Fast and artifact-free circular dichroism measurement of solid-state structural changes.

A novel chiroptical spectrophotometer, Multichannel (MC)-circular dichroism (CD), which does not require a wavelength scan and provides artifact-free CD spectra in seconds, was developed by solving the incompatibility problem between data acquisition and modulation timescales. The design and instrumentation are compared with the Universal Chiroptical Spectrophotometer (UCS)-1, which can measure artifact-free CD spectra in the solid state. Enantiomeric single crystals composed of achiral components, α-Ni(H2 O)6 ・SO4 , and achiral films with substantial macroscopic anisotropies were measured on both MC-CD and UCS-1 and compared.

Machine learning and multiple linear regression models can predict ascorbic acid and polyphenol contents, and antioxidant activity in strawberries.

BACKGROUND: Strawberry is a rich source of antioxidants, including ascorbic acid (ASA) and polyphenols, which have numerous health benefits. Antioxidant content and activity are often determined manually using laboratory equipment, which is destructive and time-consuming. This study constructs a prediction model for antioxidant compounds utilizing machine learning (ML) and multiple linear regression based on environmental, plant growth and agronomic fruit quality-related parameters as well as antioxidant levels. These were studied in three farms at two-week intervals during two years of cultivation. RESULTS: During the ML model screening, artificial neural network (ANN)-boosted models displayed a moderate coefficient of determination (R2) at 0.68-0.78 and relative root mean square error (RRMSE) at 3.8-4.8% in polyphenols and total ASA levels, as well as a high R2 of 0.96 and low RRMSE at <3.0% in antioxidant activity. Additionally, we developed variable selection models regarding the antioxidant activity, and variables two and five (environmental parameters and leaf length, respectively) with high accuracy were selected. The linear regression analysis between the actual and predicted data of antioxidants in the ANN-boosted models revealed high fitness with all parameters in almost all training, validation and test sets. Furthermore, environmental parameters are essential in developing such reliable models. CONCLUSION: We conclude that ANN-boosted, stepwise and double-Lasso regression models can predict antioxidant compounds with enhanced accuracy, and the relevant parameters can be easily acquired on-site without the need for any specific equipment. © 2024 Society of Chemical Industry.

Synthesis and properties of fully-modified 4'-selenoRNA, an endonuclease-resistant RNA analog.

A large number of chemically modified oligonucleotides (ONs) have been developed for RNA-based technologies. In most modified RNAs, the characteristic 2'-hydroxyl (2'-OH) groups are removed to enhance both nuclease resistance and hybridization ability. However, the importance of the 2'-OH group in RNA structure and function is well known. Here, we report the synthesis and properties of 4'-selenoRNA in which all four nucleoside units retain the 2'-OH groups but contain a selenium atom instead of an oxygen atom at the 4'-position of the furanose ring. 4'-SelenoRNA has enhanced ability to form duplexes with RNA, and high endonuclease resistance despite the presence of the 2'-OH groups. X-ray crystallography analysis showed that the 4'-selenoRNA duplex adopts an A-conformation, similar to natural RNA, although one 4'-selenocytidine residue has unusual South-type sugar puckering. Furthermore, preliminary studies using 4'-seleno-modified siRNAs suggest that 4'-selenoRNA may be applicable to RNA interference technology. Collectively, our results raise the possibility of a new class of modified RNA in which 2'-OH groups do not need to be masked.

Are tri-pronuclear embryos that show two normal-sized pronuclei and additional smaller pronuclei useful for embryo transfer?

Purpose: This study aimed to analyze whether tripronuclear (3PN) zygotes, with two normal-sized PNs and an additional smaller PN (2.1PN), can be used for embryo transfer. Methods: A retrospective embryo cohort study was conducted on 695 patients who underwent intracytoplasmic sperm injection treatment. Blastocyst formation rates were compared between 2.1PN and 2PN zygotes and PGT-A analysis was performed on 15 blastocysts derived from 2.1PN zygotes. Results: Blastocyst formation rates were comparable between 2.1PN (43.8%) and 2PN zygotes (54.8%; p = 0.212). The rates of blastocysts with good morphology derived from 2.1 PN and 2PN zygotes were 18.8% and 25.5%, respectively. No significant differences were detected (p = 0.383). All of the analyzed blastocysts were diploid; however, 13 of these were found to be aneuploid, with a further two being mosaic. Conclusion: Our results suggest that 2.1PN embryos can reach blastocyst stage. These blastocysts were diploid, however, predominantly aneuploid, and therefore could not be used for embryo transfer.

Construction of Ultrathin Layer-by-Layer Films of Oligothiophene Derivatives on an Electrode.

Oligothiophene derivatives, which are known as p-type materials, have been synthesized, and their ultrathin layer-by-layer films have been constructed on an electrode using a simple and convenient dipping method. The stepwise deposition behavior of quaterthiophene and sexithiophene derivatives on the electrode via hydrogen bonding was monitored by electronic spectra measurement, and the constructed films were evaluated by X-ray photoelectron spectroscopy, grazing-incidence small-angle X-ray scattering, and cyclic voltammetry. It has been clarified that the constructed layer-by-layer films were electroactive and photoelectroactive.

Microheater-integrated zinc oxide nanowire microfluidic device for hybridization-based detection of target single-stranded DNA.

Detection of cell-free DNA (cfDNA) has an impact on DNA analysis in liquid biopsies. However, current strategies to detect cfDNA have limitations that should be overcome, such as having low sensitivity and requiring much time and a specialized instrument. Thus, non-invasive and rapid detection tools are needed for disease prevention and early-stage treatment. Here we developed a device having a microheater integrated with zinc oxide nanowires (microheater-ZnO-NWs) to detect target single-stranded DNAs (ssDNAs) based on DNA probe hybridization. We confirmed experimentally that our device realizedin-situannealed DNA probes by which we subsequently detected target ssDNAs. We envision that this device can be utilized for fundamental studies related to nanobiodevice-based DNA detection.

How Viscous Is the Solidlike Structure at the Interface of Ionic Liquids? A Study Using Total Internal Reflection Fluorescence Spectroscopy with a Fluorescent Molecular Probe Sensitive to High Viscosity.

Aiming at the evaluation of the viscosity of the interfacial solidlike structure of ionic liquids (ILs), we performed total internal reflection fluorescence (TIRF) spectroscopy for N,N-diethyl-N'-phenyl-rhodamine (Ph-DER), a fluorescent probe that is sensitive to viscosity in a high-viscosity range. TIRF spectra at the glass interface of trioctylmethylammonium bis(nonafluorobutanesulfonyl)amide (TOMAC4C4N), a hydrophobic IL, showed that the fluorescence intensity of Ph-DER increases with the decrease of the evanescence penetration depth, suggesting that there exists a high-viscosity region at the interface. In contrast, glycerol, which is a molecular liquid with a bulk viscosity similar to that of TOMAC4C4N, did not show such a fluorescence increase, supporting that the formation of a highly viscous solidlike structure at the interface is intrinsic to ILs. A model analysis suggested that the high viscous region at the glass interface of TOMAC4C4N is at least twice thicker than the ionic multilayers at the air interface, implying that the solid substrate enhances the ordering of the interfacial structure of ILs. The viscosity at the glass interface of TOMAC4C4N was found to be at least 40 times higher than that of the liquid bulk.

Photoelectric Conversion System Composed of Gene-Recombined Photosystem I and Platinum Nanoparticle Nanosheet.

Photosynthesis is one of the most vital processes in nature, which consists of two main photoreaction centers called photosystem I and photosystem II. The high quantum yield of photosystem I (PSI) makes it attractive for bioelectronic applications. However, the native PSI (N-PSI) loses its robust photochemical properties once fabricated into devices. This property degradation results from the difficulty in controlling the orientation of PSI. With the optimal orientation of PSI, photoexcited electrons can easily reach the electrode, yielding good photoelectric conversion efficiency. We developed a novel photoelectrode by integrating a newly designed gene-recombined PSI (G-PSI) with platinum nanoparticles (PtNPs) on substrates using a simple stacking method, which can control the orientation of PSI on the electrode. The target orientation of the attached G-PSI toward the substrate was confirmed by the absorption spectra of polarized light. An approximately 2-fold increase in the internal quantum yield (IQY) was observed for the G-PSI-attached electrode under 680 nm irradiation compared with that of the N-PSI-modified electrode. In addition, a 4-fold enhancement of the IQY was detected for cytochrome c (Cyt c) stacking on the G-PSI because of the electrostatic interaction, suggesting that Cyt c successfully secured the electron-transfer pathway.

Decomposition of amyloid fibrils by NIR-active upconversion nanoparticles.

We demonstrate amyloid fibril (AF) decomposition induced by NIR-active upconversion nanoparticles complexed with photosensitisers. The process is triggered by upconversion, which initiates a photochemical reaction cascade that culminates in the generation of the highly reactive singlet-oxygen product 1O2 close to the amyloid superstructures, resulting in AF decomposition.

Clinical outcomes of MII oocytes with refractile bodies in patients undergoing ICSI and single frozen embryo transfer.

PURPOSE: This study aimed to analyze whether the presence of refractile bodies (RFs) negatively affects fertilization, embryo development, and/or implantation rates following intracytoplasmic sperm injection (ICSI). METHODS: This retrospective embryo cohort study involved a total of 272 patients undergoing ICSI treatment of blastocyst cryopreservation. RESULTS: In the study, no significant differences were found regarding 2PN formation rates between RF(+) (76.5%) and RF(-) oocytes (77.2%). However, the blastocyst formation rate on Day 5 in RF(+) oocytes was 45.8%, which was significantly lower than that of 52.2% in RF(-) oocytes (aOR 0.74, 95% CI 0.59-0.93, P = .011). Implantation rates were also significantly lower in RF(+) oocytes (24.2%) as compared to RF(-) oocytes (42.2%) (aOR 0.46, 95% CI 0.26-0.78, P = .005). Furthermore, the implantation rate of RF(+) oocytes (28.6%), when high-quality blastocysts were transferred, was significantly lower than that of RF(-) oocytes (46.1%) (aOR 0.50, 95% CI 0.25-0.96, P = .043). CONCLUSION: Our results suggest that oocytes with the presence of RFs have a lower potential for blastocyst development. Even when they develop into high-quality blastocysts, the chances of implantation are reduced.

GSK3ß controls the timing and pattern of the fifth spiral cleavage at the 2-4 cell stage in Lymnaea stagnalis.

Establishment of the body plan of multicellular organisms by the primary body axis determination and cell-fate specification is a key issue in biology. We have examined the mRNA localization of three Wnt pathway components gsk3ß, ß-catenin, and disheveled and investigated the effects of four selective inhibitors of these proteins on the early developmental stages of the spiral cleavage embryo of the fresh water snail Lymnaea (L.) stagnalis. mRNAs for gsk3ß and ß-catenin were distributed uniformly throughout the embryo during development whereas disheveled mRNA showed specific localization with intra- and inter-blastomere differences in concentration along the A-V axis during spiral cleavages. Remarkably, through inhibitor studies, we identified a short sensitive period from the 2- to 4-cell stage in which GSK3ß inhibition by the highly specific 1-azakenpaullone (AZ) and by LiCl induced a subsequent dramatic developmental delay and alteration of the cleavage patterns of blastomeres at the fifth cleavage (16- to 24-cell stage) resulting in exogastrulation and other abnormalities in later stages. Inhibition of ß-Catenin or Disheveled had no effect. Our inhibitor experiments establish a novel role for GSK3ß in the developmental timing and orientated cell division of the snail embryo. Further work will be needed to identify the downstream targets of the kinase.

A higher incidence of smooth endoplasmic reticulum clusters with aromatase inhibitors.

PURPOSE: This study aimed to analyze whether a regimen of aromatase inhibitor (AI) could reduce the occurrence of smooth endoplasmic reticulum clusters (sERCs) in oocytes. METHODS: The AI and the clomiphene citrate (CC) regimens were compared, regarding the sERC (+) rates and the serum estradiol and progesterone levels on the date of hCG administration, and the duration of AI, CC, and hMG administration. RESULTS: The occurrence of sERCs in oocytes from patients treated with AI was significantly higher than that in oocytes from those treated with CC. Both the serum estradiol and progesterone levels were found to be significantly higher in sERC (+) than in sERC (-) cycles. With regard to the CC cycles, no significant differences were detected. The duration of AI and hMG administration was longer for sERC (+) than for sERC (-) cycles. CONCLUSION: As AI did not reduce the occurrence of sERCs, the elevation of estradiol may not be the cause of sERC occurrence but a consequence. Considering the higher levels of progesterone and longer duration of hMG in sERC (+) cycles, the negative effects of premature luteinization, which frequently occur with the AI protocol, should be investigated further.

Tube feeding decreases pneumonia rate in patients with severe dementia: comparison between pre- and post-intervention.

BACKGROUND: It is widely supposed that there is no benefit, including extended survival and decreased rate of pneumonia, in patients with severe dementia receiving enteral tube feeding (TF). However, there have been few studies comparing the frequency of pneumonia before and after TF in severe dementia. METHODS: Nine psychiatric hospitals in Okayama Prefecture participated in this retrospective survey. All inpatients fulfilling the entry criteria were evaluated. All subjects suffered from difficulty in oral intake. Attending physicians thought that the patients could not live without long-term artificial nutrition, and they decided whether or not to make use of long-term artificial nutrition from January 1, 2014 to December 31, 2014. RESULTS: We evaluated 58 patients including 46 with TF and 12 without. The mean age of all patients was 79.6 ± 9.0 years old. Patients with probable Alzheimer's disease (n = 38) formed the biggest group, and those with vascular dementia the second (n = 14). Median survival times were 23 months among patients with TF and two months among patients without TF. The start of TF decreased the frequency of pneumonia and the use of intravenous antibiotics. CONCLUSIONS: TF decreased pneumonia and antibiotic use, even in patients with severe dementia. The results of this study do not necessarily indicate that we should administer TF to patients with severe dementia. We should consider the quality of life of patients carefully before deciding the use or disuse of TF for patients with severe dementia.

[Accelerating drug discovery process with genome editing technology from Tokushima].

In a series of drug development processes from basic research to non-clinical and clinical trials, genome editing technologies have been making innovative breakthroughs. Genome editing using CRISPR/Cas9 system, which was awarded the Novel Prize in Chemistry in 2020, has greatly streamlined the production of genetically modified mice and cells, which have been used in a variety of drug discovery research and non-clinical trials. Setsuro Tech Inc. (Setsurotech) established in 2017 is a biotech startup originated in Tokushima University. In this paper, we will briefly review genome editing technology using CRISPR/Cas9 system, and then introduce our company, our fundamental technologies; GEEP method (Genome Editing by Electroporation of Cas9 Protein) developed by Takemoto et al., and VIKING method (Versatile NHEJ-based Knock-in using Genome Editing) established by Sawatsubashi et al. Also, we will introduce our contribution to the field of drug discovery research and industrial application of genome editing technology.

Inference of transcriptome signatures of Escherichia coli in long-term stationary phase.

"Non-growing" is a dominant life form of microorganisms in nature, where available nutrients and resources are limited. In laboratory culture systems, Escherichia coli can survive for years under starvation, denoted as long-term stationary phase, where a small fraction of cells manages to survive by recycling resources released from nonviable cells. Although the physiology by which viable cells in long-term stationary phase adapt to prolonged starvation is of great interest, their genome-wide response has not been fully understood. In this study, we analyzed transcriptional profiles of cells exposed to the supernatant of 30-day long-term stationary phase culture and found that their transcriptome profiles displayed several similar responses to those of cells in the 16-h short-term stationary phase. Nevertheless, our results revealed that cells in long-term stationary phase supernatant exhibit higher expressions of stress-response genes such as phage shock proteins (psp), and lower expressions of growth-related genes such as ribosomal proteins than those in the short-term stationary phase. We confirmed that the mutant lacking the psp operon showed lower survival and growth rate in the long-term stationary phase culture. This study identified transcriptional responses for stress-resistant physiology in the long-term stationary phase environment.

Mutation detection of urinary cell-free DNA via catch-and-release isolation on nanowires for liquid biopsy.

Cell-free DNA (cfDNA) and extracellular vesicles (EVs) are molecular biomarkers in liquid biopsies that can be applied for cancer detection, which are known to carry information on the necessary conditions for oncogenesis and cancer cell-specific activities after oncogenesis, respectively. Analyses for both cfDNA and EVs from the same body fluid can provide insights into screening and identifying the molecular subtypes of cancer; however, a major bottleneck is the lack of efficient and standardized techniques for the isolation of cfDNA and EVs from clinical specimens. Here, we achieved catch-and-release isolation by hydrogen bond-mediated binding of cfDNA in urine to zinc oxide (ZnO) nanowires, which also capture EVs by surface charge, and subsequently we identified genetic mutations in urinary cfDNA. The binding strength of hydrogen bonds between single-crystal ZnO nanowires and DNA was found to be equal to or larger than that of conventional hydrophobic interactions, suggesting the possibility of isolating trace amounts of cfDNA. Our results demonstrated that nanowire-based cancer screening assay can screen cancer and can identify the molecular subtypes of cancer in urine from brain tumor patients through EV analysis and cfDNA mutation analysis. We anticipate our method to be a starting point for more sophisticated diagnostic models of cancer screening and identification.

Force plate analysis of ground reaction forces in relation to gait velocity of healthy Beagles.

OBJECTIVE: To evaluate changes in ground reaction forces (GRFs) in relation to gait velocity using 2 force plates (FPs) for healthy Beagles. ANIMALS: 18 healthy Beagles were included (body weight, 10.45 ± 1.28 kg; age, 26 ± 11 months). PROCEDURES: Ten GRF parameters were measured at three gait velocities (walk, 0.9 to 1.2 m/s; trot 1, 1.6 to 2.0 m/s; and trot 2, 2.1 to 2.5 m/s): peak lateral force (PLF), peak medial force (PMF), lateral impulse (LI), medial impulse (MI), peak propulsive force (PPF), peak braking force (PBF), propulsive impulse (PI), braking impulse (BI), peak vertical force (PVF), and vertical impulse (VI). RESULTS: As velocity increased, the PVF of all limbs increased, the VI of all limbs decreased, and the PPF of the forelimbs increased. At all velocities, PBF and BI were significantly higher than the PPF and PI in forelimbs; however, PBF and BI were significantly lower than the PPF and PI in hindlimbs. There were no significant differences in the PLF, PMF, LI, and MI of the forelimbs and hindlimbs among all velocities. The PLF was significantly higher than the PMF of forelimbs during trot 1 and trot 2. CLINICAL RELEVANCE: These results may be useful when comparing healthy Beagles with diseased ones when premorbid data are not available. Because the forelimbs are mainly responsible for the braking force, it is suggested that weight bearing is more stable in the forelimbs than in the hindlimbs, which are mainly responsible for the propulsive force, and that a greater force is generated laterally than medially during trot.

Efficacy of cancer-specific anti-podoplanin CAR-T cells and oncolytic herpes virus G47Δ combination therapy against glioblastoma.

Glioblastoma is a devastating malignant brain tumor with a poor prognosis despite standard therapy. Podoplanin (PDPN), a type I transmembrane mucin-like glycoprotein that is overexpressed in various cancers, is a potential therapeutic target for the treatment of glioblastoma. We previously reported the efficacy of chimeric antigen receptor (CAR)-T cells using an anti-pan-PDPN monoclonal antibody (mAb; NZ-1)-based third-generation CAR in a xenograft mouse model. However, NZ-1 also reacted with PDPN-expressing normal cells, such as lymphatic endothelial cells, pulmonary alveolar type I cells, and podocytes. To overcome possible on-target-off-tumor effects, we produced a cancer-specific mAb (CasMab, LpMab-2)-based CAR. LpMab-2 (Lp2) reacted with PDPN-expressing cancer cells but not with normal cells. In this study, Lp2-CAR-transduced T cells (Lp2-CAR-T) specifically targeted PDPN-expressing glioma cells while sparing the PDPN-expressing normal cells. Lp2-CAR-T also killed patient-derived glioma stem cells, demonstrating its clinical potential against glioblastoma. Systemic injection of Lp2-CAR-T cells inhibited the growth of a subcutaneous glioma xenograft model in immunodeficient mice. Combination therapy with Lp2-CAR-T and oncolytic virus G47Δ, a third-generation recombinant herpes simplex virus (HSV)-1, further inhibited the tumor growth and improved survival. These findings indicate that the combination therapy of Lp2-CAR-T cells and G47Δ may be a promising approach to treat glioblastoma.

Functional analysis of guinea pig ß1-adrenoceptor.

Although similarity of pharmacological responses to certain stimuli between guinea pigs and humans has been reported, this has been poorly defined by a molecular biological approach. In this study, we cloned the gene of guinea pig ?1-adrenoceptor (ADRB1). The deduced amino acid sequence of guinea pig ADRB1 (467-aa) showed 91% and 92% identity with the human and rat ADRB1 sequences, respectively. Using HEK293T cells expressing guinea pig, human and rat ADRB1s independently, we elucidated the functional characteristics of each ADRB1. The ligand-binding profiles and the concentration-response relationships for isoprenaline-induced cyclic adenosine monophosphate (cAMP) production were similar among the three ADRB1s. Isoprenaline also induced phosphorylation of extracellular-signal related kinases (ERK) through ADRB1s in a concentration-dependent manner. The minimum effective concentration of isoprenaline for phosphorylation of ERK, through guinea pig ADRB1 was the same as through human ADRB1, but markedly lower than that of through rat ADRB1. ERK phosphorylation through guinea pig ADRB1 was sensitive to pertussis toxin, a dominant-negative ras and PD98059, indicating that a G(i)-mediated pathway is involved in the ADRB1/ERK signaling loop. These results suggest that the G(i)-coupling efficacy of guinea pig and human ADRB1s may be higher than that of rat ADRB1.

Oxide nanowire microfluidics addressing previously-unattainable analytical methods for biomolecules towards liquid biopsy.

Nanowire microfluidics using a combination of self-assembly and nanofabrication technologies is expected to be applied to various fields due to its unique properties. We have been working on the fabrication of nanowire microfluidic devices and the development of analytical methods for biomolecules using the unique phenomena generated by the devices. The results of our research are not just limited to the development of nanospace control with "targeted dimensions" in "targeted arrangements" with "targeted materials/surfaces" in "targeted spatial locations/structures" in microfluidic channels, but also cover a wide range of analytical methods for biomolecules (extraction, separation/isolation, and detection) that are impossible to achieve with conventional technologies. Specifically, we are working on the extraction technology "the cancer-related microRNA extraction method in urine," the separation technology "the ultrafast and non-equilibrium separation method for biomolecules," and the detection technology "the highly sensitive electrical measurement method." These research studies are not just limited to the development of biomolecule analysis technology using nanotechnology, but are also opening up a new academic field in analytical chemistry that may lead to the discovery of new pretreatment, separation, and detection principles.