Predictive risk factors for pneumothorax after transbronchial biopsy using endobronchial ultrasonography with a guide sheath.

BACKGROUND: Pneumothorax is one complication of transbronchial biopsy (TBB) using endobronchial ultrasonography with a guide sheath (EBUS-GS-TBB). We sought to clarify the risk factors for pneumothorax after EBUS-GS-TBB under fluoroscopic guidance. METHODS: We retrospectively reviewed data from 916 patients who underwent EBUS-GS-TBB at Fujita Health University Hospital. We evaluated the following risk factors for pneumothorax after EBUS-GS-TBB: patient characteristics (sex, age, and pulmonary comorbidities); lesion data (location, size, existence of ground-glass opacities [GGOs], pleural involvement, computed tomography [CT] bronchus sign, visibility on fluoroscopy, and EBUS findings); final diagnosis; years of bronchoscopist experience; and guide sheath size. Univariate and multivariate logistic regression analyses were performed. RESULTS: Among the 916 patients, 30 (3.28%) presented with pneumothorax. With a univariate analysis, factors that independently predisposed to pneumothorax included lesions containing GGOs, lesions in sagittal lung segments on fluoroscopy, lesions that were not visible on fluoroscopy, and infectious lesions. A univariate analysis also showed that lesions in the right upper lobe or left upper division, as well as malignant lesions, were less likely to lead to pneumothorax. Age, underlying pulmonary disease, CT bronchus sign, EBUS findings, bronchoscopist experience, and guide sheath size did not influence the incidence of pneumothorax. A multivariate analysis revealed that only lesions containing GGOs (odds ratio [OR] 6.47; 95% confidence interval [CI] 2.13-19.6, P = 0.001) and lesions in lung segments with a sagittal orientation on fluoroscopy (OR 2.47; 95% CI 1.09-5.58, P = 0.029) were significant risk factors for EBUS-GS-TBB-related pneumothorax. CONCLUSIONS: EBUS-GS-TBB of lesions containing GGOs or lesions located in sagittal lung segments on fluoroscopy correlate with a higher pneumothorax risk.

Genetic knockout and pharmacologic inhibition of NCX2 cause natriuresis and hypercalciuria.

The Na(+)/Ca(2+) exchanger (NCX) is a bidirectional transporter that is controlled by membrane potential and transmembrane gradients of Na(+) and Ca(2+). Although two isoforms of NCX1 and NCX2 are coexpressed on the basolateral membrane of the distal nephron, the functional significance of these isoforms is not entirely clear. Therefore, we used NCX1- and NCX2-heterozygote knockout mice (KO) and their double KO, as well as isoform-selective NCX inhibitors, to determine the roles of NCX isoforms in urine formation and electrolyte excretion in mice. NCX inhibitors, particularly NCX2-sensitive inhibitors, caused a dose-dependent natriuresis and in a higher dose, moreover, hypercalciuria. Consistently, NCX1-KO possessed normal renal function similar to wild-type mice (WT), whereas NCX2-KO and double KO exhibited moderate natriuresis and hypercalciuria. Notably, renal responses to YM-244769 were equivalently observed in NCX1-KO and WT, but disappeared in NCX2-KO and double KO. Thus, functional inhibition of NCX2 initially causes natriuresis, and further inhibition of NCX2 produces hypercalciuria, suggesting that the functional significance of NCX2 lies in Na(+) and Ca(2+) reabsorption of the kidney.

New screening strategy and analysis for identification of allosteric modulators for glucagon-like peptide-1 receptor using GLP-1 (9-36) amide.

The glucagon-like peptide-1 receptor (GLP-1R) is an important physiologic regulator of insulin secretion and a major therapeutic target for diabetes mellitus. GLP-1 (7-36) amide (active form of GLP-1) is truncated to GLP-1 (9-36) amide, which has been described as a weak agonist of GLP-1R and the major form of GLP-1 in the circulation. New classes of positive allosteric modulators (PAMs) for GLP-1R may offer improved therapeutic profiles. To identify these new classes, we developed novel and robust primary and secondary high-throughput screening (HTS) systems in which PAMs were identified to enhance the GLP-1R signaling induced by GLP-1 (9-36) amide. Screening enabled identification of two compounds, HIT-465 and HIT-736, which possessed new patterns of modulation of GLP-1R. We investigated the ability of these compounds to modify GLP-1R signaling enhanced GLP-1 (9-36) amide- and/or GLP-1 (7-36) amide-mediated cyclic adenosine monophosphate (cAMP) accumulation. These compounds also had unique profiles with regard to allosteric modulation of multiple downstream signaling (PathHunter ß-arrestin signaling, PathHunter internalization signaling, microscopy-based internalization assay). We found allosteric modulation patterns to be obviously different among HIT-465, HIT-736, and Novo Nordisk compound 2. This work may enable the design of new classes of drug candidates by targeting modulation of GLP-1 (7-36) amide and GLP-1 (9-36) amide.

A Novel Drosophila-based Drug Repurposing Platform Identified Fingolimod As a Potential Therapeutic for TDP-43 Proteinopathy.

Pathogenic changes to TAR DNA-binding protein 43 (TDP-43) leading to alteration of its homeostasis are a common feature shared by several progressive neurodegenerative diseases for which there is no effective therapy. Here, we developed Drosophila lines expressing either wild type TDP-43 (WT) or that carrying an Amyotrophic Lateral Sclerosis /Frontotemporal Lobar Degeneration-associating G384C mutation that recapitulate several aspects of the TDP-43 pathology. To identify potential therapeutics for TDP-43-related diseases, we implemented a drug repurposing strategy that involved three consecutive steps. Firstly, we evaluated the improvement of eclosion rate, followed by the assessment of locomotive functions at early and late developmental stages. Through this approach, we successfully identified fingolimod, as a promising candidate for modulating TDP-43 toxicity. Fingolimod exhibited several beneficial effects in both WT and mutant models of TDP-43 pathology, including post-transcriptional reduction of TDP-43 levels, rescue of pupal lethality, and improvement of locomotor dysfunctions. These findings provide compelling evidence for the therapeutic potential of fingolimod in addressing TDP-43 pathology, thereby strengthening the rationale for further investigation and consideration of clinical trials. Furthermore, our study demonstrates the utility of our Drosophila-based screening pipeline in identifying novel therapeutics for TDP-43-related diseases. These findings encourage further scale-up screening endeavors using this platform to discover additional compounds with therapeutic potential for TDP-43 pathology.

Preferential involvement of Na⁺/Ca²âº exchanger type-1 in the brain damage caused by transient focal cerebral ischemia in mice.

The Na(+)/Ca(2+) exchanger (NCX), an ion-transporter located in the plasma membrane of neuronal cells, contributes to intracellular Ca(2+) homeostasis. Within the brain, three isoforms (NCX1, NCX2, and NCX3) are widely distributed. However, it is not clear to what extent these isoforms are involved in ischemic brain damage in mammals. We therefore used genetically altered mice and isoform-selective NCX inhibitors in a model of transient focal ischemia to investigate the role of each NCX isoform in ischemic brain damage. NCX isoform-mutant mice (NCX1(+/-), NCX2(+/-), and NCX3(+/-)) and wild-type mice were subjected to 90min of middle cerebral artery occlusion (MCAO) followed by 24h of reperfusion. One of three NCX inhibitors [SN-6, KB-R7943, or SEA0400 (3 or 10mgkg(-1), i.p.)] was administered to ddY mice at 30min before more prolonged (4-h) MCAO followed by 24h of reperfusion. After transient MCAO reperfusion, the cerebral infarcts in NCX1(+/-) mice, but not those in NCX2(+/-) or NCX3(+/-) mice, were significantly smaller than those in wild-type mice. SN-6 and SEA0400, which are more selective for the NCX1 isoform, significantly reduced the infarct volume at 10mg/kg. In contrast, KB-R7943, which is more selective for NCX3, did not. These results suggest that the NCX1 isoform may act preferentially (vs. the NCX2 and NCX3 isoforms) to exacerbate the cerebral damage caused by ischemic insult in mice, and that NCX1-selective inhibitors warrant investigation as a potential therapeutic agents for stroke.

Availability of Home sleep apnea test equipment LS-140 on a comparison with Polysomnography.

OBJECTIVE: The prevalence of obstructive sleep apnea (OSA) in Japan is 9% among males and 3% among females. Up to 2.5 million patients are estimated to suffer from the disease, but limited number of facilities are capable of carrying out polysomnography (PSG), leaving more than 80% of these individuals are undiagnosed. In recent years, the development of new portable sleep monitoring (PMs) devices has been remarkable. We evaluate the correlation between the results of the LS-140 PMs device (Fukuda Denshi Tech Co. Ltd.), released in 2017, and those of PSG. METHODS: We obtained contemporaneous data from the same patients by equipping 58 patients with PMs (LS-140) devices while they underwent PSG. Our primary outcome was Case 2 of the intraclass correlation coefficient (ICC), i.e., the ICC (2.1). And we used a Bland-Altman analysis to compare the apnea-hypopnea index (AHI) given by PSG and the respiratory event index (REI) given by LS-140 and examined the sensitivity and specificity of the REI relative to the AHI in the diagnosis of OSA. We also carried out the same comparison but in terms of the presence or absence of periodic limb movements (PLMs). RESULTS: The ICC (2.1) between The REI and the AHI was 0.944, a rather high value (p<0.0001). The mean difference between AHI and REI values was -3.6 (p<0.0001), indicating a negative fixed bias. Sensitivity may decrease in groups with PLMs. CONCLUSION: The REI and the AHI are highly correlated, giving LS-140 sufficient diagnostic sensitivity and specificity to screen for OSA.

A homogeneous time-resolved fluorescence-based high-throughput screening system for discovery of inhibitors of IKKbeta-NEMO interaction.

The nuclear transcription factor NF-kappaB is crucial to the expression of numerous cytokines, enzymes, and cell adhesion molecules, all of which can drive inflammatory and autoimmune disorders such as rheumatoid arthritis. The IKK complex plays the most important role in the signal cascade leading to NF-kappaB activation. Recently, inhibition of the interaction between NEMO (NF-kappaB essential modulator) and the catalytic subunits of IKK, especially IKKbeta, has received particular attention as a possible new therapeutic approach to treatment of inflammatory disorders, and several reports have shown the efficacy of cell permeable NEMO binding domain (NBD)-containing peptides in blocking the IKK/NF-kappaB pathway. In this article, we describe in detail the development and validation of two novel binding assays, a homogeneous time-resolved fluorescence (HTRF)-based assay and an enzyme-linked immunosorbent assay (ELISA)-based assay, suitable for the discovery of small molecules that inhibit IKKbeta-NEMO interaction. Using the HTRF-based assay, we screened approximately 15,000 compounds from our chemical library and eliminated false positive hits by the ELISA-based assay and IKK complex kinase assay. As a result, seven positive hit compounds that inhibit IKK complex activity through inhibition of IKKbeta-NEMO interaction were identified. These hit compounds may have a good potential in the treatment of inflammatory and autoimmune disorders such as rheumatoid arthritis.

Negative regulation of constitutive NF-kappaB and JNK signaling by PKN1-mediated phosphorylation of TRAF1.

Inhibitor of NF-kappaB (IkappaB) kinase (IKK) and c-Jun NH(2)-terminal kinase (JNK) are stress inducible kinases that critically regulate numerous physiological and pathological processes. Transient activation of the downstream transcription factors NF-kappaB and AP-1, allows for stress inducible, inflammatory and innate immune gene expression programs. However, elevated chronic activity is associated with cancer and chronic inflammatory disease. Despite its relevance to human health, little is known about the molecular mechanisms that control constitutive activity of IKK and JNK. Here, we demonstrate that the serine/threonine kinase PKN1 plays a critical role in regulating constitutive IKK/JNK activity in unstimulated cells and report on the molecular mechanism. We identify TRAF1 as a substrate of PKN1 kinase activity in vitro and in vivo, and show that this phosphorylation event is required for attenuating downstream kinase activities. Furthermore, this silencing was dependent on TNFR2. Mutagenesis of the phospho-acceptor residue in TRAF1 abrogated PKN1-dependent recruitment to TNFR2. Our results suggest a model by which the stoichiometric ratio of TRAF1 and TRAF2 heteromeric complexes associated with TNFR2 control the tonic activity of JNK and IKK. TRAF1 phosphorylation by the ubiquitously expressed kinase PKN1 thereby plays a critical role in the negative regulation of tonic activity of the two central inflammatory signaling pathways.

Gastrointestinal perforation during treatment with erlotinib plus bevacizumab in two patients with non-small cell lung cancer exhibiting epidermal growth factor receptor mutations: A case report.

A previous randomized phase II study in patients with non-small cell lung cancer (NSCLC) identified that combination treatment with erlotinib plus bevacizumab prolonged progression-free survival compared with erlotinib alone. However, combination bevacizumab and erlotinib treatment generally increased the risk of severe adverse events, including hemorrhage, thrombosis, fistula formation and gastrointestinal perforation. The present report describes two patients with NSCLC harboring epidermal growth factor receptor (EGFR) mutations, who experienced gastrointestinal perforation associated with erlotinib plus bevacizumab combination therapy. The first patient, a 67-year-old male with stage IIIB lung adenocarcinoma harboring a L858R point mutation in EGFR exon 21, received concurrent chemoradiotherapy. However, seven months later, the patient experienced a relapse and was administered erlotinib plus bevacizumab treatment. A total of two months subsequent to commencing treatment, the patient developed a perforated duodenal ulcer. The second patient, a 66-year-old male with lung adenocarcinoma harboring a deletion in EGFR exon 19 and multiple pulmonary metastases, demonstrated a partial response to erlotinib plus bevacizumab treatment. A total of seven months subsequent to starting treatment, the patient experienced lower abdominal pain, and abdominal computed tomography confirmed a diagnosis of colocutaneous fistula complicating sigmoid diverticulitis. Following repair of the perforation, both patients were restarted on erlotinib treatment alone. Gastrointestinal perforation may be a potentially severe adverse event of erlotinib plus bevacizumab combination therapy, even in the absence of tumor metastasis in the abdomen.

A basic study on self-reconstitution of alveolar epithelium-like cells by tissue stem cells in mouse lung.

In recent research on regenerative medicine, three-dimensional (3D) tissue reconstruction using the induced pluripotent stem cell (iPS cell) differentiated cells has attracted attention. In this study, mouse lungs at 1.5, 10, and 20 d old were subjected to enzyme treatment, and aggregates formed in serum-free suspension culture (3D-culture) were observed. The number of aggregates formed was the highest in 1.5 d. The cell aggregates in which the interior of the aggregate is filled and form small vacuoles and the organoid-like aggregates having a relatively large vacuole inside and forming the alveolar-like structure were observed. At 1.5 d, the formation ratio of the organoid-like aggregates was the highest and aggregate size was small at 20 d. For the cell aggregates derived from 1.5 d, positive cells of SSEA-1, CD29, CD90, CD105, alveolar epithelial stem cell marker of SP-C, and Sca-1 were observed in the center. In the cell aggregates derived from 10 d, the expression level of 1.5 d each protein markers and OCT4 gene of transcription factor was decreased, and furthermore, markers were hardly observed in the organoid-like aggregates derived from 10 d. In addition, cells surrounding the vacuole of organoid-like aggregate obtained over 10 d differentiated into periodic acid-Schiff (PAS), podoplanin-positive cells. When the formed cell aggregates were dispersed, cell aggregates and organoid-like aggregates were reformed. Comparing 3D-culture and adhesion culture (2D-culture), SP-C expression of 10 d of cells was maintained. Expression of markers of undifferentiated markers and alveolar tissue stem cells decreased when cell aggregates were cultured with the addition of fetal bovine serum.

Regulation of a mitogen-activated protein kinase kinase kinase, MLTK by PKN.

PKNalpha is a fatty acid- and Rho-activated serine/threonine protein kinase having a catalytic domain homologous to members of the protein kinase C family. Recently it was reported that PKNalpha is involved in the p38 mitogen-activated protein kinase (MAPK) signaling pathway. To date, however, how PKNalpha regulates the p38gamma MAPK signaling pathway is unclear. Here we demonstrate that PKNalpha efficiently phosphorylates MLTKalpha (MLK-like mitogen-activated protein triple kinase), which was recently identified as a MAPK kinase kinase (MAPKKK) for the p38 MAPK cascade. Phosphorylation of MLTKalpha by PKNalpha enhances its kinase activity in vitro. Expression of the kinase-negative mutant of PKNalpha inhibited the mobility shift of MLTKalpha caused by osmotic shock in SDS-PAGE. Furthermore, PKNalpha associates with each member of the p38gamma MAPK signaling pathway (p38gamma, MKK6, and MLTKalpha). These results suggest that PKNalpha functions as not only an upstream activator of MLTKalpha but also a putative scaffold protein for the p38gamma MAPK signaling pathway.

Protein kinase PKN1 associates with TRAF2 and is involved in TRAF2-NF-kappaB signaling pathway.

PKN1 is a fatty acid and Rho-activated serine/threonine protein kinase whose catalytic domain is highly homologous to protein kinase C (PKC) family. In yeast two-hybrid screening for PKN1 binding proteins, we identified tumor necrosis factor alpha (TNFalpha) receptor-associated factor 2 (TRAF2). TRAF2 is one of the major mediators of TNF receptor superfamily transducing TNF signal to various functional targets, including activation of NF-kappaB, JNK, and apoptosis. FLAG-tagged PKN1 was co-immunoprecipitated with endogenous TRAF2 from HEK293 cell lysate, and in vitro binding assay using the deletion mutants of TRAF2 showed that PKN1 directly binds to the TRAF domain of TRAF2. PKN1 has the TRAF2-binding consensus sequences PXQX (S/T) at amino acid residues 580-584 (PIQES), and P580AQ582A mutant was not co-immunoprecipitated with TRAF2. Furthermore, the reduced expression of PKN1 by RNA interference (RNAi) down-regulated TRAF2-induced NF-kappaB activation in HEK293T cells. These results suggest that PKN1 is involved in TRAF2-NF-kappaB signaling pathway.