Integrator complex subunit 15 controls mRNA splicing and is critical for eye development.

The eye and brain are composed of elaborately organized tissues, development of which is supported by spatiotemporally precise expression of a number of transcription factors and developmental regulators. Here we report the molecular and genetic characterization of Integrator complex subunit 15 (INTS15). INTS15 was identified in search for the causative gene(s) for an autosomal-dominant eye disease with variable individual manifestation found in a large pedigree. While homozygous Ints15 knockout mice are embryonic lethal, mutant mice lacking a small C-terminal region of Ints15 show ocular malformations similar to the human patients. INTS15 is highly expressed in the eye and brain during embryogenesis and stably interacts with the Integrator complex to support small nuclear RNA 3' end processing. Its knockdown resulted in missplicing of a large number of genes, probably as a secondary consequence, and substantially affected genes associated with eye and brain development. Moreover, studies using human iPS cells-derived neural progenitor cells showed that INTS15 is critical for axonal outgrowth in retinal ganglion cells. This study suggests a new link between general transcription machinery and a highly specific hereditary disease.

Applications and Limitations of Equilibrium Density Gradient Analytical Ultracentrifugation for the Quantitative Characterization of Adeno-Associated Virus Vectors.

Adeno-associated virus (AAV) vectors are produced as a mixture of the desired particle (full particle, FP), which is filled with the designed DNA, product-related impurities such as particle without DNA (empty particle, EP), and aggregates. Cesium chloride or iodixanol equilibrium density gradient ultracentrifugation (DGE-UC) has been used for the purification of AAV vectors. DGE-UC can separate FP from impurities based on the difference in their buoyant densities. Here, we report the applications and limitations of equilibrium density gradient analytical ultracentrifugation (DGE-AUC) using a modern AUC instrument that employs DGE-UC principles for the characterization and quantitation of AAV vectors. We evaluated the quantitative ability of DGE-AUC in comparison with sedimentation velocity AUC (SV-AUC) or band sedimentation AUC (BS-AUC) using AAVs with different DNA lengths and different serotypes. DGE-AUC enabled the accurate quantification of the ratio of FP to EP when the AAV vector primarily contains these particles. Furthermore, we developed a new workflow to identify the components of separated peaks in addition to FP and EP. Ultraviolet absorption spectra obtained by multiwavelength detection can also support peak assignment following component identification. DGE-AUC experiments for AAV vectors have limitations with regard to minor components with low absorption at the detected wavelength or those with a density similar to that of major components of AAV vectors. DGE-AUC is the only analytical method that can evaluate particle density heterogeneity; therefore, SV-AUC or BS-AUC and DGE-AUC are complementary methods for reliable assessment of the purity of AAV vectors.

Topical therapy and skin care for transplant-associated atopic dermatitis in children and adolescents.

BACKGROUND: New-onset allergic diseases, such as food allergy or atopic dermatitis, can develop after allogeneic transplantation. There are limited reports of new-onset atopic dermatitis after allogeneic hematopoietic stem cell transplantation in children and adolescents, and its treatment is yet to be established. The pathogenesis may differ from typical atopic dermatitis in terms of alloimmunity including graft-versus-host disease. METHODS: We present five children and adolescents with new-onset atopic dermatitis after allogeneic hematopoietic stem cell transplantation. The characteristics and clinical profiles of skin treatment after hematopoietic stem cell transplantation are summarized. RESULTS: Graft-versus-host disease prophylaxis included systemic tacrolimus for all patients. After hematopoietic stem cell transplantation, all patients achieved complete donor chimerism of the bone marrow and had acute graft-versus-host disease of the skin. After engraftment, all patients had skin lesions that met the international consensus diagnostic criteria for atopic dermatitis. None of the patients met the diagnostic criteria for chronic graft-versus-host disease. Topical therapy and skin care based on atopic dermatitis guidelines improved skin condition and atopic dermatitis severity scores in all patients. In addition, type 2 inflammatory markers improved accordingly. CONCLUSION: Topical therapy and skin care may be effective for transplant-related atopic dermatitis after hematopoietic stem cell transplantation. When extensive dermatitis is observed after hematopoietic stem cell transplantation, this treatment may avoid excessive immunosuppressive therapy if it meets the diagnostic criteria for atopic dermatitis.

Three human RNA polymerases interact with TFIIH via a common RPB6 subunit.

In eukaryotes, three RNA polymerases (RNAPs) play essential roles in the synthesis of various types of RNA: namely, RNAPI for rRNA; RNAPII for mRNA and most snRNAs; and RNAPIII for tRNA and other small RNAs. All three RNAPs possess a short flexible tail derived from their common subunit RPB6. However, the function of this shared N-terminal tail (NTT) is not clear. Here we show that NTT interacts with the PH domain (PH-D) of the p62 subunit of the general transcription/repair factor TFIIH, and present the structures of RPB6 unbound and bound to PH-D by nuclear magnetic resonance (NMR). Using available cryo-EM structures, we modelled the activated elongation complex of RNAPII bound to TFIIH. We also provide evidence that the recruitment of TFIIH to transcription sites through the p62-RPB6 interaction is a common mechanism for transcription-coupled nucleotide excision repair (TC-NER) of RNAPI- and RNAPII-transcribed genes. Moreover, point mutations in the RPB6 NTT cause a significant reduction in transcription of RNAPI-, RNAPII- and RNAPIII-transcribed genes. These and other results show that the p62-RPB6 interaction plays multiple roles in transcription, TC-NER, and cell proliferation, suggesting that TFIIH is engaged in all RNAP systems.

Inflammatory Cytokine-Induced Muscle Atrophy and Weakness Can Be Ameliorated by an Inhibition of TGF-ß-Activated Kinase-1.

Chronic inflammation causes muscle wasting. Because most inflammatory cytokine signals are mediated via TGF-ß-activated kinase-1 (TAK1) activation, inflammatory cytokine-induced muscle wasting may be ameliorated by the inhibition of TAK1 activity. The present study was undertaken to clarify whether TAK1 inhibition can ameliorate inflammation-induced muscle wasting. SKG/Jcl mice as an autoimmune arthritis animal model were treated with a small amount of mannan as an adjuvant to enhance the production of TNF-α and IL-1ß. The increase in these inflammatory cytokines caused a reduction in muscle mass and strength along with an induction of arthritis in SKG/Jcl mice. Those changes in muscle fibers were mediated via the phosphorylation of TAK1, which activated the downstream signaling cascade via NF-κB, p38 MAPK, and ERK pathways, resulting in an increase in myostatin expression. Myostatin then reduced the expression of muscle proteins not only via a reduction in MyoD1 expression but also via an enhancement of Atrogin-1 and Murf1 expression. TAK1 inhibitor, LL-Z1640-2, prevented all the cytokine-induced changes in muscle wasting. Thus, TAK1 inhibition can be a new therapeutic target of not only joint destruction but also muscle wasting induced by inflammatory cytokines.

Synthesis and biological evaluation of coumarin derivatives as selective CYP2A6 inhibitors.

Cytochrome P450 2A6 (CYP2A6) inhibitors are expected to be suitable as smoking cessation aids and for cancer prevention. Because the typical coumarin-based CYP2A6 inhibitor methoxsalen also inhibits CYP3A4, unintended drug-drug interactions are still a concern. Therefore, the development of selective CYP2A6 inhibitors is desirable. In this study, we synthesized coumarin-based molecules, determined the IC50 values for CYP2A6 inhibition, verified the possibility of mechanism-based inhibition, and compared the selectivity for CYP2A6 versus CYP3A4. The results demonstrated that we developed CYP2A6 inhibitors that were more potent and selective than methoxsalen.

Discovery of CRBN as a target of thalidomide: a breakthrough for progress in the development of protein degraders.

Progress in strategies aimed at breaking down therapeutic target proteins has led to a paradigm shift in drug discovery. Thalidomide and its derivatives are the only protein degraders currently used in clinical practice. Our understanding of the molecular mechanism of action of thalidomide and its derivatives has advanced dramatically since the identification of cereblon (CRBN) as their direct target. The binding of thalidomide derivatives to CRBN, a substrate recognition receptor for Cullin 4 RING E3 ubiquitin ligase (CRL4), induces the recruitment of non-native substrates to CRL4CRBN and their subsequent degradation. This discovery was a breakthrough in the current rapid development of protein-degrading agents because clarification of the mechanism of action of thalidomide derivatives has demonstrated the clinical value of these compounds. This review provides an overview of the mechanism of action of thalidomide and its derivatives and describes perspectives for protein degraders.

ARID2 is a pomalidomide-dependent CRL4CRBN substrate in multiple myeloma cells.

The immunomodulatory drug (IMiD) thalidomide and its derivatives lenalidomide and pomalidomide are therapeutic agents used in the treatment of multiple myeloma. Although pomalidomide offers considerable clinical benefits to patients with lenalidomide-resistant multiple myeloma, the molecular mechanisms underlying its superior efficacy remain unclear. Here we show that ARID2, a component of the polybromo-associated BAF (PBAF) chromatin-remodeling complex, is a pomalidomide-induced neosubstrate of CRL4CRBN. BRD7, another subunit of PBAF, is critical for pomalidomide-induced ARID2 degradation. ARID2 is involved in transcriptional regulation of pomalidomide target genes including MYC. Pomalidomide is more effective than lenalidomide in degrading ARID2 and is capable of inhibiting MYC expression and proliferation in lenalidomide-resistant cell lines. Notably, ARID2 expression is associated with a poor prognosis and is higher in chemoresistant minimal residual disease (MRD) populations, and in patients with relapsed/refractory multiple myeloma. These findings suggest that ARID2 is a promising target for overcoming lenalidomide resistance in patients with multiple myeloma.

p63 is a cereblon substrate involved in thalidomide teratogenicity.

Cereblon (CRBN) is a primary target of thalidomide and mediates its multiple pharmacological activities, including teratogenic and antimyeloma activities. CRBN functions as a substrate receptor of the E3 ubiquitin ligase CRL4, whose substrate specificity is modulated by thalidomide and its analogs. Although a number of CRL4CRBN substrates have recently been identified, the substrate involved in thalidomide teratogenicity is unclear. Here we show that p63 isoforms are thalidomide-dependent CRL4CRBN neosubstrates that are responsible, at least in part, for its teratogenic effects. The p53 family member p63 is associated with multiple developmental processes. ∆Np63α is essential for limb development, while TAp63α is important for cochlea development and hearing. Using a zebrafish model, we demonstrate that thalidomide exerts its teratogenic effects on pectoral fins and otic vesicles by inducing the degradation of ∆Np63α and TAp63α, respectively. These results may contribute to the invention of new thalidomide analogs lacking teratogenic activity.

Prostanoid receptor genes confer poor prognosis in head and neck squamous cell carcinoma via epigenetic inactivation.

BACKGROUND: Chronic inflammation is a risk factor for head and neck squamous cell carcinoma (HNSCC) and other diseases. Prostanoid receptors are clearly involved in the development of many types of cancer. However, their role is not simple and is poorly understood in HNSCC. METHODS: Methylation profiles of prostanoid receptor family genes were generated for tumour samples obtained from 274 patients with HNSCC, including 69 hypopharynx, 51 larynx, 79 oral cavity, and 75 oropharynx tumour samples, by quantitative methylation-specific PCR. Promoter methylation was then evaluated with respect to various clinical characteristics and patient survival. RESULTS: The mean number of methylated genes per sample was 2.05 ± 2.59 (range 0 to 9). Promoters of PTGDR1, PTGDR2, PTGER1, PTGER2, PTGER3, PTGER4, PTGFR, PTGIR, and TBXA2R were methylated in 43.8%, 18.2%, 25.5%, 17.5%, 41.2%, 8.0%, 19.3%, 20.4%, and 11.3% of the samples, respectively. Methylation indices for prostanoid receptor family genes tended to be higher as the number of TET methylation events increased. Patients with 5-9 methylated genes had a significantly lower survival rate than that of patients with 0-4 methylated genes (log-rank test, P= 0.007). In multivariate analyses, PTGDR1 methylation was most highly correlated with recurrence in patients with hypopharyngeal cancer (P = 0.014). A similar correlation was observed for PTGER4 in patients with laryngeal cancer (P = 0.046). Methylation of the PTGIR and TBXA2R promoters was positively correlated with recurrence in oropharyngeal cancer (P = 0.028 and P = 0.006, respectively). Moreover, Patients with 5-9 methylated genes were extremely lower of 5hmC levels (P = 0.035) and was correlated with increasing expression of DNMT3A and DNMT3B (P < 0.05 and P < 0.05, respectively). CONCLUSION: We characterised the relationship between the methylation status of prostanoid receptor genes and recurrence in HNSCC. These results provide new perspectives for the development of molecular targeted treatment approaches.

Evaluation of Synthesized Ester or Amide Coumarin Derivatives on Aromatase Inhibitory Activity.

Aromatase inhibitors are effective for the treatment of diseases such as breast cancer, which has led to an increase in their demand. However, only a limited number of aromatase inhibitor drugs are currently being marketed. In addition, considering the important aspect of drug resistance, the development of newer drug types is required. We have been developing inhibitors with backbone structures that differ from existing aromatase inhibitors. In this regard, we previously reported that diethylaminocoumarin dimers and thiazolyl coumarin derivatives possess strong aromatase inhibiting capabilities. In this study, we further examined the structure-activity relationships of coumarin derivatives synthesized from thiazolyl coumarin derivatives and their aromatase inhibiting capabilities. Consequently, amide coumarin N-benzhydryl-7-(diethylamino)-2-oxo-2H-chromene-3-carboxamide (IC50 values 4.5 µM) is inhibitor of aromatase. This inhibitor was found to be comparable aromatase inhibitory activity to the 1st generation aromatase inhibitor aminoglutethimide (3.2 µM). Substitution of the amide group on the amide coumarin derivative affects the aromatase inhibiting activity. Our findings suggest that the structure of each substituent changes the orientation of the compound in the active site of aromatase, thus creating a difference in their activities.

Risk Factors for Skin Toxicities Associated with Bendamustine-Based Chemotherapy in Patients with Non-Hodgkin Lymphoma.

Bendamustine plays an especially important role as a treatment for non-Hodgkin lymphoma (NHL). However, patients administered bendamustine alone or in combination with rituximab (BR) may experience drug-associated skin toxicities that can profoundly impact their health-related QOL through both physical discomfort and psychological distress. Moreover, worsening skin symptoms may lead to dose reduction or termination in the management of cancer chemotherapy. We retrospectively investigated patient backgrounds and pretreatment characteristics from medical records of NHL patients treated with bendamustine alone or BR therapy and identified predictive factors for skin toxicities at the start of chemotherapy. Patients were eligible for the study if they were 20 years older, diagnosed with NHL, and received bendamustine alone or BR therapy at the Department of Hematology, Kobe City Medical Center General Hospital, between April 1, 2011, and March 31, 2018. This study included 95 patients with newly diagnosed or refractory or relapsed NHL. Multivariate stepwise logistic regression analysis with backward selection revealed that baseline non-prior chemotherapy (odds ratio (OR), 15.72; 95% confidence interval (CI), 4.24-83.13, p < 0.001) was a significant factor influencing the occurrence of skin toxicity. Our results demonstrated that non-prior chemotherapy was a significant risk factor for skin toxicities in patients with NHL receiving bendamustine alone or BR therapy. No patient experience serious side effects of grade 3 or higher and that bendamustine is very useful as a first-line treatment.

Risk Factors for Febrile Neutropenia Induced by Docetaxel Chemotherapy in Patients with Non-small Cell Lung Cancer.

We retrospectively obtained data of patient background and pretreatment characteristics from medical records and identified the predictive factors of febrile neutropenia (FN) in patients with non-small cell lung cancer (NSCLC) treated with docetaxel alone or in combination with the anti-vascular endothelial growth factor (VEGF) antibody bevacizumab. Patients were eligible for inclusion in the study if they were 20 years or older, diagnosed with NSCLC, and received docetaxel monotherapy alone or in combination with bevacizumab at the Department of Respiratory Medicine, Kobe City Medical Center General Hospital, between July 1, 2011, and March 31, 2018. Eighty-one patients with recurrent or advanced NSCLC were included. Multivariate stepwise logistic regression analysis with backward selection revealed that lower baseline Eastern Cooperative Oncology Group performance status (ECOG-PS) scores of 1 and 2 (odds ratio (OR), 5.098; 95% confidence interval (CI), 1.045-24.879, p = 0.021) and baseline platelet count below 18.8 × 104/µL (OR, 3.861; 95% CI, 1.211-12.311, p = 0.022) were significant factors influencing the FN occurrence rate. Our results demonstrated that ECOG-PS 1-2 and lower baseline platelet count were significant risk factors of FN in patients with NSCLC receiving docetaxel-based chemotherapy. Moreover, the combination of anti-VEGF antibodies and docetaxel might be associated with increased FN frequency. Despite the limitations of this study including its retrospective design, single-center site, and small sample size, baseline ECOG-PS score and platelet count may be regarded as important indices to identify patients for prophylactic granulocyte-colony stimulating factor (G-CSF) treatment before docetaxel-based chemotherapy.

Application of high-performance magnetic nanobeads to biological sensing devices.

Nanomaterials have extensive applications in the life sciences and in clinical diagnosis. We have developed magnetic nanoparticles with high dispersibility and extremely low nonspecific binding to biomolecules and have demonstrated their application in chemical biology (e.g., for the screening of drug receptor proteins). Recently, the excellent properties of nanobeads have made possible the development of novel rapid immunoassay systems and high-precision technologies for exosome detection. For immunoassays, we developed a technology to encapsulate a fluorescent substance in magnetic nanobeads. The fluorescent nanobeads allow the rapid detection of a specific antigen in solution or in tissue specimens. Exosomes, which are released into the blood, are expected to become markers for several diseases, including cancer, but techniques for measuring the absolute quantity of exosomes in biological fluids are lacking. By integrating magnetic nanobead technology with an optical disc system, we developed a novel method for precisely quantifying exosomes in human serum with high sensitivity and high linearity without requiring enrichment procedures. This review focuses on the properties of our magnetic nanobeads, the development of novel biosensors using these nanobeads, and their broad practical applications. Graphical abstract ᅟ.

Evaluation of synthesized coumarin derivatives on aromatase inhibitory activity.

In women across the world, the most common type of cancer is breast cancer. Among medical treatments, endocrine therapy based on aromatase inhibitors (AI) is expected to be effective against not only post-menopausal but also pre-menopausal breast cancer. In this study, we examined the structure-activity relationship between the aromatase inhibitory effects of 7-diethylaminocoumarin derivatives with a substituent at position 3 and coumarin derivatives with a substituent at position 7. Consequently, we found that 7-(pyridin-3-yl)coumarin (IC50 values 30.3nM) and 7,7'-diethylamino-3,3'-biscoumarin (28.7nM) are the most potent inhibitors of aromatase. These inhibitors were found to be comparable to the existing CYP19 inhibitor exemestane (42.5nM).

DSIF, the Paf1 complex, and Tat-SF1 have nonredundant, cooperative roles in RNA polymerase II elongation.

Transcription elongation factor DSIF/Spt4-Spt5 is capable of promoting and inhibiting RNA polymerase II elongation and is involved in the expression of various genes. While it has been known for many years that DSIF inhibits elongation in collaboration with the negative elongation factor NELF, how DSIF promotes elongation is largely unknown. Here, an activity-based biochemical approach was taken to understand the mechanism of elongation activation by DSIF. We show that the Paf1 complex (Paf1C) and Tat-SF1, two factors implicated previously in elongation control, collaborate with DSIF to facilitate efficient elongation. In human cells, these factors are recruited to the FOS gene in a temporally coordinated manner and contribute to its high-level expression. We also show that elongation activation by these factors depends on P-TEFb-mediated phosphorylation of the Spt5 C-terminal region. A clear conclusion emerging from this study is that a set of elongation factors plays nonredundant, cooperative roles in elongation. This study also shows unambiguously that Paf1C, which is generally thought to have chromatin-related functions, is involve directlyd in elongation control.

Possible alternative treatment for mandibular asymmetry by local unilateral IGF-1 injection into the mandibular condylar cavity: Experimental study in mice.

INTRODUCTION: The purpose of this study was to investigate whether a local unilateral IGF-1 injection into the mandibular condylar cavity can induce unilateral endochondral mandibular growth without any systemic adverse effects. METHODS: Seventy-five 3-week-old male Jcl:ICR mice were used in this study. The mice were divided into 2 groups: control group (n = 22) and IGF-1 group (n = 53). In the IGF-1 group, human IGF-1 was injected into the right mandibular condylar cavity, and phosphate-buffered saline solution was injected into the left cavity, 3 times per week for 10 weeks. RESULTS: There was no significant difference in body weight, serum human IGF-1 concentration, and soft tissue thickness of the cheeks including the masseter muscles between the 2 groups. Unilateral IGF-1 injection induced a lateral shift of the mandible to the contralateral side, and microcomputed tomogtraphy analysis showed that unilateral IGF-1 injection induced endochondral growth in the condyle. Col2, Ihh, and Runx2 were extensively upregulated by the local unilateral IGF-1 injection in real-time reverse transcription polymerase chain reaction analysis. Proliferation marker KI67, IGF-1 signaling molecule AKT1, and chondrogenic differentiation marker Col2 were strongly expressed in the IGF-1 injected condyle by immunohistochemistry. Vital labeling showed that the distance between the labels was increased in the IGF-1 injection group compared with that of the control group. CONCLUSIONS: The results verified in this study indicated that local unilateral IGF-1 injection into the mandibular condylar cavity successfully induced unilateral endochondral mandibular growth in mice without any systemic adverse effects. Thus, local unilateral IGF-1 injection into the mandibular condylar cavity could be a useful alternative for mandibular asymmetry therapy during the growth period. However, additional experimental and clinical studies will be necessary to prove the real effect of this new therapy.

Measurement of optical loss in nanophotonic waveguides using integrated cavities.

Measurement of optical loss in nanophotonic waveguides is necessary for monitoring the properties of integrated photonic devices. We propose a simple method of measuring the optical loss using integrated nanocavities. It is shown theoretically that weak coupling between the waveguide and cavities leads to a direct estimation of the optical loss by measuring light radiated from the cavities. In addition, we experimentally demonstrate the optical loss in a fabricated photonic crystal waveguide. Our method gives not only a degree of freedom in real-time monitoring of the optical properties of nanophotonic structures, but it also can be used for various waveguide-based applications.

Discovery of a 2-hydroxyacetophenone derivative as an outstanding linker to enhance potency and ß-selectivity of liver X receptor agonist.

Our research found that the 2-hydroxyacetophenone derivative is an outstanding linker between the 1,1-bistrifluoromethylcarbinol moiety and the imidazolidine-2,4-dione moiety to enhance the potency and ß-selectivity of liver X receptor (LXR) agonist in our head-to-tail molecular design. The incorporation of this linker is 20-fold more potent than our previous compound (2) for LXR ß agonistic activity (EC50) in a GAL-4 luciferase assay. Furthermore, we also identified 5-[5-(1-methylethoxy)pyridyl-2-yl]-5-methylimidazoline-2,4-dione (54), which lowers the lipophilicity of 2-hydroxyacetophenone derivative. We revealed that a combination of our newly developed linker and hydantoin (54) plays a pivotal role in improving the potency and selectivity of LXRß. The optically separated (-)-56 increases high-density lipoprotein cholesterol levels without elevating plasma triglyceride levels and results in a decrease of the lipid accumulation area in the aortic arch in a high-fat- and cholesterol-fed low-density lipoprotein receptor knock-out mice. In this manuscript, we report that (-)-56 is a highly potent and ß-selective LXR agonist for use in the treatment of atherosclerosis.

Multiple-channel wavelength conversions in a photonic crystal cavity.

We demonstrate multiple-channel wavelength conversions of second harmonic and sum frequency generations in a silicon carbide photonic crystal cavity. The cavity is designed to have multiple modes including a nanocavity mode and Fabry-Pérot modes. Multiple-channel wavelength conversions in the nanocavity and Fabry-Pérot modes are shown experimentally. Furthermore, we investigate the polarization characteristics of wavelength-converted light. The experimental results of the polarization are in good agreement with calculation.