A Supramolecular Biosensor for Rapid and High-Throughput Quantification of a Disease-Associated Niacin Metabolite.

Metabolic abnormalities play a pivotal role in various pathological conditions, necessitating the quantification of specific metabolites for diagnosis. While mass spectrometry remains the primary method for metabolite measurement, its limited throughput underscores the need for biosensors capable of rapid detection. Previously, we reported that pillar[6]arene with 12 carboxylate groups (P6AC) forms host-guest complexes with 1-methylnicotinamide (1-MNA), which is produced in vivo by nicotinamide N-methyltransferase (NNMT). P6AC acts as a biosensor by measuring the fluorescence quenching caused by photoinduced electron transfer upon 1-MNA binding. However, the low sensitivity of P6AC makes it impractical for detecting 1-MNA in unpurified biological samples. In this study, we found that P6A with 12 sulfonate groups (P6AS) is a specific and potent supramolecular host for 1-MNA interactions even in biological samples. The 1-MNA binding affinity of P6AS in water was found to be (5.68 ± 1.02) × 106 M-1, which is approximately 700-fold higher than that of P6AC. Moreover, the 1-MNA detection limit of P6AS was determined to be 2.84 × 10-7 M, which is substantially lower than that of P6AC. Direct addition of P6AS to culture medium was sufficient to quantify 1-MNA produced by cancer cells. Furthermore, this sensor was able to specifically detect 1-MNA even in unpurified human urine. P6AS therefore enables rapid and high-throughput quantification of 1-MNA, and further improvement of our strategy will contribute to the establishment of high-throughput screening of NNMT inhibitors, diagnosis of liver diseases, and imaging of human cancer cells in vivo.

Coracoclavicular and acromioclavicular ligament reconstruction with a double-bundle semitendinosus autograft and cortical buttons for chronic acromioclavicular joint dislocations: clinical and imaging outcomes.

BACKGROUND: There are few clinical and radiographic studies of coracoclavicular (CC) ligament reconstruction in chronic acromioclavicular (AC) joint dislocation. Additionally, reported AC joint reduction rates vary. HYPOTHESIS: Arthroscopically assisted double-bundle semitendinosus tendon autografts with CC and AC ligament reconstruction for AC joint reconstruction provide AC joint stability and improved function at the final visit. METHODS: In this retrospective study of prospectively collected data, 21 patients surgically treated for chronic AC joint dislocation (Rockwood III-V) were assessed clinically and radiographically preoperatively, and at day 1, 3 months, 12 months, and at a final visit (>24 months) postoperatively. Clinical assessments included Constant and American Shoulder and Elbow Surgeons scores. The CC vertical distance (CCD) on the affected and unaffected sides [CCD ratio (%)] on the anterosuperior view were measured. AC joint vertical reduction loss was defined as an increase in the CCD ratio of >25%. Horizontal AC joint instability was evaluated on axillary views. Pearsons' correlation coefficients were generated to examine the relationships among postoperative clinical scores, CCD ratio, interval from injury to surgery, and age at the time of surgery. RESULTS: Twenty-one shoulders in 21 patients (mean age, 40.0 years at the time of surgery; 16 men, 5 women) were evaluated with a mean 31.7-month follow-up period. The mean Constant scores, American Shoulder and Elbow Surgeons scores, and CCD ratios significantly improved from preoperatively to the final visit (57.4 ± 10.1, 49.1 ± 12.1, 101.6 ± 64.1 preoperatively; 89.6 ± 5.3, 96.5 ± 4.2, 9.9 ± 34.5 at the final visit, respectively [P < .001 for all]). Vertical AC and horizontal AC joint instability were observed in 4 shoulders (19.0%) and in 1 shoulder (4.8%), respectively. However, there was no significant correlation between the increase in CCD and clinical scores at the final visit (Constant score; r = 0.179, P = .438: American Shoulder and Elbow Surgeons score; r = -0.260, P = .256) or the interval from injury to surgery (r = 0.099, P = .669) or age at the time of surgery (r = 0.019, P = .935). No clinical complications were associated with clinical symptoms. CONCLUSIONS: Patients who underwent the index procedure achieved significant improvement in shoulder function without complications related clinical symptom after a mean follow-up interval of 31.7 months. In contrast, the rates of total ACJ instability in the vertical and horizontal planes were unsatisfactory but compatible with those in previous studies.

Therapeutic potential of pentamidine for glioma-initiating cells and glioma cells through multimodal antitumor effects.

Glioma-initiating cells, which comprise a heterogeneous population of glioblastomas, contribute to resistance against aggressive chemoradiotherapy. Using drug reposition, we investigated a therapeutic drug for glioma-initiating cells. Drug screening was undertaken to select candidate agents that inhibit proliferation of two different glioma-initiating cells lines. The alteration of proliferation and stemness of the two glioma-initiating cell lines, and proliferation, migration, cell cycle, and survival of these two differentiated glioma-initiating cell lines and three different glioblastoma cell lines treated with the candidate agent were evaluated. We also used a xenograft glioma mouse model to evaluate anticancer effects of treated glioma cell lines. Among the 1301 agents, pentamidine-an antibiotic for Pneumocystis jirovecii-emerged as a successful antiglioma agent. Pentamidine treatment suppressed proliferation and stemness in glioma-initiating cell lines. Proliferation and migration were inhibited in all differentiated glioma-initiating cells and glioblastoma cell lines, with cell cycle arrest and caspase-dependent apoptosis induction. The in vivo study reproduced the same findings as the in vitro studies. Pentamidine showed a stronger antiproliferative effect on glioma-initiating cells than on differentiated cells. Western blot analysis revealed pentamidine inhibited phosphorylation of signal transducer and activator of transcription 3 in all cell lines, whereas Akt expression was suppressed in glioma-initiating cells but not in differentiated lines. In the present study, we identified pentamidine as a potential therapeutic drug for glioma. Pentamidine could be promising for the treatment of glioblastomas by targeting both glioma-initiating cells and differentiated cells through its multifaceted antiglioma effects.

Therapeutic advantage of targeting lysosomal membrane integrity supported by lysophagy in malignant glioma.

Lysosomes function as the digestive system of a cell and are involved in macromolecular recycling, vesicle trafficking, metabolic reprogramming, and progrowth signaling. Although quality control of lysosome biogenesis is thought to be a potential target for cancer therapy, practical strategies have not been established. Here, we show that lysosomal membrane integrity supported by lysophagy, a selective autophagy for damaged lysosomes, is a promising therapeutic target for glioblastoma (GBM). In this study, we found that ifenprodil, an FDA-approved drug with neuromodulatory activities, efficiently inhibited spheroid formation of patient-derived GBM cells in a combination with autophagy inhibition. Ifenprodil increased intracellular Ca2+ level, resulting in mitochondrial reactive oxygen species-mediated cytotoxicity. The ifenprodil-induced Ca2+ elevation was due to Ca2+ release from lysosomes, but not endoplasmic reticulum, associated with galectin-3 punctation as an indicator of lysosomal membrane damage. As the Ca2+ release was enhanced by ATG5 deficiency, autophagy protected against lysosomal membrane damage. By comparative analysis of 765 FDA-approved compounds, we identified another clinically available drug for central nervous system (CNS) diseases, amoxapine, in addition to ifenprodil. Both compounds promoted degradation of lysosomal membrane proteins, indicating a critical role of lysophagy in quality control of lysosomal membrane integrity. Importantly, a synergistic inhibitory effect of ifenprodil and chloroquine, a clinically available autophagy inhibitor, on spheroid formation was remarkable in GBM cells, but not in nontransformed neural progenitor cells. Finally, chloroquine dramatically enhanced effects of the compounds inducing lysosomal membrane damage in a patient-derived xenograft model. These data demonstrate a therapeutic advantage of targeting lysosomal membrane integrity in GBM.

RHEB is a potential therapeutic target in T cell acute lymphoblastic leukemia.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of immature T lymphocytes. Although various therapeutic approaches have been developed, refractoriness of chemotherapy and relapse cause a poor prognosis of the disease and further therapeutic strategies are required. Here, we report that Ras homolog enriched in brain (RHEB), a critical regulator of mTOR complex 1 activity, is a potential target for T-ALL therapy. In this study, we established an sgRNA library that comprehensively targeted mTOR upstream and downstream pathways, including autophagy. CRISPR/Cas9 dropout screening revealed critical roles of mTOR-related molecules in T-ALL cell survival. Among the regulators, we focused on RHEB because we previously found that it is dispensable for normal hematopoiesis in mice. Transcriptome and metabolic analyses revealed that RHEB deficiency suppressed de novo nucleotide biosynthesis, leading to human T-ALL cell death. Importantly, RHEB deficiency suppressed tumor growth in both mouse and xenograft models. Our data provide a potential strategy for efficient therapy of T-ALL by RHEB-specific inhibition.

Arthroscopic evaluation after osteochondral autogenous transfer with osteotomy of medial malleolus for osteochondral lesion of the talar dome.

BACKGROUND: The purpose of this study was to investigate the second-look arthroscopic evaluation after osteochondral autogenous transfer (OAT) for osteochondral lesion of the talar dome (OLT) with the criteria of the International Cartilage Repair Society (ICRS). METHODS: Ten patients (twelve ankles) with OLT underwent OAT with osteotomy of the medial malleolus. Clinical outcomes were evaluated using the American Orthopedic Foot & Ankle Society (AOFAS) ankle-hindfoot scale. The condition of the transplanted cartilage was evaluated at the time of second-look arthroscopy using the ICRS Cartilage Repair Assessment. RESULTS: The AOFAS ankle-hindfoot scale was significantly improved from 65.1 ± 1.9 points before surgery to 98.1 ± 2.8 points at the time of second-look arthroscopy (p < 0.01). The ICRS Cartilage Repair Assessment was 11.4 points on average (9-12 points). CONCLUSIONS: The OAT for OLT is considered to be a useful treatment even if invasion by medial malleolus osteotomy is added. LEVEL OF EVIDENCE: Level IV, Case series.

Vocal practice regulates singing activity-dependent genes underlying age-independent vocal learning in songbirds.

The development of highly complex vocal skill, like human language and bird songs, is underlain by learning. Vocal learning, even when occurring in adulthood, is thought to largely depend on a sensitive/critical period during postnatal development, and learned vocal patterns emerge gradually as the long-term consequence of vocal practice during this critical period. In this scenario, it is presumed that the effect of vocal practice is thus mainly limited by the intrinsic timing of age-dependent maturation factors that close the critical period and reduce neural plasticity. However, an alternative, as-yet untested hypothesis is that vocal practice itself, independently of age, regulates vocal learning plasticity. Here, we explicitly discriminate between the influences of age and vocal practice using a songbird model system. We prevented zebra finches from singing during the critical period of sensorimotor learning by reversible postural manipulation. This enabled to us to separate lifelong vocal experience from the effects of age. The singing-prevented birds produced juvenile-like immature song and retained sufficient ability to acquire a tutored song even at adulthood when allowed to sing freely. Genome-wide gene expression network analysis revealed that this adult vocal plasticity was accompanied by an intense induction of singing activity-dependent genes, similar to that observed in juvenile birds, rather than of age-dependent genes. The transcriptional changes of activity-dependent genes occurred in the vocal motor robust nucleus of the arcopallium (RA) projection neurons that play a critical role in the production of song phonology. These gene expression changes were accompanied by neuroanatomical changes: dendritic spine pruning in RA projection neurons. These results show that self-motivated practice itself changes the expression dynamics of activity-dependent genes associated with vocal learning plasticity and that this process is not tightly linked to age-dependent maturational factors.

Does TMJ Function and Imaging Tools Help Differentiate Between Condylar Resorption and Mandibular Hypoplasia?

PURPOSE: Differentiating between bilateral condylar resorption (CR) and mandibular hypoplasia (MH) can be challenging owing to the difficulty in chronological observation and establishing standardized measurements. The purpose of the present study was to assess whether temporomandibular joint (TMJ) function can distinguish between CR and MH and clarify the essential diagnostic imaging tools for CR. MATERIALS AND METHODS: We performed a cross-sectional study of patients with mandibular retrognathia. The primary predictor variables were a clinical dysfunction score for the TMJ, mandibular plane angle (MPA), SNA angle, SNB angle, and cortical erosion score in the condylar heads. The demographic variables were age, anterior disc displacement, and previous orthodontic treatment. The anatomic variable was the condylar height (CH). The primary outcome variable was the disease status (CR or MH). The patients were divided into the CR group and MH group. The patients with CR were selected on the basis of a CH value of less than 22 mm. TMJ function was assessed using the Helkimo clinical dysfunction index. The CH on panoramic radiographs was measured using the Kjellberg method. The MPA, SNA angle, and SNB angle were analyzed using cephalometric analysis. Cortical erosion in the condylar head was assessed using computed tomography and magnetic resonance imaging. RESULTS: A total of 23 female participants were enrolled in the present study. The average clinical dysfunction score for the TMJ was 4.4 in the CR group and 0.4 in the MH group (P < .05). The average MPA was 41.2° in the CR group and 35.5° in the MH group (P < .05). CONCLUSIONS: The present investigation has shown that assessing TMJ function and analyzing MPA using a cephalometric radiograph can differentiate CR from MH.

Bone healing potential of fascia lata autografts to the humeral head footprint in rotator cuff reconstruction based on magnetic resonance imaging and histologic evaluations.

BACKGROUND: The purpose of the study was to evaluate the bone healing potential of fascia lata autograft (FLA) by magnetic resonance imaging (MRI) and histologic analysis. METHODS: The study included 69 patients assessed by MRI after an FLA patch procedure. Three of the 69 patients underwent a revision procedure after the primary FLA procedure; 1 underwent a second-look arthroscopy and 2 underwent reverse shoulder arthroplasties (RSAs). In the 2 RSA patients, we histologically evaluated greater tuberosities with the repaired graft. Moreover, as a control, we harvested the greater tuberosity with the cuff tendon at the time of RSA for failed open reduction-internal fixation of 4-part proximal humeral fracture. Based on MRI, retear cases were divided into type 1 (the graft did not remain on the greater tuberosity) and type 2 (the graft remained on the greater tuberosity). Histologic sections were evaluated to examine fascia-bone or rotator cuff-bone interfaces. RESULTS: There were 35 intact repairs: 7 type 1 and 27 type 2 shoulders (type 1 vs. type 2, P < .001). Second-look arthroscopic findings confirmed that the graft was securely attached to the greater tuberosity. Histologic analysis of greater tuberosities in RSA patients showed solid continuity of the graft to the bone, with cells with nuclei in the collagen matrix oriented in parallel. The FLA to bone junction consisted of the FLA, fibrocartilage, and bone, which is similar to the normal cuff tendon to bone junction. CONCLUSIONS: These results indicate that a fresh cellular FLA has good to excellent bone healing potential.

[Survey of the Implementation Status of Personal Counseling among Cancer Patients in Red Cross Hospitals].

Red Cross Pharmaceutical Association conducted a questionnaire-based survey to evaluate the status of implementation of pharmaceutical intervention as well as personal counseling for outpatients with cancer undergoing chemotherapy. Based on the survey results from 93 hospitals across the country, it was found that pharmacists performed an intervention on outpatients receiving chemotherapy in 68 hospitals(73.1%)and conducted personal counseling for outpatients with cancer in 48 hospitals(51.6%). Out of the 68 hospitals, 20 did not conduct personal counseling for outpatients with cancer. This was attributable to the fact that 14 hospitals did not have a qualified pharmacist, 3 did not have sufficient manpower, and 3 did not have the required system. The results of a logistic regression analysis showed that the number of pharmacists significantly affected implementation of pharmaceutical intervention as well as personal counseling for outpatients with cancer undergoing chemotherapy(p=0.042, p=0.023, respectively). The pharmacists can receive a fee for medical services only after conducting personal counseling for outpatients with cancer undergoing chemotherapy. However, in hospitals with a small number of pharmacists, they could not claim their fees owing to lack of manpower. This survey found that lack of manpower is currently the most important issue.

Autophagy inhibition synergizes with calcium mobilization to achieve efficient therapy of malignant gliomas.

Autophagy plays a critical role in tumorigenesis, but how autophagy contributes to cancer cells' responses to chemotherapeutics remains controversial. To investigate the roles of autophagy in malignant gliomas, we used CRISPR/CAS9 to knock out the ATG5 gene, which is essential for autophagosome formation, in tumor cells derived from patients with glioblastoma. While ATG5 disruption inhibited autophagy, it did not change the phenotypes of glioma cells and did not alter their sensitivity to temozolomide, an agent used for glioblastoma patient therapy. Screening of an anticancer drug library identified compounds that showed greater efficacy to ATG5-knockout glioma cells compared to control. While several selected compounds, including nigericin and salinomycin, remarkably induced autophagy, potent autophagy inducers by mTOR inhibition did not exhibit the ATG5-dependent cytoprotective effects. Nigericin in combination with ATG5 deficiency synergistically suppressed spheroid formation by glioma cells in a manner mitigated by Ca2+ chelation or CaMKK inhibition, indicating that, in combination with autophagy inhibition, calcium-mobilizing compounds contribute to efficient anticancer therapeutics. ATG5-knockout cells treated with nigericin showed increased mitochondria-derived reactive oxygen species and apoptosis compared to controls, indicating that autophagy protects glioma cells from mitochondrial reactive oxygen species-mediated damage. Finally, using a patient-derived xenograft model, we demonstrated that chloroquine, a pharmacological autophagy inhibitor, dramatically enhanced the efficacy of compounds selected in this study. Our findings propose a novel therapeutic strategy in which calcium-mobilizing compounds are combined with autophagy inhibitors to treat patients with glioblastoma.

JLP-JNK signaling protects cancer cells from reactive oxygen species-induced cell death.

Oxidative stress, which can be caused by an overproduction of reactive oxygen species (ROS), often leads to cell death. In recent years, c-Jun NH2-terminal kinase (JNK)-associated leucine zipper protein (JLP, also known as SPAG9 or JIP4), a scaffold protein for JNK mitogen-activated protein kinase (MAPK) signaling pathways, was found to serve as a novel biomarker for cancer. However, although JNK MAPK pathways are reported to be activated in response to various stimuli, including oxidative stress, whether JLP is involved in ROS signaling remains unknown. In this study, we examined the role of JLP in hydrogen peroxide (H2O2)-induced cancer cell death, and found that JLP knockdown (KD) cells exhibit a substantially enhanced cell death response, along with increased intracellular ROS levels. This is the first demonstration of a protective role for JLP in response to cell-death stimulation. We also found that the H2O2-induced JNK activation was attenuated in JLP KD cancer cells. The decreases in cell viability and JNK activation in the JLP KD cells were almost completely reversed by expressing wild-type JLP, but not a mutant JLP lacking the JNK-binding domain. These data collectively suggest that the JLP-JNK signaling pathway counteracts ROS-induced cancer cell death.

Distinct roles of Rheb and Raptor in activating mTOR complex 1 for the self-renewal of hematopoietic stem cells.

The mammalian target of rapamycin (mTOR) complex 1 (mTORC1) senses a cell's energy status and environmental levels of nutrients and growth factors. In response, mTORC1 mediates signaling that controls protein translation and cellular metabolism. Although mTORC1 plays a critical role in hematopoiesis, it remains unclear which upstream stimuli regulate mTORC1 activity in the context of hematopoietic stem cells (HSC) maintenance in vivo. In this study, we investigated the function of Rheb, a critical regulator of mTORC1 activity controlled by the PI3K-AKT-TSC axis, both in HSC maintenance in mice at steady-state and in HSC-derived hematopoiesis post-transplantation. In contrast to the severe hematopoietic dysfunction caused by Raptor deletion, which completely inactivates mTORC1, Rheb deficiency in adult mice did not show remarkable hematopoietic failure. Lack of Rheb caused abnormalities in myeloid cells but did not have impact on hematopoietic regeneration in mice subjected to injury by irradiation. As previously reported, Rheb deficiency resulted in defective HSC-derived hematopoiesis post-transplantation. However, while Raptor is essential for HSC competitiveness in vivo, Rheb is dispensable for HSC maintenance under physiological conditions, indicating that the PI3K-AKT-TSC pathway does not contribute to mTORC1 activity for sustaining HSC self-renewal activity at steady-state. Thus, the various regulatory elements that impinge upstream of mTORC1 activation pathways are differentially required for HSC homeostasis in vivo.

Therapeutic Strategy for Targeting Aggressive Malignant Gliomas by Disrupting Their Energy Balance.

Although abnormal metabolic regulation is a critical determinant of cancer cell behavior, it is still unclear how an altered balance between ATP production and consumption contributes to malignancy. Here we show that disruption of this energy balance efficiently suppresses aggressive malignant gliomas driven by mammalian target of rapamycin complex 1 (mTORC1) hyperactivation. In a mouse glioma model, mTORC1 hyperactivation induced by conditional Tsc1 deletion increased numbers of glioma-initiating cells (GICs) in vitro and in vivo Metabolic analysis revealed that mTORC1 hyperactivation enhanced mitochondrial biogenesis, as evidenced by elevations in oxygen consumption rate and ATP production. Inhibition of mitochondrial ATP synthetase was more effective in repressing sphere formation by Tsc1-deficient glioma cells than that by Tsc1-competent glioma cells, indicating a crucial function for mitochondrial bioenergetic capacity in GIC expansion. To translate this observation into the development of novel therapeutics targeting malignant gliomas, we screened drug libraries for small molecule compounds showing greater efficacy in inhibiting the proliferation/survival of Tsc1-deficient cells compared with controls. We identified several compounds able to preferentially inhibit mitochondrial activity, dramatically reducing ATP levels and blocking glioma sphere formation. In human patient-derived glioma cells, nigericin, which reportedly suppresses cancer stem cell properties, induced AMPK phosphorylation that was associated with mTORC1 inactivation and induction of autophagy and led to a marked decrease in sphere formation with loss of GIC marker expression. Furthermore, malignant characteristics of human glioma cells were markedly suppressed by nigericin treatment in vivo Thus, targeting mTORC1-driven processes, particularly those involved in maintaining a cancer cell's energy balance, may be an effective therapeutic strategy for glioma patients.

Efficacy and Safety of Alirocumab in Japanese Patients With Heterozygous Familial Hypercholesterolemia or at High Cardiovascular Risk With Hypercholesterolemia Not Adequately Controlled With Statins　- ODYSSEY JAPAN Randomized Controlled Trial.

BACKGROUND: The ODYSSEY Japan study was designed to demonstrate the reduction in low-density lipoprotein cholesterol (LDL-C) by alirocumab as add-on to existing lipid-lowering therapy in Japanese patients with heterozygous familial hypercholesterolemia (heFH) or non-FH at high cardiovascular risk who require additional pharmacological management to achieve their LDL-C treatment goal (<2.6 or <3.1 mmol/L, depending on risk category). METHODS AND RESULTS: This randomized, double-blind, parallel-group, 52-week study was conducted in Japan. Patients (n=216) with heFH, non-FH at high cardiovascular risk with coronary disease, or classified as category III were enrolled. The prespecified safety analysis was done after the last patient completed 52 weeks. Patients were randomized (2:1, alirocumab:placebo) with stratification for heFH to s.c. alirocumab (75 mg every 2 weeks [Q2 W] with increase to 150 mg if week 8 LDL-C ≥2.6/3.1 mmol/L) or placebo for 52 weeks plus stable statin therapy. At week 24, mean±SE change in LDL-C from baseline was -62.5±1.3% in the alirocumab group and 1.6±1.8% in the placebo group (difference, -64.1±2.2%; P<0.0001); the reduction was sustained to week 52 (alirocumab, -62.5±1.4%; placebo, -3.6±1.9%). No patterns were evident between treatment groups for adverse events at 52 weeks. CONCLUSIONS: In high-risk Japanese patients with hypercholesterolemia on stable statin therapy, alirocumab markedly reduced LDL-C vs. placebo and was well tolerated over 52 weeks. (Circ J 2016; 80: 1980-1987).

Laparoscopy-Assisted Proximal Gastrectomy with the Hinged Double Flap Method.

BACKGROUND: No standard proximal gastrectomy method for gastric cancer of the upper third of the stomach (UGC) has been established because few of the current methods prevent gastro-esophageal reflux and allow easy postoperative endoscopic surveillance. In the present study, we describe laparoscopic proximal gastrectomy with the hinged double flap method (Kamikawa's method) in detail and examine the short- to medium-term outcomes of this approach, which resulted in excellent postoperative function. METHODS: Between 2011 and 2015, 24 patients with early-stage primary UGC underwent laparoscopic proximal gastrectomy with the above-mentioned method. The celiac and hepatic nerves were preserved without pyloroplasty. A hand-sewn esophagogastric anastomosis was produced intracorporeally. RESULTS: There were no in-hospital deaths, and none of the patients were converted to open surgery. Complications occurred in two patients in the early part of this study (minor anastomotic leakage in one case and an abdominal abscess in the other). None of the patients exhibited symptoms of reflux. Regarding the patients' postoperative endoscopic classifications, 17, 2, and 1 patient were considered to have grade N or M, grade A, and grade B esophagitis, respectively. All of the patients remain alive, and no cases of postoperative bowel obstruction or recurrent cancer have been observed. CONCLUSIONS: This method can be performed safely and achieves excellent outcomes in terms of preventing gastro-esophageal reflux. It deserves further evaluation in a multi-center clinical study.

An anatomical investigation of clock face landmarks around the glenoid for shoulder arthroscopy orientation.

BACKGROUND: For shoulder arthroscopy, few anatomical landmarks are available and inexperienced surgeons tend to be adrift due to the limited visual field of the scope. The purpose of this study was to demonstrate the useful landmarks around the glenoid for accurate orientation, and also the safe distance to avoid suprascapular nerve injury during surgical procedures around the glenoid. METHODS: In 15 human solution-fixed cadavers, a cross-section of the shoulder joint on the labrum surface was created. The positions of the principal anatomical structures surrounding the glenoid were marked on the labrum and measured using our clock face indication system. In 9 shoulders the distances from the labral surface to the spinoglenoid notch were recorded. As an indicator of the scapula size, the distances between the superior and inferior angles of the scapula were also measured. RESULTS: The average landmark positions in the right shoulder were as follows: center of the attachment of the long tendon 11:59, anterior edge of the supraspinatus 11:59, posterior edge of the base of the coracoid process 12:13, superior edge of the subscapularis 1:03, anterior edge of the base of the coracoid process 1:25, inferior edge of the subscapularis 5:27, inferior edge of the teres minor 6:21, border of the infraspinatus and teres minor 7:43, center of the scapula spine 10:06, border of the supra and infraspinatus 10:27. The average distance from the labral surface to the spinoglenoid notch was 23.17 mm, and that from the superior to inferior angle was 144.93 mm. The Pearson correlation coefficient for these distances was 0.007. CONCLUSIONS: The locations of anatomical landmarks surrounding the glenoid were reliably demonstrated using our clock face indication system. The expected distance from the labral surface to the suprascapular nerve was approximately 23 mm, irrespective of the size of the scapula.

A novel interplay between the Fanconi anemia core complex and ATR-ATRIP kinase during DNA cross-link repair.

When DNA replication is stalled at sites of DNA damage, a cascade of responses is activated in the cell to halt cell cycle progression and promote DNA repair. A pathway initiated by the kinase Ataxia teleangiectasia and Rad3 related (ATR) and its partner ATR interacting protein (ATRIP) plays an important role in this response. The Fanconi anemia (FA) pathway is also activated following genomic stress, and defects in this pathway cause a cancer-prone hematologic disorder in humans. Little is known about how these two pathways are coordinated. We report here that following cellular exposure to DNA cross-linking damage, the FA core complex enhances binding and localization of ATRIP within damaged chromatin. In cells lacking the core complex, ATR-mediated phosphorylation of two functional response targets, ATRIP and FANCI, is defective. We also provide evidence that the canonical ATR activation pathway involving RAD17 and TOPBP1 is largely dispensable for the FA pathway activation. Indeed DT40 mutant cells lacking both RAD17 and FANCD2 were synergistically more sensitive to cisplatin compared with either single mutant. Collectively, these data reveal new aspects of the interplay between regulation of ATR-ATRIP kinase and activation of the FA pathway.

Association of a murine leukaemia stem cell gene signature based on nucleostemin promoter activity with prognosis of acute myeloid leukaemia in patients.

Acute myeloid leukaemia (AML) is a heterogeneous neoplastic disorder in which a subset of cells function as leukaemia-initiating cells (LICs). In this study, we prospectively evaluated the leukaemia-initiating capacity of AML cells fractionated according to the expression of a nucleolar GTP binding protein, nucleostemin (NS). To monitor NS expression in living AML cells, we generated a mouse AML model in which green fluorescent protein (GFP) is expressed under the control of a region of the NS promoter (NS-GFP). In AML cells, NS-GFP levels were correlated with endogenous NS mRNA. AML cells with the highest expression of NS-GFP were very immature blast-like cells, efficiently formed leukaemia colonies in vitro, and exhibited the highest leukaemia-initiating capacity in vivo. Gene expression profiling analysis revealed that cell cycle regulators and nucleotide metabolism-related genes were highly enriched in a gene set associated with leukaemia-initiating capacity that we termed the 'leukaemia stem cell gene signature'. This gene signature stratified human AML patients into distinct clusters that reflected prognosis, demonstrating that the mouse leukaemia stem cell gene signature is significantly associated with the malignant properties of human AML. Further analyses of gene regulation in leukaemia stem cells could provide novel insights into diagnostic and therapeutic approaches to AML.

NBS1 directly activates ATR independently of MRE11 and TOPBP1.

NBS1 plays unique and essential roles in ATM activation in response to DNA double-strand breaks. We found that CHK1 phosphorylation and FANCD2 ubiquitination induced by various DNA replication-stalling agents were abrogated in Nbs1 knockout DT40 cells but not in conditional Mre11 knockout cells, indicating an MRE11-independent role for NBS1 in ATR activation. The results of in vitro ATR kinase assay indicated that the N-terminal region of NBS1 directly activates ATR independently of TOPBP1, consistent with the findings that this region of NBS1 directly interacts with ATR. This conclusion was furthermore supported by the results of in vivo experiments; the expression of the N-terminal region of NBS1 fused to PCNA induces ATR activation in Rad17 knockout cells, and the expression of the ATR activation domain of TOPBP1 fused to PCNA induces ATR activation in Nbs1 knockout cells. These results therefore indicate that NBS1 and TOPBP1 have the potential to activate ATR independently, although both are required for functional activation of ATR in vivo.