Regulation of Collective Metastasis by Nanolumenal Signaling.

Collective metastasis is defined as the cohesive migration and metastasis of multicellular tumor cell clusters. Disrupting various cell adhesion genes markedly reduces cluster formation and colonization efficiency, yet the downstream signals transmitted by clustering remain largely unknown. Here, we use mouse and human breast cancer models to identify a collective signal generated by tumor cell clusters supporting metastatic colonization. We show that tumor cell clusters produce the growth factor epigen and concentrate it within nanolumina-intercellular compartments sealed by cell-cell junctions and lined with microvilli-like protrusions. Epigen knockdown profoundly reduces metastatic outgrowth and switches clusters from a proliferative to a collective migratory state. Tumor cell clusters from basal-like 2, but not mesenchymal-like, triple-negative breast cancer cell lines have increased epigen expression, sealed nanolumina, and impaired outgrowth upon nanolumenal junction disruption. We propose that nanolumenal signaling could offer a therapeutic target for aggressive metastatic breast cancers.

Metastasis from the tumor interior and necrotic core formation are regulated by breast cancer-derived angiopoietin-like 7.

Necrosis in the tumor interior is a common feature of aggressive cancers that is associated with poor clinical prognosis and the development of metastasis. How the necrotic core promotes metastasis remains unclear. Here, we report that emergence of necrosis inside the tumor is correlated temporally with increased tumor dissemination in a rat breast cancer model and in human breast cancer patients. By performing spatially focused transcriptional profiling, we identified angiopoietin-like 7 (Angptl7) as a tumor-specific factor localized to the perinecrotic zone. Functional studies showed that Angptl7 loss normalizes central necrosis, perinecrotic dilated vessels, metastasis, and reduces circulating tumor cell counts to nearly zero. Mechanistically, Angptl7 promotes vascular permeability and supports vascular remodeling in the perinecrotic zone. Taken together, these findings show that breast tumors actively produce factors controlling central necrosis formation and metastatic dissemination from the tumor core.

Interpersonal counseling versus active listening in the treatment of mild depression: a randomized controlled trial.

[Purpose] Healthcare workers, such as physical therapists, need to be equipped in dealing with patients' psychological problems. The three-session interpersonal counseling (three-session IPC) is a constructed counseling method that can be performed even by non-mental health professionals. This study examined the efficacy of the three-session IPC for treating depression. Immediate efficacy and efficacy up to 12 weeks post-intervention were examined. [Participants and Methods] In this randomized controlled trial of the two groups, one group (n=24) received the three-session IPC therapy (IPC group) while the other (n=24) received three sessions of active listening (active listening group). Depression was assessed using the Self-Rating Depression Scale (SDS) at baseline, post-intervention, and at 4, 8, and 12 weeks. [Results] There was a significant difference in total SDS scores between the IPC and active listening groups from baseline to 4 weeks after counseling, although no significant differences were observed at other time points. [Conclusion] The three-session IPC may be effective for 4 weeks after counseling. However, further studies are warranted in this regard.

Pluripotent stem cell-derived myogenic progenitors remodel their molecular signature upon in vivo engraftment.

Optimal cell-based therapies for the treatment of muscle degenerative disorders should not only regenerate fibers but provide a quiescent satellite cell pool ensuring long-term maintenance and regeneration. Conditional expression of Pax3/Pax7 in differentiating pluripotent stem cells (PSCs) allows the generation of myogenic progenitors endowed with enhanced regenerative capacity. To identify the molecular determinants underlying their regenerative potential, we performed transcriptome analyses of these cells along with primary myogenic cells from several developmental stages. Here we show that in vitro-generated PSC-derived myogenic progenitors possess a molecular signature similar to embryonic/fetal myoblasts. However, compared with fetal myoblasts, following transplantation they show superior myofiber engraftment and ability to seed the satellite cell niche, respond to multiple reinjuries, and contribute to long-term regeneration. Upon engraftment, the transcriptome of reisolated Pax3/Pax7-induced PSC-derived myogenic progenitors changes toward a postnatal molecular signature, particularly in genes involved in extracellular matrix remodeling. These findings demonstrate that Pax3/Pax7-induced myogenic progenitors remodel their molecular signature and functionally mature upon in vivo exposure to the adult muscle environment.

Sulfated Hyaluronan Binds to Heparanase and Blocks Its Enzymatic and Cellular Actions in Carcinoma Cells.

We examined whether sulfated hyaluronan exerts inhibitory effects on enzymatic and biological actions of heparanase, a sole endo-beta-glucuronidase implicated in cancer malignancy and inflammation. Degradation of heparan sulfate by human and mouse heparanase was inhibited by sulfated hyaluronan. In particular, high-sulfated hyaluronan modified with approximately 2.5 sulfate groups per disaccharide unit effectively inhibited the enzymatic activity at a lower concentration than heparin. Human and mouse heparanase bound to immobilized sulfated hyaluronan. Invasion of heparanase-positive colon-26 cells and 4T1 cells under 3D culture conditions was significantly suppressed in the presence of high-sulfated hyaluronan. Heparanase-induced release of CCL2 from colon-26 cells was suppressed in the presence of sulfated hyaluronan via blocking of cell surface binding and subsequent intracellular NF-κB-dependent signaling. The inhibitory effect of sulfated hyaluronan is likely due to competitive binding to the heparanase molecule, which antagonizes the heparanase-substrate interaction. Fragment molecular orbital calculation revealed a strong binding of sulfated hyaluronan tetrasaccharide to the heparanase molecule based on electrostatic interactions, particularly characterized by interactions of (-1)- and (-2)-positioned sulfated sugar residues with basic amino acid residues composing the heparin-binding domain-1 of heparanase. These results propose a relevance for sulfated hyaluronan in the blocking of heparanase-mediated enzymatic and cellular actions.

Depression Among University Students With Attention-Deficit Hyperactivity Disorder Symptoms: A Study of Interpersonal Counseling.

OBJECTIVE: University students with symptoms of attention-deficit hyperactivity disorder (ADHD) often experience depression. This study examined whether interpersonal counseling (IPC) could be an effective treatment for depression among students with ADHD symptoms. METHODS: Participants were assigned to either an IPC (N=5) or control (N=7) group. Depression was assessed by using the Self-Rating Depression Scale (SDS) at baseline, postintervention, and at 4-, 8-, and 12-week follow-ups. RESULTS: No significant changes in the SDS total score were observed for either group at each postintervention point. However, the IPC group showed a large effect size at the 4- and 12-week follow-ups. A significant intergroup difference was observed after 4 weeks. No significant intergroup difference was observed after 12 weeks, but there was a large effect size. CONCLUSIONS: IPC appeared to have effects at 4 weeks postintervention. Because this was an exploratory study, further research is necessary.

Special Features of COVID-19 in the FMODB: Fragment Molecular Orbital Calculations and Interaction Energy Analysis of SARS-CoV-2-Related Proteins.

SARS-CoV-2 is the causative agent of coronavirus (known as COVID-19), the virus causing the current pandemic. There are ongoing research studies to develop effective therapeutics and vaccines against COVID-19 using various methods and many results have been published. The structure-based drug design of SARS-CoV-2-related proteins is promising, however, reliable information regarding the structural and intra- and intermolecular interactions is required. We have conducted studies based on the fragment molecular orbital (FMO) method for calculating the electronic structures of protein complexes and analyzing their quantitative molecular interactions. This enables us to extensively analyze the molecular interactions in residues or functional group units acting inside the protein complexes. Such precise interaction data are available in the FMO database (FMODB) (https://drugdesign.riken.jp/FMODB/). Since April 2020, we have performed several FMO calculations on the structures of SARS-CoV-2-related proteins registered in the Protein Data Bank. We have published the results of 681 structures, including three structural proteins and 11 nonstructural proteins, on the COVID-19 special page (as of June 8, 2021). In this paper, we describe the entire COVID-19 special page of the FMODB and discuss the calculation results for various proteins. These data not only aid the interpretation of experimentally determined structures but also the understanding of protein functions, which is useful for rational drug design for COVID-19.

Increased prefrontal cortex activity on near-infrared spectroscopy after interpersonal counseling in individuals with attention deficit hyperactivity disorder.

[Purpose] The number of patients with attention deficit hyperactivity disorder has been increasing. These patients show low activity in the prefrontal cortex, which can be improved by pharmacotherapy and neurofeedback training. This exploratory study aimed to examine whether the hemodynamic response in the prefrontal cortex during an inhibition response in patients with attention deficit hyperactivity disorder tendencies increased after interpersonal counseling. [Participants and Methods] Participants (n=5) received three interpersonal counseling sessions. Interpersonal counseling focuses on the patient's current problems and devises specific coping strategies, and it can be performed by healthcare personnel such as physiotherapists. Prefrontal cortex activity during a suppression reaction task was measured by using near-infrared spectroscopy at baseline and post-interpersonal counseling. The outcome was a difference in the oxyhemoglobin level from baseline to post-interpersonal counseling. [Results] The oxyhemoglobin level in the prefrontal cortex significantly increased post-interpersonal counseling. [Conclusion] These results suggested that interpersonal counseling could improve the hemodynamic response in the prefrontal cortex under inhibition in individuals with attention deficit hyperactivity disorder tendencies, suggesting that interpersonal counseling may be effective for treating attention deficit hyperactivity disorder symptoms.

Endoglin: a novel target for therapeutic intervention in acute leukemias revealed in xenograft mouse models.

Endoglin (CD105), a receptor of the transforming growth factor-ß superfamily, has been reported to identify functional long-term repopulating hematopoietic stem cells, and has been detected in certain subtypes of acute leukemias. Whether this receptor plays a functional role in leukemogenesis remains unknown. We identified endoglin expression on the majority of blasts from patients with acute myeloid leukemia (AML) and acute B-lymphoblastic leukemia (B-ALL). Using a xenograft model, we find that CD105+ blasts are endowed with superior leukemogenic activity compared with the CD105- population. We test the effect of targeting this receptor using the monoclonal antibody TRC105, and find that in AML, TRC105 prevented the engraftment of primary AML blasts and inhibited leukemia progression following disease establishment, but in B-ALL, TRC105 alone was ineffective due to the shedding of soluble CD105. However, in both B-ALL and AML, TRC105 synergized with reduced intensity myeloablation to inhibit leukemogenesis, indicating that TRC105 may represent a novel therapeutic option for B-ALL and AML.

Mechanism of microtubule lumen entry for the α-tubulin acetyltransferase enzyme αTAT1.

Microtubules are structural polymers inside of cells that are subject to posttranslational modifications. These posttranslational modifications create functionally distinct subsets of microtubule networks in the cell, and acetylation is the only modification that takes place in the hollow lumen of the microtubule. Although it is known that the α-tubulin acetyltransferase (αTAT1) is the primary enzyme responsible for microtubule acetylation, the mechanism for how αTAT1 enters the microtubule lumen to access its acetylation sites is not well understood. By performing biochemical assays, fluorescence and electron microscopy experiments, and computational simulations, we found that αTAT1 enters the microtubule lumen through the microtubule ends, and through bends or breaks in the lattice. Thus, microtubule structure is an important determinant in the acetylation process. In addition, once αTAT1 enters the microtubule lumen, the mobility of αTAT1 within the lumen is controlled by the affinity of αTAT1 for its acetylation sites, due to the rapid rebinding of αTAT1 onto highly concentrated α-tubulin acetylation sites. These results have important implications for how acetylation could gradually accumulate on stable subsets of microtubules inside of the cell.

Targeted mutagenesis of intergenic regions in the Neisseria gonorrhoeae gonococcal genetic island reveals multiple regulatory mechanisms controlling type IV secretion.

Gonococci secrete chromosomal DNA into the extracellular environment using a type IV secretion system (T4SS). The secreted DNA acts in natural transformation and initiates biofilm development. Although the DNA and its effects are detectable, structural components of the T4SS are present at very low levels, suggestive of uncharacterized regulatory control. We sought to better characterize the expression and regulation of T4SS genes and found that the four operons containing T4SS genes are transcribed at very different levels. Increasing transcription of two of the operons through targeted promoter mutagenesis did not increase DNA secretion. The stability and steady-state levels of two T4SS structural proteins were affected by a homolog of tail-specific protease. An RNA switch was also identified that regulates translation of a third T4SS operon. The switch mechanism relies on two putative stem-loop structures contained within the 5' untranslated region of the transcript, one of which occludes the ribosome binding site and start codon. Mutational analysis of these stem loops supports a model in which induction of an alternative structure relieves repression. Taken together, these results identify multiple layers of regulation, including transcriptional, translational and post-translational mechanisms controlling T4SS gene expression and DNA secretion.

The iron content and ferritin contribution in fresh, dried, and toasted nori, Pyropia yezoensis.

Iron is one of the essential trace elements for humans. In this study, the iron contents in fresh, dried, and toasted nori (Pyropia yezoensis) were analyzed. The mean iron content of fresh, dried, and toasted nori were 19.0, 22.6, and 26.2 mg/100 g (dry weight), respectively. These values were superior to other food of plant origin. Furthermore, most of the iron in nori was maintained during processing, such as washing, drying, and toasting. Then, the form of iron in fresh, dried, and toasted nori was analyzed. As a result, an iron storage protein ferritin contributed to iron storage in raw and dried nori, although the precise rate of its contribution is yet to be determined, while ferritin protein cage was degraded in the toasted nori. It is the first report that verified the ferritin contribution to iron storage in such edible macroalgae with commercial importance.

Phase III, multicenter, open-label, long-term study of the safety of abatacept in Japanese patients with rheumatoid arthritis and an inadequate response to conventional or biologic disease-modifying antirheumatic drugs.

OBJECTIVES: To examine the long-term safety of intravenous (IV) abatacept treatment in Japanese patients with rheumatoid arthritis (RA) and an inadequate response to methotrexate (MTX) or other conventional or biologic disease-modifying antirheumatic drugs. METHODS: This Phase III, open-label, long-term study (NCT00484289) comprised Japanese patients with RA who had completed abatacept Phase I or Phase II studies, and new patients intolerant to MTX. Patients from Phase I and Phase II studies received a weight-tiered dosing equivalent of 10 mg/kg abatacept, with MTX at doses up to 8 mg/week; newly enrolled patients received weight-tiered 10 mg/kg abatacept monotherapy. Safety and efficacy were assessed. RESULTS: A total of 217 patients (Phase I, n = 13; Phase II, n = 178; newly enrolled, n = 26) were treated with IV abatacept for a mean of 3 years. Serious adverse events occurred in 67/217 (30.9%) patients. Most adverse events were mild or moderate. For all cohorts combined, American College of Rheumatology 20% response rates ranged from 61.3 to 81.8% for as-observed and last observation carried forward analyses over 192 weeks. Following initial response, clinical and functional outcomes were maintained for up to 3 years. CONCLUSIONS: In Japanese patients with RA, IV abatacept with and without background MTX showed tolerable safety and sustained efficacy over 3 years.

Dexamethasone reduces cisplatin-induced hair cell damage by inducing cisplatin resistance through metallothionein-2.

PURPOSE: Hair cell damage is a common side effect caused by the anticancer drug cisplatin (CDDP), which reduces patient quality of life. One CDDP resistance mechanism that occurs in recurrent cancers is heavy metal detoxification by metallothionein-2 (mt2). Here, we show that in zebrafish larvae, dexamethasone (DEX) reduces CDDP-induced hair cell damage by enhancing mt2 expression. METHODS: Transgenic zebrafish (cldn: gfp; atoh1: rfp) that express green and red fluorescent proteins in neuromasts and hair cells, respectively, were used. The zebrafish were pretreated with DEX at 52 h post-fertilization (hpf) for 8 h, followed by CDDP treatment for 12 h. The lateral line hair cells of CDDP-treated zebrafish at 72 hpf were observed by fluorescence microscopy. RESULTS: Reporting odds ratio (ROR) analysis using an adverse event database indicated an association between a decrease in CDDP-induced ototoxicity and DEX as an antiemetic treatment for cancer chemotherapy. Pretreatment with DEX protected 72 hpf zebrafish hair cells from CDDP-induced damage. The expression of mt2 mRNA was significantly increased by the combination of 10 µM DEX with CDDP. Gene editing of mt2 reversed the protective effect of DEX against CDDP-induced damage in hair cells. CONCLUSION: DEX protects hair cells from CDDP-induced damage through increased mt2 expression, which is a resistance mechanism for platinum-based anticancer drugs.

Phase II dose-response study of abatacept in Japanese patients with active rheumatoid arthritis with an inadequate response to methotrexate.

OBJECTIVE: The objective of this study was to assess the response to abatacept at doses of 2 mg/kg and 10 mg/kg compared to placebo in patients with active rheumatoid arthritis (RA) with an inadequate clinical response to methotrexate (MTX). METHODS: In this multicenter, placebo-controlled, double-blind, parallel-group, dose-response study, 195 Japanese patients with active RA with an inadequate response to MTX were randomized 1:1:1 to receive 10 mg/kg or 2 mg/kg abatacept plus MTX, or placebo plus MTX, for 24 weeks. RESULTS: Abatacept demonstrated a dose-response relationship when given at 2 and 10 mg/kg. Based on the American College of Rheumatology criteria (20, 50, and 70 %), the responses to 10 mg/kg abatacept were significantly greater than those to placebo at week 24 (p < 0.001). Smaller yet statistically significant responses were also seen in the 2 mg/kg abatacept group. Overall rates of adverse events, serious adverse events, and treatment discontinuations because of adverse events were comparable in all three groups. CONCLUSIONS: Abatacept (2 mg/kg and 10 mg/kg) showed a dose-response relationship in Japanese patients with active RA with an inadequate clinical response to MTX. Administration of abatacept in combination with MTX for 24 weeks was well tolerated.

Tolerability and efficacy of abatacept in Japanese patients with rheumatoid arthritis: a phase I study.

OBJECTIVE: The primary objective of this study was to evaluate the tolerability of single and multiple doses of abatacept in Japanese patients with rheumatoid arthritis. Secondary objectives included evaluating its pharmacokinetics, pharmacodynamics, immunogenicity, and efficacy. METHODS: This dose-escalation, single- and multiple-dose, multicenter, open-label study was conducted at nine sites in Japan. Seven patients were enrolled at each of three dose levels (2, 8 and 16 mg/kg) and received a single intravenous dose of abatacept on day 1 of the single-dose phase. The multiple-dose phase, at the same dose, started once the patients had completed the single-dose phase and when it was confirmed that there were no safety issues. RESULTS: Twenty patients started the single-dose phase. Single and multiple doses of abatacept were well tolerated, and adverse events were of mild to moderate intensity. There were no discontinuations or deaths due to adverse events. The pharmacokinetics of abatacept were linear, with no notable accumulation. There were no immunogenic effects on the safety, efficacy, or pharmacokinetics of abatacept. Multiple doses of abatacept improved individual items of the American College of Rheumatology core set. CONCLUSION: Single and multiple doses of abatacept showed favorable tolerability and efficacy in Japanese patients with rheumatoid arthritis.

Orchestration of Collective Migration and Metastasis by Tumor Cell Clusters.

Metastatic dissemination has lethal consequences for cancer patients. Accruing evidence supports the hypothesis that tumor cells can migrate and metastasize as clusters of cells while maintaining contacts with one another. Collective metastasis enables tumor cells to colonize secondary sites more efficiently, resist cell death, and evade the immune system. On the other hand, tumor cell clusters face unique challenges for dissemination particularly during systemic dissemination. Here, we review recent progress toward understanding how tumor cell clusters overcome these disadvantages as well as mechanisms they utilize to gain advantages throughout the metastatic process. We consider useful models for studying collective metastasis and reflect on how the study of collective metastasis suggests new opportunities for eradicating and preventing metastatic disease.

Pluripotent stem cell-derived skeletal muscle fibers preferentially express myosin heavy-chain isoforms associated with slow and oxidative muscles.

BACKGROUND: Skeletal muscle function is essential for health, and it depends on the proper activity of myofibers and their innervating motor neurons. Each adult muscle is composed of different types of myofibers with distinct contractile and metabolic characteristics. The proper balance of myofiber types is disrupted in most muscle degenerative disorders, representing another factor compromising muscle function. One promising therapeutic approach for the treatment of these diseases is cell replacement based on the targeted differentiation of pluripotent stem cells (PSCs) towards the myogenic lineage. We have previously shown that transient induction of Pax3 or Pax7 in PSCs allows for the generation of skeletal myogenic progenitors endowed with myogenic regenerative potential, but whether they contribute to different fiber types remains unknown. RESULTS: Here, we investigate the fiber type composition of mouse PSC-derived myofibers upon their transplantation into dystrophic and non-dystrophic mice. Our data reveal that PSC-derived myofibers express slow and oxidative myosin heavy-chain isoforms, along with developmental myosins, regardless of the recipient background. Furthermore, transplantation of the mononuclear cell fraction re-isolated from primary grafts into secondary recipients results in myofibers that maintain preferential expression of slow and oxidative myosin heavy-chain isoforms but no longer express developmental myosins, thus indicating postnatal composition. CONCLUSIONS: Considering oxidative fibers are commonly spared in the context of dystrophic pathogenesis, this feature of PSC-derived myofibers could be advantageous for therapeutic applications.

Transplantation studies reveal internuclear transfer of toxic RNA in engrafted muscles of myotonic dystrophy 1 mice.

BACKGROUND: Stem cell transplantation represents a potential therapeutic option for muscular dystrophies (MD). However, to date, most reports have utilized mouse models for recessive types of MD. Here we performed studies to determine whether myotonic dystrophy 1 (DM1), an autosomal dominant type of MD, could benefit from cell transplantation. METHODS: We injected human pluripotent stem (PS) cell-derived myogenic progenitors into the muscles of a novel mouse model combining immunodeficiency and skeletal muscle pathology of DM1 and investigated transplanted mice for engraftment as well as for the presence of RNA foci and alternative splicing pattern. FINDINGS: Engraftment was clearly observed in recipient mice, but unexpectedly, we detected RNA foci in donor-derived engrafted myonuclei. These foci proved to be pathogenic as we observed MBNL1 sequestration and abnormal alternative splicing in donor-derived transcripts. INTERPRETATION: It has been assumed that toxic CUG repeat-containing RNA forms foci in situ in the nucleus in which it is expressed, but these data suggest that CUG repeat-containing RNA may also exit the nucleus and traffic to other nuclei in the syncytial myofiber, where it can exert pathological effects. FUND: This project was supported by funds from the LaBonte/Shawn family and NIH grants R01 AR055299 and AR071439 (R.C.R.P.). R.M-G. was funded by CONACyT-Mexico (#394378).

Growth hormone deficiency by growth hormone releasing hormone-arginine testing criteria predicts increased cardiovascular risk markers in normal young overweight and obese women.

CONTEXT: Little is known about the relationship between GH and cardiovascular risk markers in women without organic hypothalamic/pituitary disease. OBJECTIVE: The objective of the study was to determine whether healthy young overweight and obese women, who would be classified as having GH deficiency (GHD) based on standard criteria used in hypopituitarism (peak GH after stimulation with GHRH and arginine < 5 ng/ml), have increased cardiovascular risk markers. DESIGN: This was a cross-sectional study. SETTING: The study was conducted at the General Clinical Research Center. STUDY PARTICIPANTS: Forty-five women of reproductive age, mean age 33.1 +/- 1.2 yr and mean body mass index (BMI) 30.9 +/- 1.0 kg/m(2). INTERVENTION: There was no intervention. MAIN OUTCOME MEASURES: Measures included carotid intima-medial thickness, high-sensitivity C-reactive protein (hsCRP), total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein, triglycerides, E-selectin, soluble intercellular adhesion molecule-1, TNF-alpha receptor I, TNF-alpha receptor II, fasting insulin levels, and oral glucose tolerance testing. RESULTS: Twenty-six percent of overweight or obese subjects and none with BMI less than 25 kg/m(2) met criteria for GHD. Subjects who met GHD criteria had a mean BMI of 37.0 +/- 1.7 kg/m(2) (range 28.6-43.6 kg/m(2)), and their mean waist circumference (110.1 +/- 3.5 cm) was higher than in overweight/obese women with GH sufficiency (P = 0.007). Mean carotid intima-media thickness, hsCRP, soluble intercellular adhesion molecule-1, TNF-alpha receptor I, and TNF-alpha receptor II levels were higher, and HDL lower, in women meeting GHD criteria than in GH sufficiency. Differences in HDL, hsCRP, and TNF-alpha receptor II remained after controlling for age plus BMI, waist circumference, or trunk fat. There were no differences in measures of insulin resistance. CONCLUSIONS: There may be a relative GHD syndrome in overweight and obese women without organic pituitary or hypothalamic disease that confers increased cardiovascular risk, independent of weight.