Conversion efficiency improvement of terahertz wave generation laterally emitted by a ridge-type periodically poled lithium niobate.

We demonstrate terahertz (THz) wave generation by wavelength conversion in a ridge-type/bulk periodically poled lithium niobate (RT-/bulk-PPLN) under almost the same experimental conditions. When using the RT-PPLN, the ridge structure works as a slab waveguide for the incident pump beam (wavelength: ∼1 µm), and the generated THz wave (∼200 µm) was emitted uniformly from the entire side surface of the crystal. The RT-PPLN has a much higher conversion efficiency from the pumping beam to the THz wave than the bulk-PPLN, and the ratio improved several ten times compared with those of previous studies.

R3HDM1 haploinsufficiency is associated with mild intellectual disability.

R3HDM1 (R3H domain containing 1) is an uncharacterized RNA-binding protein that is highly expressed in the human cerebral cortex. We report the first case of a 12-year-old Japanese male with haploinsufficiency of R3HDM1. He presented with mild intellectual disability (ID) and developmental delay. He had a pericentric inversion of 46,XY,inv(2)(p16.1q21.3)dn with breakpoints in intron 19 of R3HDM1 (2q21.3) and the intergenic region (2p16.1). The R3HDM1 levels in his lymphoblastoid cells were reduced to approximately half that of the healthy controls. However, the expression of MIR128-1, in intron 18 of R3HDM1, was not affected via the pericentric inversion. Knockdown of R3HDM1 in mouse embryonic hippocampal neurons suppressed dendritic growth and branching. Notably, the Database of Genomic Variants reported the case of a healthy control with a 488-kb deletion that included both R3HDM1 and MIR128-1. miR-128 has been reported to inhibit dendritic growth and branching in mouse brain neurons, which directly opposes the novel functions of R3HDM1. These findings suggest that deleting both R3HDM1 and MIR128-1 alleviates the symptoms of the disease caused by loss-of-function mutations in R3HDM1 only. Thus, haploinsufficiency of R3HDM1 in the patient may be the cause of the mild ID due to the genetic imbalance between R3HDM1 and MIR128-1.

Zero-spindle spectral drill: real-time spectral measurement in a fixed Fabry-Pérot cavity.

An optical configuration for Fabry-Pérot cavity scanning using a geometric phase shifter, known as the "spectral drill," is improved to acquire a spectrum in real-time. Previously, the resonance condition of the spectral drill is swept by the mechanical rotation of a phase plate comprising a geometric phase shifter, and the acquisition time is limited. In this work, using a q-plate and a camera instead of phase plate rotation and a photo detector, we remove all the spinning mechanics and increase the acquisition rate by a factor 720. This technique will be applied to locking laser frequency.

Kit-independent mast cell adhesion mediated by Notch.

Kit/CD117 plays a crucial role in the cell-cell and cell-matrix adhesion of mammalian mast cells (MCs); however, it is unclear whether other adhesion molecule(s) perform important roles in the adhesion of MCs. In the present study, we show a novel Kit-independent adhesion mechanism of mouse cultured MCs mediated by Notch family members. On stromal cells transduced with each Notch ligand gene, Kit and its signaling become dispensable for the entire adhesion process of MCs from tethering to spreading. The Notch-mediated spreading of adherent MCs involves the activation of signaling via phosphatidylinositol 3-kinases and mitogen-activated protein kinases, similar to Kit-mediated spreading. Despite the activation of the same signaling pathways, while Kit supports the adhesion and survival of MCs, Notch only supports adhesion. Thus, Notch family members are specialized adhesion molecules for MCs that effectively replace the adhesion function of Kit in order to support the interaction of MCs with the surrounding cellular microenvironments.

Genetic variants in C5 and poor response to eculizumab.

BACKGROUND: Eculizumab is a humanized monoclonal antibody that targets complement protein C5 and inhibits terminal complement-mediated hemolysis associated with paroxysmal nocturnal hemoglobinuria (PNH). The molecular basis for the poor response to eculizumab in a small population of Japanese patients is unclear. METHODS: We assessed the sequences of the gene encoding C5 in patients with PNH who had either a good or poor response to eculizumab. We also evaluated the functional properties of C5 as it was encoded in these patients. RESULTS: Of 345 Japanese patients with PNH who received eculizumab, 11 patients had a poor response. All 11 had a single missense C5 heterozygous mutation, c.2654G → A, which predicts the polymorphism p.Arg885His. The prevalence of this mutation among the patients with PNH (3.2%) was similar to that among healthy Japanese persons (3.5%). This polymorphism was also identified in a Han Chinese population. A patient in Argentina of Asian ancestry who had a poor response had a very similar mutation, c.2653C → T, which predicts p.Arg885Cys. Nonmutant and mutant C5 both caused hemolysis in vitro, but only nonmutant C5 bound to and was blocked by eculizumab. In vitro hemolysis due to nonmutant and mutant C5 was completely blocked with the use of N19-8, a monoclonal antibody that binds to a different site on C5 than does eculizumab. CONCLUSIONS: The functional capacity of C5 variants with mutations at Arg885, together with their failure to undergo blockade by eculizumab, account for the poor response to this agent in patients who carry these mutations. (Funded by Alexion Pharmaceuticals and the Ministry of Health, Labor, and Welfare of Japan.).

SNP array screening of cryptic genomic imbalances in 450 Japanese subjects with intellectual disability and multiple congenital anomalies previously negative for large rearrangements.

Intellectual disability (ID) is a heterogeneous condition affecting 2-3% of the population, often associated with multiple congenital anomalies (MCA). The genetic cause remains largely unexplained for most cases. To investigate the causes of ID/MCA of unknown etiology in the Japanese population, 645 subjects have been recruited for the screening of pathogenic copy-number variants (CNVs). Two screenings using bacterial artificial chromosome (BAC) arrays were previously performed, which identified pathogenic CNVs in 133 cases (20.6%; Hayashi et al., J. Hum. Genet., 2011). Here, we present the findings of the third screening using a single-nucleotide polymorphism (SNP) array, performed in 450 negative cases from our previous report. Pathogenic CNVs were found in 22 subjects (4.9%), in which 19 CNVs were located in regions where clinical significance had been previously established. Among the 22 cases, we identified PPFIA2 as a novel candidate gene for ID. Analysis of copy-neutral loss of heterozygosity (CNLOH) detected one case in which the CNLOH regions seem to be significant. The SNP array detected a modest fraction of small causative CNVs, which is explained by the fact that the majority of causative CNVs have larger sizes, and those had been mostly identified in the two previous screenings.

Identical deletion at 14q13.3 including PAX9 and NKX2-1 in siblings from mosaicism of unaffected parent.

By screening patients with undiagnosed multiple congenital anomalies and intellectual disability using array-comparative genomic hybridization, we identified an 884 kb heterozygous microdeletion at 14q13.3 in two siblings presenting with oligodontia, hypothyroidism and persistent pulmonary hypertension of the newborn, resulting from their parental gonosomal mosaicism. Among the six genes included in the deletion, haploinsufficiency of PAX9 and NKX2-1 was probably associated with their phenotypes. These results highlighted a possibility of recurrence of pathogenic copy-number variants associated with parental mosaicism, which requires careful genetic counseling.

Mechanisms of hormonal therapy resistance in breast cancer.

Whilst estrogen receptor (ER)-positive breast cancers are preferentially treated with hormone therapy, approximately one-third of them relapse. The mechanisms of refractoriness have been investigated by numerous studies but have not been fully clarified. Hormonal therapy resistance, particularly aromatase inhibitor (AI) resistance, may be related to the acquisition of alternative intracellular ER signaling. We have been investing the mechanisms using cancer specimens and cell lines by monitoring the transcription activity of ERs. AI refractory specimens showed diverse ER activity in the adenovirus estrogen receptor element-green fluorescent protein (ERE-GFP) assay and varied sensitivity to anti-estrogens, indicating the existence of multiple resistant mechanisms. We established six different types of cell lines mimicking AI resistance from ERE-GFP-introduced ER-positive cell lines. They revealed that multiple and alternative ER activating pathways were involved in the resistance, such as phosphorylation-dependent or androgen metabolite-dependent mechanisms. The response to fulvestrant and mammalian target of rapamycin inhibitor also varied among individual resistant cell lines. These results indicate that further subclassification of ER-positive breast cancer is extremely important to decide the therapeutic management of not only hormonal therapy but also new molecular target therapy.

Related factors for worsening renal function following percutaneous transluminal renal angioplasty (PTRA) in patients with atherosclerotic renal artery stenosis.

PURPOSE: To identify candidates for PTRA in terms of the preservation of renal function, we herein evaluated factors that caused worsening renal function (WRF) after PTRA. METHODS: We evaluated 92 patients with atherosclerotic renal artery stenosis (mean age 70.7 ± 8.4 years). WRF was defined as a ≥0.3 mg/dL increase in creatinine levels after PTRA compared to before PTRA. RESULTS: A total of 92 patients exhibited non-WRF 83 (90.2%), WRF 9 (9.8%). Significant differences were observed in serum creatinine levels between two groups both before (non-WRF 1.34 ± 0.49 versus WRF 1.70 ± 0.68 mg/dL, p = 0.0462) and after PTRA (non-WRF 1.31 ± 0.43 versus WRF 2.42 ± 1.12 mg/dL, p < 0.0001). Patients with WRF had higher comorbidity rate of diabetes mellitus (DM) (non-WRF 31.3% versus WRF 66.7%, p = 0.0345) and proteinuria (non-WRF 27.7% versus WRF 66.7%, p = 0.0169), and had higher systolic blood pressure (non-WRF 143.6 ± 18.7 versus WRF 157.1 ± 19.9 mmHg, p = 0.0436), higher plasma B-type natriuretic peptide (BNP) levels, and larger left atrial and left ventricular end-diastolic dimensions before PTRA. Patients with WRF had a higher rate of taking diuretics (non-WRF 27.7% versus WRF 66.7%, p = 0.0169) after PTRA. Multiple logistic regression analysis revealed that comorbidity of DM was an independent related factor for WRF (comorbidity of DM, yes: OR 31.0, 95% CI 2.44-1024.62, p = 0.0055). CONCLUSIONS: Comorbidity of DM, coexisting of proteinuria, high creatinine level, high blood pressure, high BNP levels, and large left atrial and ventricular dimensions were related to WRF after PTRA in patients with atherosclerotic renal artery stenosis.

Possible role of the aromatase-independent steroid metabolism pathways in hormone responsive primary breast cancers.

Aromatase inhibitors (AIs) exert antiproliferative effects by reducing local estrogen production from androgens in postmenopausal women with hormone-responsive breast cancer. Previous reports have shown that androgen metabolites generated by the aromatase-independent enzymes, 5α-androstane-3ß, 17ß-diol (3ß-diol), androst-5-ene-3ß, and 17ß-diol (A-diol), also activate estrogen receptor (ER) α. Estradiol (E2) can also reportedly be generated from estrone sulfate (E1S) pooled in the plasma. Estrogenic steroid-producing aromatase-independent pathways have thus been proposed as a mechanism of AI resistance. However, it is unclear whether these pathways are functional in clinical breast cancer. To investigate this issue, we assessed the transcriptional activities of ER in 45 ER-positive human breast cancers using the adenovirus estrogen-response element-green fluorescent protein assay and mRNA expression levels of the ER target gene, progesterone receptor, as indicators of ex vivo and in vivo ER activity, respectively. We also determined mRNA expression levels of 5α-reductase type 1 (SRD5A1) and 3ß-hydroxysteroid dehydrogenase type 1 (3ß-HSD type 1; HSD3B1), which produce 3ß-diol from androgens, and of steroid sulfatase (STS) and 17ß-hydroxysteroid dehydrogenase type 1 (17ß-HSD type 1; HSD17B1), which produce E2 or A-diol from E1S or dehydroepiandrosterone sulfate. SRD5A1 and HSD3B1 expression levels were positively correlated with ex vivo and in vivo ER activities. STS and HSD17B1 expression levels were positively correlated with in vivo ER activity alone. Elevated expression levels of these steroid-metabolizing enzymes in association with high in vivo ER activity were particularly notable in postmenopausal patients. Analysis of the expression levels of steroid-metabolizing enzymes revealed positive correlations between SRD5A1 and HSD3B1, and STS and HSD17B1. These findings suggest that the SRD5A1-HSD3B1 as well as the STS-HSD17B pathways, could contributes to ER activation, especially postmenopause. These pathways might function as an alternative estrogenic steroid-producing, aromatase-independent pathways.

Terahertz wave parametric amplifier.

The importance of terahertz (THz) wave techniques has been demonstrated in various fields, and the range of applications is now expanding rapidly. However, the practical implementation of THz science to solve the real-world problems is restricted due to the lack not only of convenient high power THz wave emitters and sensitive detectors but also of efficient quasi-optical active devices such as amplifiers. In this work, we demonstrate the direct amplification of THz waves in room temperature using magnesium oxide-doped lithium niobate (MgO:LiNbO3) crystals as the nonlinear gain medium. The input THz wave is injected as a seed beam along with the pump beam into the nonlinear crystal and it is amplified by the optical parametric process. We report gain in excess of 30 dB with an input THz pulse energy of less than 1 pJ. We believe that this demonstration will contribute to the convenience and further applicability of THz frequency techniques.

Pathophysiological significance of the p.E31G variant in RAC1 responsible for a neurodevelopmental disorder with microcephaly.

RAC1 encodes a Rho family small GTPase that regulates actin cytoskeletal reorganization and intracellular signaling pathways. Pathogenic RAC1 variants lead to a neurodevelopmental disorder with diverse phenotypic manifestations, including abnormalities in brain size and facial dysmorphism. However, the underlying pathophysiological mechanisms have yet to be elucidated. Here, we present the case of a school-aged male who exhibited global developmental delay, intellectual disability, and acquired microcephaly. Through whole exome sequencing, we identified a novel de novo variant in RAC1, (NM_006908.5): c.92 A > G,p.(E31G). We then examined the pathophysiological significance of the p.E31G variant by focusing on brain development. Biochemical analyses revealed that the recombinant RAC1-E31G had no discernible impact on the intrinsic GDP/GTP exchange activity. However, it exhibited a slight inhibitory effect on GTP hydrolysis. Conversely, it demonstrated a typical response to both a guanine-nucleotide exchange factor and a GTPase-activating protein. In transient expression analyses using COS7 cells, RAC1-E31G exhibited minimal interaction with the downstream effector PAK1, even in its GTP-bound state. Additionally, overexpression of RAC1-E31G was observed to exert a weak inhibitory effect on the differentiation of primary cultured hippocampal neurons. Moreover, in vivo studies employing in utero electroporation revealed that acute expression of RAC1-E31G resulted in impairments in axonal elongation and dendritic arborization in the young adult stage. These findings suggest that the p.E31G variant functions as a dominant-negative version in the PAK1-mediated signaling pathway and is responsible for the clinical features observed in the patient under investigation, namely microcephaly and intellectual disability.

CCR7-independent transport of skin antigens occurs in the dermis.

Under homeostatic conditions, skin DCs migrate to regional LNs transporting self-antigens (self-Ags). The transport of self-Ags is considered to be critical for maintaining peripheral tolerance. Although the chemokine receptor CCR7 potently induces the migration of skin DCs to regional LNs, Ccr7(-/-) (Ccr7-KO) mice do not show skin auto-immune diseases. To resolve this inconsistency, we examined Ccr7-KO epidermis- or dermis-hyperpigmented transgenic (Tg) mice, in which the transport of skin self-Ags is traceable by melanin granules (MGs). Under CCR7-deficient conditions, the transport of epidermal MGs to regional LNs was impaired at 7 weeks of age. However, epidermal MGs could be transported when they had accumulated in the dermis. Ccr7-KO-dermis-pigmented Tg mice confirmed the presence of CCR7-independent transport from the dermis. Compared with WT-dermis-pigmented Tg mice, the amount of transported melanin and number of MG-laden CD11c(+) cells were both approximately 40% of the WT levels, while the number of MG-laden CD205(+) or CD207(+) cells decreased to about 10% in skin regional LNs of Ccr7-KO-dermis-pigmented Tg mice. Cell sorting highlighted the involvement of CD11c(+) cells in the CCR7-independent transport. Here, we show that CCR7-independent transport of skin self-Ags occurs in the dermis. This system might contribute to the continuous transport of self-Ags, and maintain peripheral tolerance.

Androgen metabolite-dependent growth of hormone receptor-positive breast cancer as a possible aromatase inhibitor-resistance mechanism.

Aromatase inhibitors (AIs) have been reported to exert their antiproliferative effects in postmenopausal women with hormone receptor-positive breast cancer not only by reducing estrogen production but also by unmasking the inhibitory effects of androgens such as testosterone (TS) and dihydrotestosterone (DHT). However, the role of androgens in AI-resistance mechanisms is not sufficiently understood. 5α-Androstane-3ß,17ß-diol (3ß-diol) generated from DHT by 3ß-hydroxysteroid dehydrogenase type 1 (HSD3B1) shows androgenic and substantial estrogenic activities, representing a potential mechanism of AI resistance. Estrogen response element (ERE)-green fluorescent protein (GFP)-transfected MCF-7 breast cancer cells (E10 cells) were cultured for 3 months under steroid-depleted, TS-supplemented conditions. Among the surviving cells, two stable variants showing androgen metabolite-dependent ER activity were selected by monitoring GFP expression. We investigated the process of adaptation to androgen-abundant conditions and the role of androgens in AI-resistance mechanisms in these variant cell lines. The variant cell lines showed increased growth and induction of estrogen-responsive genes rather than androgen-responsive genes after stimulation with androgens or 3ß-diol. Further analysis suggested that increased expression of HSD3B1 and reduced expression of androgen receptor (AR) promoted adaptation to androgen-abundant conditions, as indicated by the increased conversion of DHT into 3ß-diol by HSD3B1 and AR signal reduction. Furthermore, in parental E10 cells, ectopic expression of HSD3B1 or inhibition of AR resulted in adaptation to androgen-abundant conditions. Coculture with stromal cells to mimic local estrogen production from androgens reduced cell sensitivity to AIs compared with parental E10 cells. These results suggest that increased expression of HSD3B1 and reduced expression of AR might reduce the sensitivity to AIs as demonstrated by enhanced androgen metabolite-induced ER activation and growth mechanisms. Androgen metabolite-dependent growth of breast cancer cells may therefore play a role in AI-resistance.

Increased gene dosage of myelin protein zero causes Charcot-Marie-Tooth disease.

OBJECTIVE: On the basis of the hypothesis that copy number mutations of the genes encoding myelin compact proteins are responsible for myelin disorders in humans, we have explored the possibility of copy number mutations in patients with Charcot-Marie-Tooth disease (CMT) whose responsible genes remain undefined. METHODS: A family with 6 affected members in 3 consecutive generations, presenting with motor and sensory demyelinating polyneuropathy, was investigated. Characteristic clinical features in this pedigree include Adie pupils and substantial intrafamilial variability in the age at onset, electrophysiological findings, and clinical severity. Nucleotide sequence analyses of PMP22, MPZ, or GJB1 and gene dosage study of PMP22 did not reveal causative mutations. Hence, we applied a custom-designed array for comparative genomic hybridization (CGH) analysis to conduct a comprehensive screening of copy number mutations involving any of the known causative genes for CMT other than PMP22. RESULTS: The array CGH analyses revealed increased gene dosage involving the whole MPZ, and the flanking genes of SDHC and C1orf192. The gene dosage is estimated to be 5 copies. This mutation showed complete cosegregation with the disease phenotype in this pedigree. INTERPRETATION: The increased gene dosage of MPZ and increased expression level of MPZ mRNA emphasize the important role of the dosage of the MPZ protein in the functional integrity of peripheral nerve myelin in humans, and provide a new insight into the pathogenic mechanisms underlying CMT.

Subporphyrins with an axial B-C bond.

Axial fabrications of subporphyrins have been conveniently accomplished by the reaction of B(methoxo)triphenylsubporphyrin with Grignard reagents such as aryl-, heteroaryl-, ferrocenyl-, ß-styryl-, phenylethynyl-, and ethylmagnesium bromides. The axial groups thus introduced are not conjugated with the subporphyrin core. This situation leads to effective fluorescence quenching of subporphyrins when the axial group is strongly electron donating such as 4-dimethylaminophenyl and ferrocenyl groups.

Two neonatal cholestasis patients with mutations in the SRD5B1 (AKR1D1) gene: diagnosis and bile acid profiles during chenodeoxycholic acid treatment.

BACKGROUND AND AIMS: In two Japanese infants with neonatal cholestasis, 3-oxo-Δ(4)-steroid 5ß-reductase deficiency was diagnosed based on mutations of the SRD5B1 gene. Unusual bile acids such as elevated 3-oxo-Δ(4) bile acids were detected in their serum and urine by gas chromatography-mass spectrometry. We studied effects of oral chenodeoxycholic acid treatment. PATIENTS AND METHODS: SRD5B1 gene analysis used peripheral lymphocyte genomic DNA. Diagnosis and treatment of these two patients were investigated retrospectively and prospectively investigated. RESULTS: With respect to SRD5B1, one patient was heterozygous (R266Q, a novel mutation) while the other was a compound heterozygote (G223E/R261C). Chenodeoxycholic acid treatment was effective in improving liver function and decreasing unusual bile acids such as 7α-hydroxy- and 7α,12α-dihydroxy-3-oxo-4-cholen-24-oic acids in serum and urine. CONCLUSION: Primary bile acid treatment using chenodeoxycholic acid was effective for these patients treated in early infancy before the late stage of chronic cholestatic liver dysfunction.

Resveratrol exhibits diverse anti-cancer activities through epigenetic regulation of E-cadherin and p21 in triple-negative breast cancer cells.

BACKGROUND: Triple-negative breast cancer (TNBC) has an aggressive phenotype and poor outcome, however no specific targeted therapy has been established for TNBC lacking germline BRCA1/2 pathogenic variants. To develop a novel therapeutic strategy, we explored the potential of resveratrol (RSV) for TNBC treatment. METHODS: We investigated the effects of RSV on malignant phenotypes of TNBC cells as well as on apoptosis induced by ABT263, a specific inhibitor of BCL-2 and BCL-xL, using morphological observation, migration assay, ß-galactosidase staining, and Hoechst staining. To elucidate the underlying mechanisms of RSV-mediated effects, expression levels and histone acetylation levels of cadherin 1 (CDH1, E-cadherin) and cyclin dependent kinase inhibitor 1A (CDKN1A, p21) were determined by RT-qPCR, western blotting, and chromatin immunoprecipitation. Furthermore, knockdown analysis was conducted to evaluate the involvement of E-cadherin and/or p21 in RSV potentiation on cytotoxic activity of ABT263. RESULTS: RSV treatment induced epithelial-like cellular morphology and suppressed the migration capacity in MDA-MB-231 and BT-549-Luc TNBC cells. ß-galactosidase-positive cells were increased after RSV treatment, indicating the induction of cellular senescence, in MDA-MB-231 cells but not in BT-549-Luc cells. RSV increased the expression and histone acetylation of CDH1 and CDKN1A in both cells. Interestingly, pre-treatment with RSV enhanced the induction of apoptosis in the ABT263-treated MDA-MB-231 and BT-549-Luc cells, and knockdown of CDKN1A decreased ABT263-induced apoptosis in RSV-treated MDA-MB-231 cells. CONCLUSIONS: RSV represses the metastatic capacity and enhances the cytotoxic activity of ABT263 in TNBC cells. Our results suggested that RSV can potentially be used as a repressor of metastasis or a sensitizer to ABT263 for TNBC treatment via up-regulation of CDH1 and CDKN1A through epigenetic mechanisms.

Pathogenicity evaluation of variants of uncertain significance at exon-intron junction by splicing assay in patients with Mowat-Wilson syndrome.

High-throughput sequencing has identified vast numbers of variants in genetic disorders. However, the significance of variants at the exon-intron junction remains controversial. Even though most cases of Mowat-Wilson syndrome (MOWS) are caused by heterozygous loss-of-function variants in ZEB2, the pathogenicity of variants at exon-intron junction is often indeterminable. We identified four intronic variants in 5/173 patients with clinical suspicion for MOWS, and evaluated their pathogenicity by in vitro analyses. The minigene analysis showed that c.73+2T>G caused most of the transcripts skipping exon 2, while c.916+6T>G led to partial skipping of exon 7. No splicing abnormalities were detected in both c.917-21T>C and c.3067+6A>T. The minigene analysis reproduced the splicing observed in the blood cells of the patient with c.73+2T>G. The degree of the exon skipping was concordant with the severity of MOWS; while the patient with c.73+2T>G was typical MOWS, the patient with c.916+6T>G showed milder phenotype which has been seldom reported. Our results demonstrate that mRNA splicing assays using the minigenes are valuable for determining the clinical significance of intronic variants in patients with not only MOWS but also other genetic diseases with splicing aberrations and may explain atypical or milder cases, such as the current patient.