NMR Line Shape Analysis of a Multi-state Ligand Binding Mechanism in Chitosanase

Chitosan interaction with chitosanase was examined through analysis of spectral line shapes in the NMR HSQC titration experiments. We established that the substrate, chitosan hexamer, binds to the enzyme through the three-state induced-fit mechanism with fast formation of the encounter complex followed by slow isomerization of the bound-state into the final conformation. Mapping of the chemical shift perturbations in two sequential steps of the mechanism highlighted involvement of the substrate-binding subsites and the hinge region in the binding reaction. Equilibrium parameters of the three-state model agreed with the overall thermodynamic dissociation constant determined by ITC. This study presented the first kinetic evidence of the induced-fit mechanism in the glycoside hydrolases

production of chitooligosaccharides from Rhizopus oligosporus NRRL2710 cells by chitosanase digestion

The intact cells of Rhizopus oligosporus NRRL2710, whose cell walls are abundant source of N-acetylglu- cosamine (GlcNAc) and glucosamine (GlcN), were digested with three chitinolytic enzymes, a GH-46 chitosanase from Streptomyces sp. N174 (CsnN174), a chitinase from Pyrococcus furiosus, and a chitinase from Trichoderma viride, respectively. Solubilization of the intact cells by CsnN174 was found to be the most efficient from solid state CP/MAS 13C NMR spectroscopy. Chitosanase products from Rhizopus cells were purified by cation exchange chromatography on CM-Sephadex C-25 and gel-filtration on Cellulofine Gcl-25 m. NMR and MALDI-TOF-MS analyses of the purified products revealed that GlcN–GlcNAc, (GlcN)2–GlcNAc, and (GlcN)2 were produced by the enzymatic digestion of the intact cells. The chitosan- ase digestion of Rhizopus cells was found to be an excellent system for the conversion of fungal biomass without any environmental impact

Functional visualization of NK Cell-mediated killing of metastatic single tumor cells

ナチュラルキラー（NK）細胞による転移がん細胞殺傷の可視化 --NK細胞とがん細胞の肺毛細血管上での戦いを実況中継する--. 京都大学プレスリリース. 2022-02-07.Natural killer (NK) cells lyse invading tumor cells to limit metastatic growth in the lung, but how some cancers evade this host protective mechanism to establish a growing lesion is unknown. Here we have combined ultra-sensitive bioluminescence imaging with intravital two-photon microscopy involving genetically-encoded biosensors to examine this question. NK cells eliminated disseminated tumor cells from the lung within 24 hrs of arrival, but not thereafter. Intravital dynamic imaging revealed that 50% of NK-tumor cell encounters lead to tumor cell death in the first 4 hrs after tumor cell arrival, but after 24 hrs of arrival, nearly 100% of the interactions result in the survival of the tumor cell. During this 24 hrs period, the probability of ERK activation in NK cells upon encountering the tumor cells was decreased from 68% to 8%, which correlated with the loss of the activating ligand CD155/PVR/Necl5 from the tumor cell surface. Thus, by quantitatively visualizing the NK-tumor cell interaction at the early stage of metastasis, we have revealed the crucial parameters of NK cell immune surveillance in the lung

Brain structure alterations in girls with central precocious puberty

PurposeCentral precocious puberty (CPP) is puberty that occurs at an unusually early age with several negative psychological outcomes. There is a paucity of data on the morphological characteristics of the brain in CPP. This study aimed to determine the structural differences in the brain of patients with CPP.MethodsWe performed voxel- and surface-based morphometric analyses of 1.5 T T1-weighted brain images scanned from 15 girls with CPP and 13 age-matched non-CPP controls (NC). All patients with CPP were diagnosed by gonadotropin-releasing hormone (GnRH) stimulation test. The magnetic resonance imaging (MRI) data were evaluated using Levene’s test for equality of variances and a two-tailed unpaired t-test for equality of means. False discovery rate correction for multiple comparisons was applied using the Benjamini–Hochberg procedure.ResultsMorphometric analyses of the brain scans identified 33 candidate measurements. Subsequently, increased thickness of the right precuneus was identified in the patients with CPP using general linear models and visualizations of cortical thickness with a t-statistical map and a random field theory map.ConclusionThe brain scans of the patients with CPP showed specific morphological differences to those of the control. The features of brain morphology in CPP identified in this study could contribute to further understanding the association between CPP and detrimental psychological outcomes

The Human Anatomic Gene Expression Library (H-ANGEL), the H-Inv integrative display of human gene expression across disparate technologies and platforms

The Human Anatomic Gene Expression Library (H-ANGEL) is a resource for information concerning the anatomical distribution and expression of human gene transcripts. The tool contains protein expression data from multiple platforms that has been associated with both manually annotated full-length cDNAs from H-InvDB and RefSeq sequences. Of the H-Inv predicted genes, 18 897 have associated expression data generated by at least one platform. H-ANGEL utilizes categorized mRNA expression data from both publicly available and proprietary sources. It incorporates data generated by three types of methods from seven different platforms. The data are provided to the user in the form of a web-based viewer with numerous query options. H-ANGEL is updated with each new release of cDNA and genome sequence build. In future editions, we will incorporate the capability for expression data updates from existing and new platforms. H-ANGEL is accessible at http://www.jbirc.aist.go.jp/hinv/h-angel/

Complete Genetic Correction of iPS Cells From Duchenne Muscular Dystrophy

Human artificial chromosome (HAC) has several advantages as a gene therapy vector, including stable episomal maintenance that avoids insertional mutations and the ability to carry large gene inserts including the regulatory elements. Induced pluripotent stem (iPS) cells have great potential for gene therapy, as such cells can be generated from the individual's own tissues, and when reintroduced can contribute to the specialized function of any tissue. As a proof of concept, we show herein the complete correction of a genetic deficiency in iPS cells derived from Duchenne muscular dystrophy (DMD) model (mdx) mice and a human DMD patient using a HAC with a complete genomic dystrophin sequence (DYS-HAC). Deletion or mutation of dystrophin in iPS cells was corrected by transferring the DYS-HAC via microcell-mediated chromosome transfer (MMCT). DMD patient- and mdx-specific iPS cells with the DYS-HAC gave rise to differentiation of three germ layers in the teratoma, and human dystrophin expression was detected in muscle-like tissues. Furthermore, chimeric mice from mdx-iPS (DYS-HAC) cells were produced and DYS-HAC was detected in all tissues examined, with tissue-specific expression of dystrophin. Therefore, the combination of patient-specific iPS cells and HAC-containing defective genes represents a powerful tool for gene and cell therapies

ジョセイカイゴシャ ニ オケル ヨウツウ ノ ジッタイ ト カンレンヨウイン ニ カンスル オウダンケンキュウ

本研究は,横断研究により,女性介護者における腰痛との関連要因を明らかにすることを目的とした。4カ所の特別養護老人ホームに勤務する女性介護職員88人を対象として,腰痛の有無,経験年数,Body Mass Index(BMI),握力,長座体前屈,指床間距離(Finger-Floor Distance : FFD),抑うつ自己評価スコア(the Center for Epidemiologic Study Depression Scale : CES-D),SF-8による身体的健康度(Physical Health Score : PHS)と精神的健康度(Mental Health Score : MHS),疲労の自覚症状を調査した。腰痛の有無を目的変数とし,その他の変数を説明変数とした多重ロジスティック回帰分析を行った。49人(55.7%)が,慢性的な腰痛を訴えていた。多重ロジスティック回帰分析の結果,腰痛の発生は,身長がオッズ比(OR)1.12,95%信頼区間(95%CI)1.02-1.23,SF-8による身体的健康度(PHS)が,OR : 0.89(95%CI : 0.86-0.97),精神的健康度(MHS)がOR : 0.90(95%CI : 0.81-0.99)で有意であった。本研究は,身長が高いことは中腰姿勢を助長し,腰痛を起こす原因になるかもしれないこと,身体・精神的な健康の程度は,腰痛と関係があることが明らかになった。今後,交代勤務に伴う睡眠障害や慢性疲労との関連性も含めて検討すべきであることが示唆された。The objective of this cross-sectional study was to reveal the actual conditions and related factors of low back pain among female caregivers in special nursing homes for the elderly.The participants were 88 female caregivers in four facilities for the elderly. We surveyed the existence of low back pain, employment period, Body Mass Index (BMI), grip power, sitting forward flexion, Finger-Floor Distance (FFD), the Center for Epidemiologic Study Depression Scale (CES-D), Physical Health Score (PHS) and Mental Health Score (MHS) by using SF-8 and subjective symptoms of fatigue. Multinomial logistic regression model was used to investigate how far low back pain was related to mental and physical health status, flexibility, power, employment period, and BMI.55.7% (N=46) of the participants notified the presence of chronic lumbago. The result of multinomial logistic regression model showed that the existence of low back pain was significant in the following variables : 1) Odds ratio (OR)=1.12 (95% confidence interval : 95%CI=1.02-1.23) for height, 2) OR=0.89 (95% CI=0.81-0.97) for PHS, 3) OR=0.90 (95% CI=0.81-0.99) for MHS.Our findings suggest that height may reinforce a half-crouching position, thus it can be a cause of low back pain, and that the degree of physical and mental health is related to low back pain. It was also suggested that lumbago prevention and/or reduction strategy should include the relevance of sleep interruption and chronic fatigue with shift work when this problem is examined in future

Noise peak filtering in multi-dimensional NMR spectra using convolutional neural networks

Motivation: Multi-dimensional NMR spectra are generally used for NMR signal assignment and structure analysis. There are several programs that can achieve highly automated NMR signal assignments and structure analysis. On the other hand, NMR spectra tend to have a large number of noise peaks even for data acquired with good sample and machine conditions, and it is still difficult to eliminate these noise peaks. Results: We have developed a method to eliminate noise peaks using convolutional neural networks, implemented in the program package Filt_Robot. The filtering accuracy of Filt_Robot was around 90–95% when applied to 2D and 3D NMR spectra, and the numbers of resulting non-noise peaks were close to those in corresponding manually prepared peaks lists. The filtering can strongly enhance automated NMR spectra analysis. Availability and implementation: The full package of the program, documents and example data are available from http://bmrbdep.pdbj.org/en/nmr_tool_box/Filt_Robot.html.ISSN:1367-4803ISSN:1460-205

Breast Cancer Detection from a Urine Sample by Dog Sniffing: A Preliminary Study for the Development of a New Screening Device, and a Literature Review

Background: Breast cancer is a leading cause of cancer death worldwide. Several studies have demonstrated that dogs can sniff and detect cancer in the breath or urine sample of a patient. This study aims to assess whether the urine sample can be used for breast cancer screening by its fingerprints of volatile organic compounds using a single trained sniffer dog. This is a preliminary study for developing the “electronic nose” for cancer screening. Methods: A nine-year-old female Labrador Retriever was trained to identify cancer from urine samples of breast cancer patients. Urine samples from patients histologically diagnosed with primary breast cancer, those with non-breast malignant diseases, and healthy volunteers were obtained, and a double-blind test was performed. Total of 40 patients with breast cancer, 142 patients with non-breast malignant diseases, and 18 healthy volunteers were enrolled, and their urine samples were collected. Results: In 40 times out of 40 runs of a double-blind test, the trained dog could correctly identify urine samples of breast cancer patients. Sensitivity and specificity of this breast cancer detection method using dog sniffing were both 100%. Conclusions: The trained dog in this study could accurately detect breast cancer from urine samples of breast cancer patients. These results indicate the feasibility of a method to detect breast cancer from urine samples using dog sniffing in the diagnosis of breast cancer. Although the methodological standardization is still an issue to be discussed, the current result warrants further study for developing a new breast cancer screening method based on volatile organic compounds in urine samples