APC/PIK3CA mutations and ß-catenin status predict tankyrase inhibitor sensitivity of patient-derived colorectal cancer cells.

BACKGROUND: Aberrant WNT/ß-catenin signaling drives carcinogenesis. Tankyrases poly(ADP-ribosyl)ate and destabilize AXINs, ß-catenin repressors. Tankyrase inhibitors block WNT/ß-catenin signaling and colorectal cancer (CRC) growth. We previously reported that 'short' APC mutations, lacking all seven ß-catenin-binding 20-amino acid repeats (20-AARs), are potential predictive biomarkers for CRC cell sensitivity to tankyrase inhibitors. Meanwhile, 'Long' APC mutations, which possess more than one 20-AAR, do not predict inhibitor-resistant cells. Thus, additional biomarkers are needed to precisely predict the inhibitor sensitivity. METHODS: Using 47 CRC patient-derived cells (PDCs), we examined correlations between the sensitivity to tankyrase inhibitors (G007-LK and RK-582), driver mutations, and the expressions of signaling factors. NOD.CB17-Prkdcscid/J and BALB/c-nu/nu xenograft mice were treated with RK-582. RESULTS: Short APC mutant CRC cells exhibited high/intermediate sensitivities to tankyrase inhibitors in vitro and in vivo. Active ß-catenin levels correlated with inhibitor sensitivity in both short and long APC mutant PDCs. PIK3CA mutations, but not KRAS/BRAF mutations, were more frequent in inhibitor-resistant PDCs. Some wild-type APC PDCs showed inhibitor sensitivity in a ß-catenin-independent manner. CONCLUSIONS: APC/PIK3CA mutations and ß-catenin predict the sensitivity of APC-mutated CRC PDCs to tankyrase inhibitors. These observations may help inform the strategy of patient selection in future clinical trials of tankyrase inhibitors.

Spiral Chiral Metamaterial Structure Shape for Optical Activity Improvements.

We report on a spiral structure suitable for obtaining a large optical response. We constructed a structural mechanics model of the shape of the planar spiral structure when deformed and verified the effectiveness of the model. As a verification structure, we fabricated a large-scale spiral structure that operates in the GHz band by laser processing. Based on the GHz radio wave experiments, a more uniform deformation structure exhibited a higher cross-polarization component. This result suggests that uniform deformation structures can improve circular dichroism. Since large-scale devices enable speedy prototype verification, the obtained knowledge can be exported to miniaturized-scale devices, such as MEMS terahertz metamaterials.

Analysis of the Factors That Affect the Detection Duration of SARS-CoV-2 in Loop Mediated Isothermal Amplification among COVID-19 Inpatients.

In COVID-19 patients who are immunocompromised or have severe COVID-19, the duration of infectious viral shedding may be longer, and a longer isolation duration is recommended. At the National Sagamihara Hospital, a decline in the viral load to end the isolation of hospitalized patients with COVID-19 was confirmed using loop-mediated isothermal amplification (LAMP). However, a subset of patients displayed LAMP positivity for more than 20 days after symptom onset. Therefore, we conducted a retrospective observational study to investigate the factors that affect the persistence of LAMP positivity. This study included a total of 102 participants. The severity of COVID-19 was mild (25.5%), moderate (67.6%), or severe (6.9%). The median number (interquartile range) of days until negative LAMP results from symptom onset were 16 (14-19) days. Multivariate logistic regression analysis showed that patients ≥55 years and/or those with the delta variant were correlated with persistent LAMP positivity for more than 20 days after symptom onset. This study identified age, the delta variant, and oxygen requirement as factors that contribute to persistently positive LAMP results. Therefore, it is posited that in these patients, the implementation of LAMP for deisolation would result in a prolonged isolation duration.

A Monolayer MoS2 FET with an EOT of 1.1 nm Achieved by the Direct Formation of a High-κ Er2 O3 Insulator Through Thermal Evaporation.

Achieving the direct growth of an ultrathin gate insulator with high uniformity and high quality on monolayer transition metal dichalcogenides (TMDCs) remains a challenge due to the chemically inert surface of TMDCs. Although the main solution for this challenge is utilizing buffer layers before oxide is deposited on the atomic layer, this method drastically degrades the total capacitance of the gate stack. In this work, we constructed a novel direct high-κ Er2 O3 deposition system based on thermal evaporation in a differential-pressure-type chamber. A uniform Er2 O3 layer with an equivalent oxide thickness of 1.1 nm was achieved as the gate insulator for top-gated MoS2 field-effect transistors (FETs). The top gate Er2 O3 insulator without the buffer layer on MoS2 exhibited a high dielectric constant that reached 18.0, which is comparable to that of bulk Er2 O3 and is the highest among thin insulators (< 10 nm) on TMDCs to date. Furthermore, the Er2 O3 /MoS2 interface (Dit  ≈ 6 × 1011 cm-2 eV-1 ) is confirmed to be clean and is comparable with that of the h-BN/MoS2 heterostructure. These results prove that high-quality dielectric properties with retained interface quality can be achieved by this novel deposition technique, facilitating the future development of 2D electronics.

Disruption of the anterior commissure in Olig2 deficient mice.

In the present study, we examined neural circuit formation in the forebrain of the Olig2 knockout (Olig2-KO) mouse model and found disruption of the anterior commissure at the late foetal stage. Axon bundles of the anterior commissure encountered the wall of the third ventricle and ceased axonal extension. L1-CAM immunohistochemistry showed that Olig2-KO mice lose decussation formation in the basal forebrain. DiI tracing revealed that the thin bundles of the anterior commissure axons crossed the midline but ceased further extension into the deep part of the contralateral side. Furthermore, some fractions of DiI-labelled axons were oriented dorsolaterally, which was not observed in the control mouse forebrain. The rostral part of the third ventricle was much wider in the Olig2-KO mice than in wild-type mice, which likely resulted in the delay of midline fusion and subsequent delay and malformation of the anterior commissure. We analysed gene expression alterations in the Olig2-KO mice using a public database and found multiple genes, which are related to axon guidance and epithelial-mesenchymal transition, showing subtle expression changes. These results suggest that Olig2 is essential for anterior commissure formation, likely by regulating multiple biological processes.

Acquired resistance to BRAF inhibitors is mediated by BRAF splicing variants in BRAF V600E mutation-positive colorectal neuroendocrine carcinoma.

Neuroendocrine carcinomas (NECs), a poorly differentiated subtype of neuroendocrine neoplasms, are aggressive and have a poor prognosis. Colorectal neuroendocrine carcinomas (CRC-NECs) are observed in about 0.6% of all patients with CRC. Interestingly, patients with CRC-NECs show higher frequencies of BRAF mutation than typical CRC. BRAF V600E mutation-positive CRC-NECs were shown to be sensitive to BRAF inhibitors and now are treated by BRAF inhibitors. Similar to the other BRAF V600E mutated cancers, resistances against BRAF inhibitors have been observed, but the resistance mechanisms are still unclear. In this study, we established BRAF V600E mutated CRC-NEC cell line directly from surgical specimens and experimentally obtained BRAF inhibitor dabrafenib resistant cell lines. The resistant cells are revealed to express at least three types of BRAF splicing variants harboring V600E-mutation, and contribute to RAF/MEK/ERK pathway activation. In these cells, MEK and ERK inhibitors but not dabrafenib significantly suppressed cell growth and survival. Thus, in BRAF V600E mutation-positive CRC-NECs, BRAF splicing variants activate the RAF/MEK/ERK pathway and contribute to acquire BRAF inhibitor resistance. Hence, MEK or ERK are potential therapeutic targets to overcome BRAF resistance.

Newt Hoxa13 has an essential and predominant role in digit formation during development and regeneration.

The 5'Hox genes play crucial roles in limb development and specify regions in the proximal-distal axis of limbs. However, there is no direct genetic evidence that Hox genes are essential for limb development in non-mammalian tetrapods or for limb regeneration. Here, we produced single to quadruple Hox13 paralog mutants using the CRISPR/Cas9 system in newts (Pleurodeles waltl), which have strong regenerative capacities, and also produced germline mutants. We show that Hox13 genes are essential for digit formation in development, as in mice. In addition, Hoxa13 has a predominant role in digit formation, unlike in mice. The predominance is probably due to the restricted expression pattern of Hoxd13 in limb buds and the strong dependence of Hoxd13 expression on Hoxa13. Finally, we demonstrate that Hox13 genes are also necessary for digit formation in limb regeneration. Our findings reveal that the general function of Hox13 genes is conserved between limb development and regeneration, and across taxa. The predominance of Hoxa13 function both in newt limbs and fish fins, but not in mouse limbs, suggests a potential contribution of Hoxa13 function in fin-to-limb transition.

Gilteritinib overcomes lorlatinib resistance in ALK-rearranged cancer.

ALK gene rearrangement was observed in 3%-5% of non-small cell lung cancer patients, and multiple ALK-tyrosine kinase inhibitors (TKIs) have been sequentially used. Multiple ALK-TKI resistance mutations have been identified from the patients, and several compound mutations, such as I1171N + F1174I or I1171N + L1198H are resistant to all the approved ALK-TKIs. In this study, we found that gilteritinib has an inhibitory effect on ALK-TKI-resistant single mutants and I1171N compound mutants in vitro and in vivo. Surprisingly, EML4-ALK I1171N + F1174I compound mutant-expressing tumors were not completely shrunk but regrew within a short period of time after alectinib or lorlatinib treatment. However, the relapsed tumor was markedly shrunk after switching to the gilteritinib in vivo model. In addition, gilteritinib was effective against NTRK-rearranged cancers including entrectinib-resistant NTRK1 G667C-mutant and ROS1 fusion-positive cancer.

Membrane-Deformation Ability of ANKHD1 Is Involved in the Early Endosome Enlargement.

Ankyrin-repeat domains (ARDs) are conserved in large numbers of proteins. ARDs are composed of various numbers of ankyrin repeats (ANKs). ARDs often adopt curved structures reminiscent of the Bin-Amphiphysin-Rvs (BAR) domain, which is the dimeric scaffold for membrane tubulation. BAR domains sometimes have amphipathic helices for membrane tubulation and vesiculation. However, it is unclear whether ARD-containing proteins exhibit similar membrane deformation properties. We found that the ARD of ANK and KH domain-containing protein 1 (ANKHD1) dimerize and deform membranes into tubules and vesicles. Among 25 ANKs of ANKHD1, the first 15 ANKs can form a dimer and the latter 10 ANKs enable membrane tubulation and vesiculation through an adjacent amphipathic helix and a predicted curved structure with a positively charged surface, analogous to BAR domains. Knockdown and localization of ANKHD1 suggested its involvement in the negative regulation of early endosome enlargement owing to its membrane vesiculation.

High-throughput SNP detection using nano-scale engineered biomagnetite.

A semi-automated system for the large-scale detection of single nucleotide polymorphisms (SNPs) has been developed based on allele-specific oligonucleotide hybridization and thermal dissociation curve analysis using nano-scale engineered biomagnetite (bacterial magnetic particles; BacMPs). For reliable detection in large numbers of samples, several conditions for the capture of target DNA on nano-sized BacMPs and the denaturation of double-stranded DNA were optimized. The most efficient target DNA capture was observed using short PCR amplicons (69 bp). Captured DNAs were denatured using 50 mM NaOH. With these optimizations, large-scale SNP detection was performed on 822 samples of the transforming growth factor (TGF)-beta1 gene, which is rich in both GC content and repetitive sequences. High reliability for the semi-automated BacMP-based SNP detection system was confirmed following comparison to traditional sequencing-based methods.

Determination of microsatellite repeats in the human thyroid peroxidase (TPOX) gene using an automated gene analysis system with nanoscale engineered biomagnetite.

The number of repeat in the microsatellite region (AATG)(5-14) of the human thyroid peroxidase gene (TOPX) was determined using an automated DNA analysis system with nano-scale engineered biomagnetite. Thermal melting curve analysis of DNA duplexes on biomagnetite indicated that shorter repeat sequences (less than 9 repeats) were easily discriminated. However, it was difficult to determine the number of repeats at more than nine. In order to improve the selectivity of this method for the longer repeats, a "double probe hybridization assay" was performed in which an intermediate probe was used to replace a target repeat sequence having more than 9 repeats with a shorter sequence possessing less than 9 repeats. Thermal probe melting curve analyses and Tm determination confirmed that the target with 10 repeats was converted to 5 repeats, 11 repeats converted to 4 and 12 to 3, respectively. Furthermore, rapid determination of repeat numbers was possible by measuring fluorescence intensities obtained by probe dissociation at 56 and 66 degrees C, and 40, 60 and 80 degrees C for signal normalization.

Detection of epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC) using a fully automated system with a nano-scale engineered biomagnetite.

A fully automated system using nano-scale engineered biomagnetite was developed to detect mutations in the epidermal growth factor receptor (EGFR) gene in non-small cell lung cancer (NSCLC). Bacterial magnetic particles (BacMPs) were isolated from the magnetic bacterium Magnetospirillum magneticum AMB-1 and conjugated to streptavidin. Biotin-labeled target PCR products were then captured with the BacMPs, hybridized with the detection probe and detected by fluorescence signaling. The process was performed using a newly designed automated processor equipped with an XYZ mobile arm containing a 96-way automated pipetter, reagent dispenser and fluorescence detector. Two types of somatic mutations (in-frame deletions and point substitutions) in the EGFR gene were successfully identified within 3.5h using this system, suggesting that this system could be used in clinical tests of EGFR gene mutations in lung cancer, and potentially other cancer, patients. Additionally, a very low mutation rate could be detected in these samples.

Capture and release of DNA using aminosilane-modified bacterial magnetic particles for automated detection system of single nucleotide polymorphisms.

Bacterial magnetic particles (BMPs) were modified with 3-[2-(2-aminoethylamino)-ethylamino]-propyltrimethoxysilane (AEEA) to produce a dense amine surface. Modification of BMPs in a toluene solution resulted in an increased amine yield, and approximately 11.3 x 10(4) surface amines were detected on a single particle. The modified BMPs were capable of efficient electrostatic capture of DNA. The maximum amount of DNA captured on 10 microg of aminosilane-modified BMPs was 600 ng. A 10 mM phosphate buffer effectively released the captured DNA. This efficiency was dramatically enhanced by incubation at 80 degrees C and DNA recovery from aminosilane-modified BMPs approached 95%. DNA extraction from whole blood using these modified BMPs, followed by PCR, was successfully performed. Furthermore, automated single nucleotide polymorphism (SNP) detection of the aldehyde dehydrogenase 2 (ALDH2) was demonstrated.

Single nucleotide polymorphism detection in aldehyde dehydrogenase 2 (ALDH2) gene using bacterial magnetic particles based on dissociation curve analysis.

Single nucleotide polymorphism (SNP) detection for aldehyde dehydrogenase 2 (ALDH2) gene based on DNA thermal dissociation curve analysis was successfully demonstrated using an automated system with bacterial magnetic particles (BMPs) by developing a new method for avoiding light scattering caused by nanometer-size particles when using commercially available fluorescent dyes such as FITC, Cy3, and Cy5 as labeling chromophores. Biotin-labeled PCR products in ALDH2, two allele-specific probes (Cy3-labeled detection probe for ALDH2*1 and Cy5-labeled detection probe for ALDH2*2), streptavidin-immobilized BMPs (SA-BMPs) were simultaneously mixed. The mixture was denatured at 70 degrees C for 3 min, cooled slowly to 25 degrees C, and incubated for 10 min, allowing the DNA duplex to form between Cy3- or Cy5-labeled detection probes and biotin-labeled PCR products on SA-BMPs. Then duplex DNA-BMP complex was heated to 58 degrees C, a temperature determined by dissociation curve analysis and a dissociated single-base mismatched detection probe was removed at the same temperature under precise control. Furthermore, fluorescence signal from the detection probe was liberated into the supernatant from completely matched duplex DNA-BMP complex by heating to 80 degrees C and measured. In the homozygote target DNA (ALDH2*1/*1 and ALDH2*2/*2), the fluorescence signals from single-base mismatched were decreased to background level, indicating that mismatched hybridization was efficiently removed by the washing process. In the heterozygote target DNA (ALDH2*1/*2), each fluorescence signals was at a similar level. Therefore, three genotypes of SNP in ALDH2 gene were detected using the automated detection system with BMPs.

Development and evaluation of an automated workstation for single nucleotide polymorphism discrimination using bacterial magnetic particles.

We designed an automated workstation for magnetic particle-based single nucleotide polymorphism (SNP) discrimination of ALDH genotypes. Bacterial magnetic particles (BMPs) extracted from Magnetospirillum magneticum AMB-1 were used as DNA carriers. The principle for SNP discrimination in this study was based on fluorescence resonance energy transfer (FRET) between FITC (donor) and POPO-3 (acceptor) bound to double-stranded DNA. The workstation is equipped with a 96-way automated pipetter which collects and dispenses fluids as it moves in x- and z-directions. The platform contains a disposable tip rack station, a reagent vessel serving as a stock for POPO-3 and FITC-labeled probes and a reaction station for a 96-well microtiter plate. BMPs were collected by attaching a neodymium iron boron sintered (Nd-Fe-B) magnet on the bottom of the microtiter plate. This system permits the simultaneous heating and magnetic separation of 96 samples per assay. The genotypes ALDH2*1 and ALDH2*2 were discriminated by calculating the relative fluorescence intensities on BMPs.

Single-nucleotide polymorphism analysis using fluorescence resonance energy transfer between DNA-labeling fluorophore, fluorescein isothiocyanate, and DNA intercalator, POPO-3, on bacterial magnetic particles.

To develop an analytical system for single-nucleotide polymorphisms (SNPs), the fluorescence resonance energy transfer (FRET) technique was employed on a bacterial magnetic particle (BMP) surface. A combination of fluorescein isothiocyanate (FITC; excitation 490 nm/emission 520 nm) labeled at the 5' end of DNA and an intercalating compound (POPO-3, excitation 534 nm/emission 570 nm) was used to avoid the interference from light scattering caused by nanoparticles. After hybridization between target DNA immobilized onto BMPs and FITC-labeled probes, fluorescence from POPO-3, which was excited by the energy from the FITC, was detected. The major homozygous (ALDH2*1), heterozygous (ALDH2*1/*2), and minor homozygous (ALDH2*2) genotypes in the blood samples were discriminated by this method. The assay described herein allows for a simple and rapid SNP analysis using a fully automated system.

Fully automated DNA extraction from blood using magnetic particles modified with a hyperbranched polyamidoamine dendrimer.

Bacterial and artificial magnetic particles were modified using a polyamidoamine (PAMAM) dendrimer and outer shell amines determined. Bacterial magnetic particles were the most consistently modified. Transmission electron microscopic (TEM) analysis showed that the artificial magnetic particles were structurally damaged by the modification process including sonication. Furthermore, laser particle analysis of the magnetite also revealed damage. Small quantities of dendrimer-modified bacterial magnetic particles were used to extract DNA from blood. The efficiency of DNA recovery was consistently about 30 ng of DNA using 2-10 microg of dendrimer-modified bacterial magnetite. This technique was fully automated using newly developed liquid handling robots and bacterial magnetic particles.