Proximity-dependent recruitment of Polycomb repressive complexes by the lncRNA Airn.

During mouse embryogenesis, expression of the long non-coding RNA (lncRNA) Airn leads to gene repression and recruitment of Polycomb repressive complexes (PRCs) to varying extents over a 15-Mb domain. The mechanisms remain unclear. Using high-resolution approaches, we show in mouse trophoblast stem cells that Airn expression induces long-range changes to chromatin architecture that coincide with PRC-directed modifications and center around CpG island promoters that contact the Airn locus even in the absence of Airn expression. Intensity of contact between the Airn lncRNA and chromatin correlated with underlying intensity of PRC recruitment and PRC-directed modifications. Deletion of CpG islands that contact the Airn locus altered long-distance repression and PRC activity in a manner that correlated with changes in chromatin architecture. Our data imply that the extent to which Airn expression recruits PRCs to chromatin is controlled by DNA regulatory elements that modulate proximity of the Airn lncRNA product to its target DNA.

A monoclonal antibody raised against human EZH2 cross-reacts with the RNA-binding protein SAFB.

The Polycomb Repressive Complex 2 (PRC2) is a conserved enzyme that tri-methylates Lysine 27 on Histone 3 (H3K27me3) to promote gene silencing. PRC2 is remarkably responsive to the expression of certain long noncoding RNAs (lncRNAs). In the most notable example, PRC2 is recruited to the X-chromosome shortly after expression of the lncRNA Xist begins during X-chromosome inactivation. However, the mechanisms by which lncRNAs recruit PRC2 to chromatin are not yet clear. We report that a broadly used rabbit monoclonal antibody raised against human EZH2, a catalytic subunit of PRC2, cross-reacts with an RNA-binding protein called Scaffold Attachment Factor B (SAFB) in mouse embryonic stem cells (ESCs) under buffer conditions that are commonly used for chromatin immunoprecipitation (ChIP). Knockout of EZH2 in ESCs demonstrated that the antibody is specific for EZH2 by western blot (no cross-reactivity). Likewise, comparison to previously published datasets confirmed that the antibody recovers PRC2-bound sites by ChIP-Seq. However, RNA-IP from formaldehyde-crosslinked ESCs using ChIP wash conditions recovers distinct peaks of RNA association that co-localize with peaks of SAFB and whose enrichment disappears upon knockout of SAFB but not EZH2. IP and mass spectrometry-based proteomics in wild-type and EZH2 knockout ESCs confirm that the EZH2 antibody recovers SAFB in an EZH2-independent manner. Our data highlight the importance of orthogonal assays when studying interactions between chromatin-modifying enzymes and RNA.

Ectopically expressed Airn lncRNA deposits Polycomb with a potency that rivals Xist.

We report that when expressed at similar levels from an isogenic locus, the Airn lncRNA induces Polycomb deposition with a potency that rivals Xist . However, when subject to the same degree of promoter activation, Xist is more abundant and more potent than Airn . Our data definitively demonstrate that the Airn lncRNA is functional and suggest that Xist achieved extreme potency in part by evolving mechanisms to promote its own abundance.

A monoclonal antibody raised against human EZH2 cross-reacts with the RNA-binding protein SAFB.

The Polycomb Repressive Complex 2 (PRC2) is a conserved enzyme that tri-methylates Lysine 27 on Histone 3 (H3K27me3) to promote gene silencing. PRC2 is remarkably responsive to the expression of certain long noncoding RNAs (lncRNAs). In the most notable example, PRC2 is recruited to the X-chromosome shortly after expression of the lncRNA Xist begins during X-chromosome inactivation. However, the mechanisms by which lncRNAs recruit PRC2 to chromatin are not yet clear. We report that a broadly used rabbit monoclonal antibody raised against human EZH2, a catalytic subunit of PRC2, cross-reacts with an RNA-binding protein called Scaffold Attachment Factor B (SAFB) in mouse embryonic stem cells (ESCs) under buffer conditions that are commonly used for chromatin immunoprecipitation (ChIP). Knockout of EZH2 in ESCs demonstrated that the antibody is specific for EZH2 by western blot (no cross-reactivity). Likewise, comparison to previously published datasets confirmed that the antibody recovers PRC2-bound sites by ChIP-Seq. However, RNA-IP from formaldehyde-crosslinked ESCs using ChIP wash conditions recovers distinct peaks of RNA association that co-localize with peaks of SAFB and whose enrichment disappears upon knockout of SAFB but not EZH2. IP and mass spectrometry-based proteomics in wild-type and EZH2 knockout ESCs confirm that the EZH2 antibody recovers SAFB in an EZH2-independent manner. Our data highlight the importance of orthogonal assays when studying interactions between chromatin-modifying enzymes and RNA.

Salvage Surgery Compared to Surgery After Induction Chemoradiation Therapy for Advanced Lung Cancer.

BACKGROUND: Salvage surgery is performed for selected patients with relapses of locally advanced lung cancer after definitive chemoradiation therapy (CRT), and it seems to be effective. To assess the feasibility of salvage surgery after definitive CRT, this study compared clinical outcomes of surgery after definitive CRT with those of surgery after induction CRT. METHODS: Medical records of patients who underwent surgery from January 2000 to January 2018 were reviewed. The study compared patients with salvage anatomic pulmonary resection after definitive CRT with patients with surgery after induction CRT in terms of perioperative and long-term outcomes. RESULTS: A total of 23 patients underwent salvage surgery after definitive CRT for locally advanced lung cancer (salvage group), and 36 underwent surgery after induction CRT for cN2 stage III non-small cell lung cancer (induction CRT group). The surgical procedures in the salvage group were 2 segmentectomies, 13 lobectomies, 1 bilobectomy, and 7 pneumonectomies, and those in the induction CRT group were 34 lobectomies and 2 bilobectomies. There was no 30-day or 90-day mortality in either group. The 5-year overall survival was 44.7% for the salvage group and 58.6% for the induction CRT group. The 5-year progression-free interval was 42.2% for the salvage group and 47.7% for the induction CRT group. CONCLUSIONS: Salvage anatomic pulmonary resection after definitive CRT for locally advanced lung cancer is feasible, with acceptable morbidity and prognosis in highly selected patients.

Women in thoracic surgery: Asian perspective.

There is a significant shortage of surgeons in Japan similar to many other countries. There are 40,267 registered surgeons for Japan Surgical Society, among them 23,723 are board certificated, and 7,246 are certified instructors. A total of 3,207 surgeons are members of The Japanese Associations for Chest Surgery, and 245 (7.6%) are female surgeons. Approximately 8,000 medical students graduate annually with medical license. The number of newly registered residents and surgeons has recently been declining overall in the last two decades, however, the percentage of female physicians entering into surgery has been increasing gradually along with increasing number of female medical students entering the medical schools and colleges. With regard to the issue of gender gap, our country is ranked as low as 121 out of 153 countries according to global gender gap report 2020 by World Economic Forum, dropping down from 110 out of 149 countries from previous report in 2018. As the numbers of female colleagues in our workplace increases, we must consider what we can do to build a better working environment for them that is more inclusive and supports their career. In this review, the brief history of surgery and female surgeons is described as well as current situations of female surgeons today in Japan.

Salvage surgery after chemotherapy and/or radiotherapy including SBRT and proton therapy: A consecutive analysis of 38 patients.

BACKGROUND: Local recurrence after definitive chemoradiation therapy, chemotherapy or radiotherapy with curative intent is often seen in patients with advanced non-small cell lung cancer. We evaluated the feasibility of salvage pulmonary resection after definitive non-surgical treatments and the postoperative morbidity and mortality rates. METHODS: We retrospectively analyzed the characteristics and medical courses of patients who had undergone salvage pulmonary resections after local relapse or progression between January 2000 and March 2018 at the National Cancer Centre Hospital, Tokyo, Japan. All the candidates were evaluated, and curability by surgical resection was assessed by a multidisciplinary tumor board. RESULTS: A total of 38 patient received salvage surgery: 26 of the patients were men, and the median age was 64.5 years (range, 20-78 years). Among these 38 patients, salvage lung resection was performed after chemoradiotherapy in 23 patients, after chemotherapy in 9 patients, and after radiotherapy with curative intent in 6 patients. The surgical resection methods were as follows: 26 lobectomies (2 bilobectomy, 15 right upper, 5 right lower, 1 right middle, 2 left lower and 1 left upper), 8 pneumonectomies (5 left and 3 right), and 4 segmentectomies. A complete resection (R0 resection) was achieved in 35 cases (92.1 %). Postoperative complications were observed in 3 patients (prolonged air leakage, bronchopleural fistula and surgical site infection in 1 patient each). No postoperative deaths occurred within 30 days after surgery. CONCLUSION: Along with better outcomes after definitive chemoradiotherapy, chemotherapy, and radiotherapy, the frequency of salvage surgery has been increasing in recent years. Salvage pulmonary resections after definitive non-surgical treatments with curative intent are feasible with an acceptable morbidity rate and oncological outcomes in thoroughly assessed patients.

Formation of Stomach Tissue by Organoid Culture Using Mouse Embryonic Stem Cells.

In this chapter, we describe a method for the induction of stomach organoids from mouse embryonic stem (ES) cells. We used an embryoid body-based differentiation method to induce gastric primordial epithelium covered with mesenchyme and further differentiate it in Matrigel by 3D culture. The differentiated organoid contains both corpus- and antrum-specific mature gastric tissue cells. This protocol may be useful for a variety of studies in developmental biology and disease modeling of the stomach.

Ground-based assessment of JAXA mouse habitat cage unit by mouse phenotypic studies.

The Japan Aerospace Exploration Agency developed the mouse Habitat Cage Unit (HCU) for installation in the Cell Biology Experiment Facility (CBEF) onboard the Japanese Experimental Module ("Kibo") on the International Space Station. The CBEF provides "space-based controls" by generating artificial gravity in the HCU through a centrifuge, enabling a comparison of the biological consequences of microgravity and artificial gravity of 1 g on mice housed in space. Therefore, prior to the space experiment, a ground-based study to validate the habitability of the HCU is necessary to conduct space experiments using the HCU in the CBEF. Here, we investigated the ground-based effect of a 32-day housing period in the HCU breadboard model on male mice in comparison with the control cage mice. Morphology of skeletal muscle, the thymus, heart, and kidney, and the sperm function showed no critical abnormalities between the control mice and HCU mice. Slight but significant changes caused by the HCU itself were observed, including decreased body weight, increased weights of the thymus and gastrocnemius, reduced thickness of cortical bone of the femur, and several gene expressions from 11 tissues. Results suggest that the HCU provides acceptable conditions for mouse phenotypic analysis using CBEF in space, as long as its characteristic features are considered. Thus, the HCU is a feasible device for future space experiments.

Effectiveness and Drug Survival of TNF Inhibitors in the Treatment of Ankylosing Spondylitis: A Prospective Cohort Study.

OBJECTIVE: The aim of this research was to describe the effectiveness and drug survival of tumor necrosis factor (TNF) inhibitors in the treatment of ankylosing spondylitis (AS) and to analyze the effect of concomitant treatment with conventional disease-modifying antirheumatic drugs. METHODS: Patients with AS identified from the National Register for Biologic Treatment in Finland starting their first TNF inhibitor treatment between July 2004 and December 2011 were included. Treatment response was measured as an improvement of 50% (or 20 mm) after 6 months of treatment onset compared to the baseline Bath AS Disease Activity Index (BASDAI) score. Treatment response and 2-year drug survival were modeled with logistic regression and time-dependent Cox proportional hazard models, respectively. RESULTS: The study comprised 543 patients, of whom 123 also commenced a second TNF inhibitor during the followup. Treatment was discontinued within 24 months by 25% and 28% of the users of the first and the second TNF inhibitors, respectively. BASDAI response at 6 months was achieved by 52% and 25% of the users of the first and the second TNF inhibitors, respectively. Etanercept (ETN; HR 0.42, 95% CI 0.29-0.62) and adalimumab (ADA; HR 0.48, 95% CI 0.30-0.77) were associated with better drug survival in comparison to infliximab (IFX). Also, concurrent use of sulfasalazine (SSZ; HR 0.70, 95% CI 0.49-0.99) decreased the hazard for treatment discontinuation. CONCLUSION: TNF inhibitors are equipotent in the treatment of AS; however, ETN and ADA were found superior to IFX in drug survival. The use of SSZ improves treatment continuation.

Generation of stomach tissue from mouse embryonic stem cells.

Successful pluripotent stem cell differentiation methods have been developed for several endoderm-derived cells, including hepatocytes, ß-cells and intestinal cells. However, stomach lineage commitment from pluripotent stem cells has remained a challenge, and only antrum specification has been demonstrated. We established a method for stomach differentiation from embryonic stem cells by inducing mesenchymal Barx1, an essential gene for in vivo stomach specification from gut endoderm. Barx1-inducing culture conditions generated stomach primordium-like spheroids, which differentiated into mature stomach tissue cells in both the corpus and antrum by three-dimensional culture. This embryonic stem cell-derived stomach tissue (e-ST) shared a similar gene expression profile with adult stomach, and secreted pepsinogen as well as gastric acid. Furthermore, TGFA overexpression in e-ST caused hypertrophic mucus and gastric anacidity, which mimicked Ménétrier disease in vitro. Thus, in vitro stomach tissue derived from pluripotent stem cells mimics in vivo development and can be used for stomach disease models.

Diagnostic approach to pupillary abnormalities.

PURPOSE OF REVIEW: This article presents an overview of the common and various kinds of pupillary disorders that can be encountered in an outpatient setting. RECENT FINDINGS: The dorsal midbrain is a site where lesions may produce either an afferent or an efferent pupillary defect. The classic pupillary syndrome secondary to a dorsal midbrain lesion is bilateral light-near dissociation. Another recognized deficit is bilateral mydriasis. Recent reports have documented unilateral mydriasis, unilateral light-near dissociation, and a relative afferent pupillary defect without visual loss in association with lesions of the dorsal midbrain. These are rare syndromes. SUMMARY: Careful history and examination can often identify and localize the pupillary disorder, as well as guide appropriate evaluation.

Prohibitin 2 regulates the proliferation and lineage-specific differentiation of mouse embryonic stem cells in mitochondria.

BACKGROUND: The pluripotent state of embryonic stem (ES) cells is controlled by a network of specific transcription factors. Recent studies also suggested the significant contribution of mitochondria on the regulation of pluripotent stem cells. However, the molecules involved in these regulations are still unknown. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we found that prohibitin 2 (PHB2), a pleiotrophic factor mainly localized in mitochondria, is a crucial regulatory factor for the homeostasis and differentiation of ES cells. PHB2 was highly expressed in undifferentiated mouse ES cells, and the expression was decreased during the differentiation of ES cells. Knockdown of PHB2 induced significant apoptosis in pluripotent ES cells, whereas enhanced expression of PHB2 contributed to the proliferation of ES cells. However, enhanced expression of PHB2 strongly inhibited ES cell differentiation into neuronal and endodermal cells. Interestingly, only PHB2 with intact mitochondrial targeting signal showed these specific effects on ES cells. Moreover, overexpression of PHB2 enhanced the processing of a dynamin-like GTPase (OPA1) that regulates mitochondrial fusion and cristae remodeling, which could induce partial dysfunction of mitochondria. CONCLUSIONS/SIGNIFICANCE: Our results suggest that PHB2 is a crucial mitochondrial regulator for homeostasis and lineage-specific differentiation of ES cells.

Novel cell surface genes expressed in the stomach primordium during gastrointestinal morphogenesis of mouse embryos.

The mechanisms of gastrointestinal morphogenesis in mammals are not well understood. This is partly due to the lack of appropriate markers that are expressed with spatiotemporal specificity in the gastrointestinal tract during development. Using mouse embryos, we surveyed markers of the prospective stomach region during gastrointestinal morphogenesis. The initiation of organ bud formation occurs at E10.5 in mice. These primordia for the digestive organs protrude from a tube-like structured endoderm and have their own distinct morphogenesis. We identified 3 cell surface genes -Adra2a, Fzd5, and Trpv6 - that are expressed in the developing stomach region during gastrointestinal morphogenesis using a microarray-based screening. These novel genes will be useful in expanding our understanding of the mechanisms of gastrointestinal development.

Evaluation of suicide by hanging : From the video recording.

Most evaluations of the pathophysiological processes of suicidal hangings are based on animal experiments and only a few have examined the hanging process. The little information available is mostly the result of animal experiments that have focused on tracheal obstructions. In the study presented here, a camcorder was set up by the victim to record the entire hanging process. According to the analysis from the recording, the complete asphyxial process lasted only 2 minutes and 43 seconds. Stage 1 (the prodromal stage) was especially short. Typical convulsions were not observed in this case. Body movements, such as clonic and tonic spasms, were not noticed in stage 2 (the dyspnea stage). Because the body did not touch the ground, the pressure on the neck by the ligature caused cerebral ischemia as a result of the obstruction of the neck vessels. Consequently, it can be hypothesized that sui cide by hanging may not be based on the effect of changed concentration in the blood of carbon dioxide and oxygen, but on the effect of cerebral ischemia.

Functional association between nicotinic acetylcholine receptor and sarcomeric proteins via actin and desmin filaments.

By affinity chromatography utilizing alpha-cobrotoxin from digitonin-solubilized fractions of rabbit skeletal muscle, we found that many proteins are associated with the nicotinic acetylcholine receptor (AChR). In addition to the proteins we previously reported to bind to AChR (including dystrophin-dystrophin-associated protein (DAP) complex, utrophin, rapsyn, and actin; Mitsui et al. [1996] Biochem. Biophys. Res. Commun.224:802-807), alpha-actinin, desmin, myosin, tropomyosin, troponin T, and titin are also identified to be associated with AChR. Alkaline treatment or Triton X-100 solubilization released dystrophin-DAP complex, utrophin, and rapsyn from the AChR fraction, while actin and desmin remained associated. These findings demonstrate that AChR is supported primarily by a submembranous organization of actin and desmin filaments, and is linked to sarcomeric proteins via these filaments. To further investigate whether the association has any functional role, we studied the effect of acetylcoline on ATPase activity of the AChR fraction. Acetylcholine (0.5-4 microM) significantly activated Mg(2+)-ATPase activity of digitonin-solubilized AChR fraction (P < 0.05). Furthermore, we found that desmin as well as actin activated myosin Mg(2+)-ATPase activity. From these findings, it is suggested that desmin and actin form a submembranous organization in the postsynaptic region, and function as mediators of excitation of AChR to the sarcomeric contraction system.

L-lactate oxidase from Aerococcus viridans crystallized as an octamer. Preliminary X-ray studies.

Crystals of flavoenzyme L-lactate oxidase from Aerococcus viridans (LOX) have been obtained that diffract to 3.0 A resolution (P2(1)2(1)2(1), a = 118.4 A, b = 138.4 A, c = 194.6 A). Crystallographic studies suggest that the enzyme may exist as an octameric form with non-crystallographic two- and four-fold axes in the center of the octamer. The four-fold axis makes the tetramer tight, and the tetramers lie upon one another by the two-fold axis.

Evidence of shallow fault zone strengthening after the 1992 M7.5 landers, california, earthquake

Repeated seismic surveys of the Landers, California, fault zone that ruptured in the magnitude (M) 7.5 earthquake of 1992 reveal an increase in seismic velocity with time. P, S, and fault zone trapped waves were excited by near-surface explosions in two locations in 1994 and 1996, and were recorded on two linear, three-component seismic arrays deployed across the Johnson Valley fault trace. The travel times of P and S waves for identical shot-receiver pairs decreased by 0.5 to 1.5 percent from 1994 to 1996, with the larger changes at stations located within the fault zone. These observations indicate that the shallow Johnson Valley fault is strengthening after the main shock, most likely because of closure of cracks that were opened by the 1992 earthquake. The increase in velocity is consistent with the prevalence of dry over wet cracks and with a reduction in the apparent crack density near the fault zone by approximately 1.0 percent from 1994 to 1996.

On the reaction mechanism of L-lactate oxidase: quantitative structure-activity analysis of the reaction with para-substituted L-mandelates.

The rate constants for reduction of the flavoenzyme, L-lactate oxidase, and a mutant (in which alanine 95 is replaced by glycine), by a series of para-substituted mandelates, in both the 2-1H- and 2-2H- forms, have been measured by rapid reaction spectrophotometry. In all cases, significant isotope effects (1H/2H = 3-7) on the rate constants of flavin reduction were found, indicating that flavin reduction is a direct measure of alpha-C-H bond breakage. The rate constants show only a small influence of the electronic characteristics of the substituents, but show a good correlation when combined with some substituent volume parameters. A surprisingly good correlation is found with the molecular mass of the substrate. The results are compatible with any mechanism in which there is little development of charge in the transition state. This could be a transfer of hydride to the flavin N(5) position or a synchronous mechanism in which the alpha-C-H is formally abstracted as a H+ while the resulting charge is simultaneously neutralized by another event.