivity to X-ray, whereas the wild-type, but not the tankyrase-unbound mutant, MERIT40 rescued the phenotype of the knockdown cells. Tankyrase inhibitors, such as G007-LK and XAV939, increased the cellular sensitivity to X-ray irradiation and anticancer drugs that induce DNA double-stranded breaks. These observations suggest that tankyrase plays a role in the DNA damage repair response and implicates a potential therapeutic utility of tankyrase inhibitors in combination treatments with DNA-damaging anticancer drugs.

Highly sensitive detection of microbending in single-mode fibers and its applications.

This paper investigates the applicability of 1-µm-band mode-detection optical time domain reflectometry (OTDR), which detects microbending in single-mode fibers with high sensitivity by conducting measurements in the two-mode region of the fibers under test and observing the second-order mode of the backscattered light. To demonstrate its feasibility, we evaluate microbending losses as determined by the technique, and comparisons are made with conventional OTDR operating at the wavelength of 1650 nm. In addition, we discuss two potential applications of the technique. One is microbend sensing of fibers and cables for detecting microbending losses that are too small to be found with conventional OTDR. The other is health monitoring of installed fiber cables. This enables us to detect the growth in microbending losses at an earlier stage. Proof of concept is demonstrated experimentally.

Coupling of dyspnea perception and occurrence of tachypnea during exercise.

During exercise, tidal volume initially contributes to ventilatory responses more than respiratory frequency, and respiratory frequency then increases rapidly while tidal volume stabilizes. Dyspnea intensity is also known to increase in a threshold-like manner. We tested the possibility that the threshold of tachypneic breathing is equal to that of dyspnea perception during cycle ergometer exercise (n = 27). Dyspnea intensity was scored by a visual analog scale. Thresholds were expressed as values of pulmonary O2 uptake at each breakpoint. Dyspnea intensity and respiratory frequency started increasing rapidly once the intensity of stimuli exceeded a threshold level. The thresholds for dyspnea intensity and for occurrence of tachypnea were significantly correlated. An intraclass correlation coefficient of 0.71 and narrow limits of agreement on the Bland-Altman plot indicated a good agreement between these thresholds. These results suggest that the start of tachypneic breathing coincides with the threshold for dyspnea intensity during cycle ergometer exercise.

G-quadruplex ligand-induced DNA damage response coupled with telomere dysfunction and replication stress in glioma stem cells.

Glioblastoma (GBM) is an invariably fatal brain tumor in which a small subpopulation of self-renewable glioma stem cells (GSCs) contributes to tumor propagation and relapse. Targeting GSCs could therefore have a significant clinical impact for GBM. Telomestatin is a naturally-occurring compound that preferentially impairs GSC growth by perturbing transcription and inducing a DNA damage response. Telomestatin stabilizes G-quadruplexes (G4s), which are guanine-rich four-strand nucleic acid structures observed in vitro and in vivo. However, the mechanism underlying the GSC-selective nature of the DNA damage response remains unknown. Here we demonstrate that GSCs are more susceptible to telomestatin-induced telomere dysfunction and replication stress when compared with GSC-derived non-stem glioma cells (NSGCs). Telomestatin induced dissociation of the telomere-capping protein TRF2 from telomeres, leading to telomeric DNA damage in GSCs-but not in NSGCs. BIBR1532, a telomerase catalytic inhibitor, did not preferentially inhibit GSC growth, suggesting that telomestatin promotes telomere dysfunction in a telomerase-independent manner. GSCs and NSGCs had comparable levels of G4s in their nuclei, and both responded to telomestatin with phosphorylation of RPA2 at Ser33-a hallmark of replication stress. However, activation of the checkpoint kinase Chk1, induction of a DNA damage response, and subsequent growth inhibition occurred only in telomestatin-treated GSCs. These observations suggest that telomestatin impairs GSC growth through removal of TRF2 from telomeres and potent activation of the replication stress response pathway. Therefore, a novel G4-directed therapeutic strategy could specifically target cancer stem cells in GBM.

Association of Nocturnal Hypertension With Disease Activity in Rheumatoid Arthritis.

OBJECTIVES: Both nocturnal hypertension (HT) and systemic inflammation underlying rheumatoid arthritis (RA) have been shown to be independent predictors of cardiovascular disease (CVD), although little is known on the relationship between nocturnal blood pressure (BP) and disease activity in RA patients. METHODS: We performed 24-hour ambulatory BP monitoring (ABPM) in 71 RA patients to examine the relationship of nocturnal fall in BP and RA disease activity based on a disease activity score of 28 joint counts with C-reactive protein (CRP, 28-joint disease activity score (DAS28)-CRP). Among them, 25 RA patients whose consent obtained were reexamined by ABPM to assess the improvement of nocturnal fall in BP after RA therapeutic intervention. RESULTS: The mean DAS28-CRP level was 4.8±1.6 in 71 RA patients. The mean nocturnal fall in BP was 5.6±8.9%. DAS28-CRP was associated significantly and independently in a negative manner with the nocturnal fall in BP (ß = -0.388, P = 0.004). In 25 RA patients, DAS28-CRP improved from 5.4±1.1 to 3.5±0.8 (P < 0.0001) and the nocturnal fall in BP increased significantly from 4.5±9.2% to 10.6±5.8% (P = 0.002) with the significant decrease of nighttime systolic BP (SBP) from 121.2±22.5mm Hg to 112.5±18.8mm Hg (P = 0.02) in spite of no change in daytime BP after 4 weeks of RA treatment. CONCLUSIONS: The present study observed that higher RA activity was associated with lower nocturnal fall in BP, but not daytime BP, in RA patients.

BRCA1 and CtIP promote alternative non-homologous end-joining at uncapped telomeres.

Loss of telomere protection occurs during physiological cell senescence and ageing, due to attrition of telomeric repeats and insufficient retention of the telomere-binding factor TRF2. Subsequently formed telomere fusions trigger rampant genomic instability leading to cell death or tumorigenesis. Mechanistically, telomere fusions require either the classical non-homologous end-joining (C-NHEJ) pathway dependent on Ku70/80 and LIG4, or the alternative non-homologous end-joining (A-NHEJ), which relies on PARP1 and LIG3. Here, we show that the tumour suppressor BRCA1, together with its interacting partner CtIP, both acting in end resection, also promotes end-joining of uncapped telomeres. BRCA1 and CtIP do not function in the ATM-dependent telomere damage signalling, nor in telomere overhang removal, which are critical for telomere fusions by C-NHEJ. Instead, BRCA1 and CtIP act in the same pathway as LIG3 to promote joining of de-protected telomeres by A-NHEJ. Our work therefore ascribes novel roles for BRCA1 and CtIP in end-processing and fusion reactions at uncapped telomeres, underlining the complexity of DNA repair pathways that act at chromosome ends lacking protective structures. Moreover, A-NHEJ provides a mechanism of previously unanticipated significance in telomere dysfunction-induced genome instability.

[Glucocorticoid and Bone. Efficacy of active vitamin D on glucocorticoid-induced osteoporosis].

Active vitamin D is a second line treatment followed by bisphosphonate in the Japanese guideline of glucocorticoid-induced osteoporosis (GIO) . Although it shows relatively mild but significant effect on BMD and fracture prevention, it is utilized as combination therapy with bisphosphonate. Vitamin D is recommended for GIO with gastrointestinal disease and hepatobiliary disorders. Vitamin D may be the first line treatment for younger GIO patients.

DUB-resistant ubiquitin to survey ubiquitination switches in mammalian cells.

The ubiquitin-modification status of proteins in cells is highly dynamic and maintained by specific ligation machineries (E3 ligases) that tag proteins with ubiquitin or by deubiquitinating enzymes (DUBs) that remove the ubiquitin tag. The development of tools that offset this balance is critical in characterizing signaling pathways that utilize such ubiquitination switches. Herein, we generated a DUB-resistant ubiquitin mutant that is recalcitrant to cleavage by various families of DUBs both in vitro and in mammalian cells. As a proof-of-principle experiment, ectopic expression of the uncleavable ubiquitin stabilized monoubiquitinated PCNA in the absence of DNA damage and also revealed a defect in the clearance of the DNA damage response at unprotected telomeres. Importantly, a proteomic survey using the uncleavable ubiquitin identified ubiquitinated substrates, validating the DUB-resistant ubiquitin expression system as a valuable tool for interrogating cell signaling pathways.

A two-step mechanism for TRF2-mediated chromosome-end protection.

Mammalian telomeres repress DNA-damage activation at natural chromosome ends by recruiting specific inhibitors of the DNA-damage machinery that form a protective complex termed shelterin. Within this complex, TRF2 (also known as TERF2) has a crucial role in end protection through the suppression of ATM activation and the formation of end-to-end chromosome fusions. Here we address the molecular properties of TRF2 that are both necessary and sufficient to protect chromosome ends in mouse embryonic fibroblasts. Our data support a two-step mechanism for TRF2-mediated end protection. First, the dimerization domain of TRF2 is required to inhibit ATM activation, the key initial step involved in the activation of a DNA-damage response (DDR). Next, TRF2 independently suppresses the propagation of DNA-damage signalling downstream of ATM activation. This novel modulation of the DDR at telomeres occurs at the level of the E3 ubiquitin ligase RNF168 (ref. 3). Inhibition of RNF168 at telomeres involves the deubiquitinating enzyme BRCC3 and the ubiquitin ligase UBR5, and is sufficient to suppress chromosome end-to-end fusions. This two-step mechanism for TRF2-mediated end protection helps to explain the apparent paradox of frequent localization of DDR proteins at functional telomeres without concurrent induction of detrimental DNA-repair activities.

Beneficial effect of risedronate on arterial thickening and stiffening with a reciprocal relationship to its effect on bone mass in female osteoporosis patients: a longitudinal study.

AIMS: A longitudinal study was performed to examine the effect of risedronate on arterial thickening and stiffening in postmenopausal female osteoporosis patients. MAIN METHODS: Patients treated with risedronate (2.5mg/day) (n=33) and those that did not receive risedronate (n=30, control group) were monitored over a 1-year period. Bone metabolic markers, bone mineral density (BMD) of the femur neck (FN), brachial-ankle pulse wave velocity (baPWV), and intima-media thickness at the carotid artery (CA-IMT) were measured. KEY FINDINGS: At baseline, there was no significant difference in blood pressure, serum lipid profiles, FN BMD, baPWV and CA-IMT between the risedronate-treated patients and the controls. Baseline levels of FN BMD were significantly negatively correlated with those of CA-IMT and baPWV. During the study, FN BMD increased significantly in the risedronate group (p=0.0097), but decreased significantly in the control group (p=0.0013). BaPWV and CA-IMT did not change significantly in the risedronate group, but both increased significantly in the control group. The percentage change in FN BMD over the study period showed a significant negative correlation with those for baPWV (r=-0.294, p=0.0262) and CA-IMT (r=-0.305, p=0.0234) in all subjects (risedronate-treated patients and controls). SIGNIFICANCE: In addition to increasing BMD, risedronate significantly suppressed the progression of arterial thickening and stiffening in postmenopausal osteoporotic patients over one year. These changes may indirectly be due to the effect of risedronate on bone.

Channel-allocation-adaptive WDM signal observation based on sequential ultrafast field sampling.

A novel technology for simultaneous WDM signal monitoring is presented based on ultrafast field sampling. Its flexibility regarding channel allocation and/or channel bandwidth is unique and attractive, as is its ultrafast nature. Two- and four-channel WDM signals, with the total bandwidth of 800 GHz, are successfully discriminated and observed with the proposed scheme.

Time-course of health status in patients with rheumatoid arthritis during the first year of treatment with infliximab.

OBJECTIVES: A longitudinal study was performed to examine changes in health status in comparison with rheumatoid arthritis (RA) inflammation in patients with RA during the first 54 weeks of infliximab (IFX) treatment. METHODS: Health status in active RA patients (n=13) was assessed monthly using the Arthritis Impact Measurement Scale 2 (AIMS2) and the VAS-GH during the first year of IFX treatment. Simultaneously, RA activity was assessed using inflammation markers, MMP-3 and the Disease Activity Score in 28 joints (DAS-28) based on CRP [DAS-28(CRP)] and ESR[DAS-28(ESR)]. RESULTS: Serum CRP and ESR decreased significantly from 2.14+/-0.52mg/dL and 56.9+/-6.96mm/h, respectively, at baseline to 0.24+/-0.11mg/dL and 31.6+/-4.39mm/h, respectively, at 2 weeks after initiation of IFX. Other inflammatory markers and MMP-3 were also suppressed significantly after 2 weeks of IFX treatment. DAS-28(CRP) and DAS-28(ESR) were also significantly decreased after 2 weeks and suppression of both DAS values remained significant until 54 weeks of IFX treatment. After initiation of IFX, patient-reported general health also showed a significant improvement based on the changes in the six summary component scores on the AIMS2 (physical, affect, symptom, role, social interaction, and patient satisfaction). These scores all improved progressively until 14-18 weeks after initiation of IFX treatment, and then exhibited a temporary but insignificant exacerbation. The six components of the physical score also improved in a time-dependent manner until 14-18 weeks, but the scores for walking and bending, hand and finger function, arm function, self-care, and household tasks showed significant exacerbation at 22-30 weeks. The score for mobility level did not show this change. CONCLUSION: IFX treatment significantly improved both RA disease activity and health status in active RA patients. Time-dependent improvement of ADL until 14-18 weeks after initiation of IFX treatment, as reflected in the six components of the physical score, might have contributed to the temporary exacerbation of health status thereafter in these patients.

TRFH domain is critical for TRF1-mediated telomere stabilization.

The telomeres are nucleoprotein complexes essential for maintaining the genomic integrity of linear chromosomes. Six telomere localizing proteins form a complex named "shelterin/telosome" to cooperatively regulate telomere length and protect chromosomal ends from DNA damage and repair responses. Mouse embryonic stem (ES) cells lacking TRF1, a shelterin component, exhibit a high-incidence of broken or lost telomere FISH signals, supporting a critical role for TRF1 in telomere maintenance. We demonstrate that these abnormal telomere structures are not caused by the inability of TRF1-deficient cells to recruit TIN2 but are due to a specific role for TRF1 at telomeres. Furthermore, we provide evidence that the mTRF1 TRF homology (TRFH) domain is crucial for this abnormal telomere FISH phenotype. These novel findings suggest that the TRFH domain is crucial not only for dimerization of TRF1 and TIN2-telomere recruitment, but also telomere stabilization.

Simultaneous WDM signal detection realized by ultrafast field sampling.

This paper proposes a novel detection technique for wavelength-division multiplexing (WDM) signals that uses the ultrafast field sampling approach. The proposed technique simultaneously samples the total field of the WDM signal with no wavelength-demultiplexing; its electrical post-processing provides a filtering function in the digital domain. As a result, the individual fields of the WDM channels, including mutual phase relationship, can be jointly reconstructed. As a preliminary demonstration, the simultaneous monitoring of a WDM signal composed of two channels with independent polarization states, is successfully performed using a dual-channel field sampling system with polarization diversity. This demonstration can be expanded to the detection of a WDM signal with N-channels by using an N-channel field sampling system. It is also experimentally-verified that the reconstructed fields preserve the mutual phase relationship of the original fields. This 'field sensitive' WDM detection may open new possibilities for advanced WDM monitoring systems and even the electrical compensation of linear and/or non-linear inter-channel cross-talk in digital coherent detection systems.