The cytosolic lncRNA dutA affects STATa signaling and developmental commitment in Dictyostelium.

STATa is a pivotal transcription factor for Dictyostelium development. dutA is the most abundant RNA transcribed by RNA polymerase II in Dictyostelium, and its functional interplay with STATa has been suggested. This study demonstrates that dutA RNA molecules are distributed as spot-like structures in the cytoplasm, and that its cell type-specific expression changes dramatically during development. dutA RNA was exclusively detectable in the prespore region of slugs and then predominantly localized in prestalk cells, including the organizer region, at the Mexican hat stage before most dutA transcripts, excluding those in prestalk O cells, disappeared as culmination proceeded. dutA RNA was not translated into small peptides from any potential open reading frame, which confirmed that it is a cytoplasmic lncRNA. Ectopic expression of dutA RNA in the organizer region of slugs caused a prolonged slug migration period. In addition, buffered suspension-cultured cells of the strain displayed reduced STATa nuclear translocation and phosphorylation on Tyr702. Analysis of gene expression in various dutA mutants revealed changes in the levels of several STATa-regulated genes, such as the transcription factors mybC and gtaG, which might affect the phenotype. dutA RNA may regulate several mRNA species, thereby playing an indirect role in STATa activation.

Protocol for the 2ND-STEP study, Japan Clinical Oncology Group study JCOG1802: a randomized phase II trial of second-line treatment for advanced soft tissue sarcoma comparing trabectedin, eribulin and pazopanib.

BACKGROUND: Soft tissue sarcomas (STS) are a rare type of malignancy comprising a variety of histological diagnoses. Chemotherapy constitutes the standard treatment for advanced STS. Doxorubicin-based regimens, which include the administration of doxorubicin alone or in combination with ifosfamide or dacarbazine, are widely accepted as first-line chemotherapy for advanced STS. Trabectedin, eribulin, pazopanib, and gemcitabine plus docetaxel (GD), which is the empirical standard therapy in Japan, are major candidates for second-line chemotherapy for advanced STS, although clear evidence of the superiority of any one regimen is lacking. The Bone and Soft Tissue Tumor Study Group of the Japan Clinical Oncology Group (JCOG) conducts this trial to select the most promising regimen among trabectedin, eribulin, and pazopanib for comparison with GD as the test arm regimen in a future phase III trial of second-line treatment for patients with advanced STS. METHODS: The JCOG1802 study is a multicenter, selection design, randomized phase II trial comparing trabectedin (1.2 mg/m2 intravenously, every 3 weeks), eribulin (1.4 mg/m2 intravenously, days 1 and 8, every 3 weeks), and pazopanib (800 mg orally, every day) in patients with unresectable or metastatic STS refractory to doxorubicin-based first-line chemotherapy. The principal eligibility criteria are patients aged 16 years or above; unresectable and/or metastatic STS; exacerbation within 6 months prior to registration; histopathological diagnosis of STS other than Ewing sarcoma, embryonal/alveolar rhabdomyosarcoma, well-differentiated liposarcoma and myxoid liposarcoma; prior doxorubicin-based chemotherapy for STS, and Eastern Cooperative Oncology Group performance status 0 to 2. The primary endpoint is progression-free survival, and the secondary endpoints include overall survival, disease-control rate, response rate, and adverse events. The total planned sample size to correctly select the most promising regimen with a probability of > 80% is 120. Thirty-seven institutions in Japan will participate at the start of this trial. DISCUSSION: This is the first randomized trial to evaluate trabectedin, eribulin, and pazopanib as second-line therapies for advanced STS. We endeavor to perform a subsequent phase III trial comparing the best regimen selected by this study (JCOG1802) with GD. TRIAL REGISTRATION: This study was registered with the Japan Registry of Clinical Trials ( jRCTs031190152 ) on December 5, 2019.

Cyclic AMP induction of Dictyostelium prespore gene expression requires autophagy.

Dictyostelium discoideum amoebas display colonial multicellularity where starving amoebas aggregate to form migrating slugs and fruiting bodies consisting of spores and three supporting cell types. To resolve the cell signalling mechanism that control sporulation, we use insertional mutagenesis of amoebas transformed with fusion constructs of spore genes and red fluorescent protein. We identified the defective gene in a mutant lacking spore gene expression as the autophagy gene Atg7. Directed knock-out of atg7 and of autophagy genes like atg5 and atg9 yielded a similar phenotype, with lack of viable spores and excessive differentiation of stalk cells. The atg7-, atg5- and atg9- cells were specifically defective in cAMP induction of prespore genes, but showed enhanced cAMP stimulation of prestalk genes at the same developmental stage. The lack of prespore gene induction in the autophagy mutants was not due to deleterious effects of loss of autophagy on known components of the cAMP pathway, such as cAMP receptors and their cAMP-induced phosphorylation and internalization, PKA and the transcription factors SpaA and GbfA, or to lack of NH3 production by proteolysis, which was previously suggested to stimulate the spore pathway. Our continued mutagenesis approach is the most likely to yield the intriguing link between autophagy and prespore gene induction.

Phylogeny-wide conservation and change in developmental expression, cell-type specificity and functional domains of the transcriptional regulators of social amoebas.

BACKGROUND: Dictyostelid social amoebas self-organize into fruiting bodies, consisting of spores and up to four supporting cell types in the phenotypically most complex taxon group 4. High quality genomes and stage- and cell-type specific transcriptomes are available for representative species of each of the four taxon groups. To understand how evolution of gene regulation in Dictyostelia contributed to evolution of phenotypic complexity, we analysed conservation and change in abundance, functional domain architecture and developmental regulation of their transcription factors (TFs). RESULTS: We detected 440 sequence-specific TFs across 33 families, of which 68% were upregulated in multicellular development and about half conserved throughout Dictyostelia. Prespore cells expressed two times more TFs than prestalk cells, but stalk cells expressed more TFs than spores, suggesting that gene expression events that define spores occur earlier than those that define stalk cells. Changes in TF developmental expression, but not in TF abundance or functional domains occurred more frequently between group 4 and groups 1-3, than between the more distant branches formed by groups 1 + 2 and 3 + 4. CONCLUSIONS: Phenotypic innovation is correlated with changes in TF regulation, rather than functional domain- or TF acquisition. The function of only 34 TFs is known. Of 12 TFs essential for cell differentiation, 9 are expressed in the cell type for which they are required. The information acquired here on conserved cell type specifity of 120 additional TFs can effectively guide further functional analysis, while observed evolutionary change in TF developmental expression may highlight how genotypic change caused phenotypic innovation.

Late presentation of arrhythmogenic right ventricular cardiomyopathy in an octogenarian associated with a pathogenic variant in the plakophilin 2 gene: a case report.

BACKGROUND: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited myocardial disease characterized by fibrofatty replacement and ventricular arrhythmias. ARVC is believed to be a disease of the young, with most cases being diagnosed before the age of 40 years. We report here a case of newly diagnosed ARVC in an octogenarian associated with a pathogenic variant in the plakophilin 2 gene (PKP2). CASE PRESENTATION: An 80-year-old Japanese man was referred for sustained ventricular tachycardia. His baseline electrocardiogram showed negative T waves in V1-V4. Right ventriculography showed right ventricular aneurysm. Because this case met three major criteria, ARVC was diagnosed. He was successfully treated with radiofrequency ablation and oral amiodarone. Genetic analysis identified an insertion mutation in exon 8 of PKP2 (1725_1728dupGATG), which caused a frameshift and premature termination of translation (R577DfsX5). CONCLUSIONS: To the best of our knowledge, this is the first report of newly diagnosed ARVC in an octogenarian associated with a loss-of-function PKP2 pathogenic variant. Although the late clinical presentation of ARVC is rare, it should be included in the differential diagnosis when treating older patients with ventricular tachyarrhythmias.

Improved overall survival over recent decades in patients with hormone-receptor-positive, HER2-negative breast cancer: a single-center retrospective analysis of prognostic factors.

BACKGROUND: Hormone receptor (HR)-positive HER2-negative breast cancer (BC) rates and associated mortality have been increasing among Japanese women. It is unclear whether the prognosis of these patients has improved. METHODS: We retrospectively analyzed 1806 Japanese women with operable invasive HR-positive HER2-negative BC, who underwent complete resection at the National Cancer Center Hospital East between July 1992 and December 2010. We investigated whether overall survival (OS) and recurrence-free survival (RFS) had improved by comparing the 4-year periods 1992-96, 1997-2001, 2002-06, and 2007-10. The prognostic factors were evaluated using uni- and multivariate analyses. RESULTS: The number of ER- and PgR-positive cancers had increased over the years (P < 0.001). Tumor sizes and numbers of involved lymph nodes both gradually decreased (P < 0.001 for both). OS and RFS of all patients significantly improved in each of the periods analyzed: 5-year OS was 92.6%, 94.8%, 95.4% and 97.6% (P < 0.001, Log-rank), and 5-year RFS was 82.1%, 82.8%, 88.6% and 94.5% (P < 0.001) in 1992-96, 1997-2001, 2002-06 and 2007-10, respectively. In multivariate analysis, the history of adjuvant AI and that of TAM had positive-correlation with RFS. CONCLUSIONS: The prognosis for HR-positive HER2-negative BC patients after surgical therapy has improved, resulting in longer OS and RFS across the study periods. These changes could be associated with early detection of tumor and history of hormone therapy.

The first multicenter, randomized, controlled trial of home telemonitoring for Japanese patients with heart failure: home telemonitoring study for patients with heart failure (HOMES-HF).

Home telemonitoring is becoming more important to home medical care for patients with heart failure. Since there are no data on home telemonitoring for Japanese patients with heart failure, we investigated its effect on cardiovascular outcomes. The HOMES-HF study was the first multicenter, open-label, randomized, controlled trial (RCT) to elucidate the effectiveness of home telemonitoring of physiological data, such as body weight, blood pressure, and pulse rate, for Japanese patients with heart failure (UMIN Clinical Trials Registry 000006839). The primary end-point was a composite of all-cause death or rehospitalization due to worsening heart failure. We analyzed 181 recently hospitalized patients with heart failure who were randomly assigned to a telemonitoring group (n = 90) or a usual care group (n = 91). The mean follow-up period was 15 (range 0-31) months. There was no statistically significant difference in the primary end-point between groups [hazard ratio (HR), 0.95; 95% confidence interval (CI), 0.548-1.648; p = 0.572]. Home telemonitoring for Japanese patients with heart failure was feasible; however, beneficial effects in addition to those of usual care were not demonstrated. Further investigation of more patients with severe heart failure, participation of home medical care providers, and use of a more integrated home telemonitoring system emphasizing communication as well as monitoring of symptoms and physiological data are required.

Multi-scale, tailor-made heart simulation can predict the effect of cardiac resynchronization therapy.

BACKGROUND: The currently proposed criteria for identifying patients who would benefit from cardiac resynchronization therapy (CRT) still need to be optimized. A multi-scale heart simulation capable of reproducing the electrophysiology and mechanics of a beating heart may help resolve this problem. The objective of this retrospective study was to test the capability of patient-specific simulation models to reproduce the response to CRT by applying the latest multi-scale heart simulation technology. METHODS AND RESULTS: We created patient-specific heart models with realistic three-dimensional morphology based on the clinical data recorded before treatment in nine patients with heart failure and conduction block treated by biventricular pacing. Each model was tailored to reproduce the surface electrocardiogram and hemodynamics of each patient in formats similar to those used in clinical practice, including electrocardiography (ECG), echocardiography, and hemodynamic measurements. We then performed CRT simulation on each heart model according to the actual pacing protocol and compared the results with the clinical data. CRT simulation improved the ECG index and diminished wall motion dyssynchrony in each patient. These results, however, did not correlate with the actual response. The best correlation was obtained between the maximum value of the time derivative of ventricular pressure (dP/dtmax) and the clinically observed improvement in the ejection fraction (EF) (r=0.94, p<0.01). CONCLUSIONS: By integrating the complex pathophysiology of the heart, patient-specific, multi-scale heart simulation could successfully reproduce the response to CRT. With further verification, this technique could be a useful tool in clinical decision making.

The effects of collaborative research-based programming on public health nurses and their practice.

The study aim was to evaluate a collaborative research-based program for public health nurses. The program was initiated by a college of nursing to address public health issues. Participants were 33 public health nurses who completed a questionnaire survey; data for 25 respondents were analyzed both quantitatively and qualitatively. To understand the experiences of nurses in depth, three group interviews were conducted with 14 nurses. Qualitative analysis revealed three major themes: (i) opportunities for learning from collaboration; (ii) developing competence of changes in practice; and (iii) openness to continuing practice improvement. Study participants reported practical changes and new openness to continued practice improvement. Thus, schools of nursing and public health nurses should welcome and invite opportunities to collaborate to address practice issues using research-based information. Because changing practice can only occur step by step, nursing educators and practitioners should cultivate an environment that expands professional development and addresses practice improvement.

[Hypertension of old people in frailty].

In older people, the therapeutic importance for hypertension is different from young people because of wide interindividual variability in organ failure or dysfunction. The association with the severity and mortality of hypertension is known to be attenuated by aging Here, we describe the therapeutic strategy for hypertension of old people in frailty.

A canine case of Ehrlichia canis infection without a history of being in an endemic area in Japan.

A mixed-breed, 8-year-old male dog developed neutropenia, thrombocytopenia, and hyperglobulinemia. Bone marrow hyperplasia and splenic plasmacytosis were cytologically observed. The dog had never been outside of Tokyo or Shizuoka Prefecture. Splenectomy was performed to confirm and remove the cause of splenic plasmacytosis. A histopathological diagnosis of splenic plasmacytoma was made; however, serum protein electrophoresis showed polyclonal gammopathy. Further screening was performed, and Ehrlichia canis infection was confirmed. The dog was treated with doxycycline for 5 weeks. After the antibiotic therapy, no relapse of neutropenia, thrombocytopenia, hyperglobulinemia, or positive polymerase chain reaction result of E. canis infection was observed for 3 years. Careful attention should be given to ehrlichiosis when exploring the cause of pancytopenia or hyperglobulinemia, regardless of the travel history.

A novel class of cysteine protease receptors that mediate lysosomal transport.

The transport of lysosomal proteins is, in general, mediated by mannose 6-phosphate receptors via carbohydrate modifications. Here, we describe a novel class of receptors that regulate the transport of lysosomal hydrolases in the enteric protozoan Entamoeba histolytica, which is a good model organism to investigate membrane traffic. A novel 110 kDa cysteine protease (CP) receptor (CP-binding protein family 1, CPBF1) was initially discovered by affinity co-precipitation of the major CP (EhCP-A5), which plays a pivotal role in the pathogenesis of E. histolytica. We demonstrated that CPBF1 regulates EhCP-A5 transport from the endoplasmic reticulum to lysosomes and its binding to EhCP-A5 is independent of carbohydrate modifications. Repression of CPBF1 by gene silencing led to the accumulation of the unprocessed form of EhCP-A5 in the non-acidic compartment and the mis-secretion of EhCP-A5, suggesting that CPBF1 is involved in the trafficking and processing of EhCP-A5. The CPBF represents a new class of transporters that bind to lysosomal hydrolases in a carbohydrate-independent fashion and regulate their trafficking, processing and activation and, thus, regulate the physiology and pathogenesis of E. histolytica.

DIF-1 regulates Dictyostelium basal disc differentiation by inducing the nuclear accumulation of a bZIP transcription factor.

Exposure of monolayer Dictyostelium cells to the signalling polyketide DIF-1 causes DimB, a bZIPtranscription factor, to accumulate in the nucleus where it induces prestalk gene expression. Here we analyse DimB signalling during normal development. In slugs DimB is specifically nuclear enriched in the pstB cells; a cluster of vital dye-staining cells located on the ventral surface of the posterior, prespore region. PstB cells move at culmination, to form the lower cup and the outer basal disc of the fruiting body, and DimB retains a high nuclear concentration in both these tissues. In a dimB null (dimB-) strain there are very few pstB or lower cup cells, as detected by neutral red staining, and it is known that the outer basal disc is absent or much reduced. In the dimB- strain ecmB, a marker of pstB differentiation, is not DIF inducible. Furthermore, ChIP analysis shows that DimB binds to the ecmB promoter in DIF-induced cells. These results suggest that the differentiation of pstB cells is caused by a high perceived level of DIF-1 signalling, leading to nuclear localization of DimB and direct activation of cell type-specific gene expression.

[Evaluation of the Accuracy of the Displayed Average Glandular Dose in Mammography].

PURPOSE: We aimed to clarify the error of displayed value against the measured value of the average glandular dose (AGD) in two-dimensional (2D) mammography and digital breast tomosynthesis (DBT) and evaluate the accuracy of the displayed AGD as an index to estimate AGD. METHODS: Polymethyl methacrylate (PMMA) phantoms with thicknesses varying from 20 to 80 mm were imaged, and the values displayed on the mammography system were used as the displayed AGD. The incident air kerma and the half-valued layer were measured, and the measured AGD in 2D mammography was calculated using the equation by Dance et al. On the other hand, the measured AGD in DBT was calculated by correcting for different projection angles. The relative error to the PMMA thickness was evaluated by assessing the relative error of the displayed AGD against the measured AGD. RESULTS: The maximum relative error of the displayed AGD against the measured AGD was 17.3% in 2D mammography, 19.1% in the standard (ST) mode, and 19.8% in the high-resolution (HR) mode. CONCLUSION: The relative error of the displayed AGD against the measured AGD tended to increase with increase in PMMA thickness. This tendency was especially noticeable for PMMA with thicknesses of 70 and 80 mm in DBT.

Establishment of large canine hepatocyte spheroids by mixing vascular endothelial cells and canine adipose-derived mesenchymal stem cells.

INTRODUCTION: Differentiation of hepatocytes and culture methods have been investigated in dogs as a tool to establish liver transplant and drug metabolism examination systems. However, mass culture techniques for canine hepatocytes (cHep) have not been investigated, and it is necessary to construct a suitable culture system. Recently, a protocol called Bud production has attracted attention, and a mixed culture of human and mouse hepatocytes, stem cells, and artificial blood vessels significantly improved the size and formation ratio of spheroids. The purpose of this study was to investigate and improve the in vitro culture of cHep by mixing canine adipose-derived mesenchymal stem cells (cASCs) and human umbilical vein endothelial cells (HUVECs). METHODS: Spheroid formation ratio and histological examination were evaluated among four culture methods, including cHep alone, two-mix (cHep + cASCs and cHep + HUVEC), and three-mix (cHep + HUVEC + cASCs), on days 0, 4, and 7. Expression levels of liver-related genes (ALB, AFP, α1-AT, CDH1, CYP2E1, CYP3A12, and TAT) were evaluated by quantitative real-time polymerase chain reaction (RT-PCR). Protein expression of albumin, vimentin, and von Willebrand Factor (vWF) was investigated to confirm the location of the hepatocytes. RESULTS: The ratio of spheroid formation was 60.2% in the three-mix culture and was significantly improved compared with cHep alone (5.9%) and two-mix; cHep + cASCs (36.2%) and cHep + HUVEC (26.4%) (P < 0.001). Histological evaluation revealed that the three-mix spheroids formed large canine hepatocyte spheroids (LcHS), and hepatocytes were distributed in the center of the spheroids. Quantitative gene expression analysis of LcHS showed that liver-related genes expression were maintained the same levels with that of a culture of cHep alone from days 4-7. CONCLUSION: These results revealed that the three-mix culture method using cHep, HUVECs, and cASCs was capable of promoting LcHS without impairing liver function in cHep, suggesting that LcHS could be used for the application of high-volume culture techniques in dogs.

Characterisation of canine CD34+/CD45 diminished cells by colony-forming unit assay and transcriptome analysis.

Haematopoietic stem and progenitor cells (HSPCs) are used for transplantation to reconstruct the haematopoietic pathways in humans receiving severe chemotherapy. However, the characteristics of canine HSPCs, such as specific surface antigens and gene expression profiles, are still unclear. This study aimed to characterise the haematopoietic ability and gene expression profiles of canine bone marrow HSPCs in healthy dogs. In this study, the CD34 positive (CD34+) cells were defined as classical HSPCs, CD34+/CD45 diminished (CD45dim) cells as more enriched HSPCs, and whole viable cells as controls. Haematopoietic abilities and gene expression profiles were evaluated using a colony-forming unit assay and RNA-sequencing analysis. Canine CD34+/CD45dim cells exhibited a significantly higher haematopoietic colony formation ability and expressed more similarity in the gene expression profiles to human and mouse HSPCs than those of the other cell fractions. Furthermore, the canine CD34+/CD45dim cells expressed candidate cell surface antigens necessary to define the canine haematopoietic hierarchy roadmap. These results indicate that the canine CD34+/CD45dim cells express the HSPC characteristics more than the other cell fractions, thereby suggesting that these cells have the potential to be used for studying haematopoietic stem cells in dogs.

A new Dictyostelium prestalk cell sub-type.

The mature fruiting body of Dictyostelium consists of stalk and spore cells but its construction, and the migration of the preceding slug stage, requires a number of specialized sub-types of prestalk cell whose nature and function are not well understood. The prototypic prestalk-specific gene, ecmA, is inducible by the polyketide DIF-1 in a monolayer assay and requires the DimB and MybE transcription factors for full inducibility. We perform genome-wide microarray analyses, on parental, mybE- and dimB- cells, and identify many additional genes that depend on MybE and DimB for their DIF-1 inducibility. Surprisingly, an even larger number of genes are only DIF inducible in mybE- cells, some genes are only inducible in DimB- cells and some are inducible when either transcription factor is absent. Thus in assay conditions where MybE and DimB function as inducers of ecmA these genes fall under negative control by the same two transcription factors. We have studied in detail rtaA, one of the MybE and DimB repressed genes. One especially enigmatic group of prestalk cells is the anterior-like cells (ALCs), which exist intermingled with prespore cells in the slug. A promoter fusion reporter gene, rtaA:gal(u), is expressed in a subset of the ALCs that is distinct from the ALC population detected by a reporter construct containing ecmA and ecmB promoter fragments. At culmination, when the ALC sort out from the prespore cells and differentiate to form three ancillary stalk cell structures: the upper cup, the lower cup and the outer basal disk, the rtaA:gal(u) expressing cells preferentially populate the upper cup region. This fact, and their virtual absence from the anterior and posterior regions of the slug, identifies them as a new prestalk sub-type: the pstU cells. PstU cell differentiation is, as expected, increased in a dimB- mutant during normal development but, surprisingly, they differentiate normally in a mutant lacking DIF. Thus genetic removal of MybE or DimB reveals an alternate DIF-1 activation pathway, for pstU differentiation, that functions under monolayer assay conditions but that is not essential during multicellular development.

Deduced RNA binding mechanism of ThiI based on structural and binding analyses of a minimal RNA ligand.

ThiI catalyzes the thio-introduction reaction to tRNA, and a truncated tRNA consisting of 39 nucleotides, TPHE39A, is the minimal RNA substrate for modification by ThiI from Escherichia coli. To examine the molecular basis of the tRNA recognition by ThiI, we have solved the crystal structure of TPHE39A, which showed that base pairs in the T-stem were almost completely disrupted, although those in the acceptor-stem were preserved. Gel shift assays and isothermal titration calorimetry experiments showed that ThiI can efficiently bind with not only tRNA(Phe) but also TPHE39A. Binding assays using truncated ThiI, i.e., N- and C-terminal domains of ThiI, showed that the N-domain can bind with both tRNA(Phe) and TPHE39A, whereas the C-domain cannot. These results indicated that the N-domain of ThiI recognizes the acceptor-stem region. Thermodynamic analysis indicated that the C-domain also affects RNA binding by its enthalpically favorable, but entropically unfavorable, contribution. In addition, circular dichroism spectra showed that the C-domain induced a conformation change in tRNA(Phe). Based on these results, a possible RNA binding mechanism of ThiI in which the N-terminal domain recognizes the acceptor-stem region and the C-terminal region causes a conformational change of RNA is proposed.