Unexpected A-form formation of 4′-thioDNA in solution, revealed by NMR, and the implications as to the mechanism of nuclease resistance

Fully modified 4′-thioDNA, an oligonucleotide only comprising 2′-deoxy-4′-thionucleosides, exhibited resistance to an endonuclease, in addition to preferable hybridization with RNA. Therefore, 4′-thioDNA is promising for application as a functional oligonucleotide. Fully modified 4′-thioDNA was found to behave like an RNA molecule, but no details of its structure beyond the results of circular dichroism analysis are available. Here, we have determined the structure of fully modified 4′-thioDNA with the sequence of d(CGCGAATTCGCG) by NMR. Most sugars take on the C3′-endo conformation. The major groove is narrow and deep, while the minor groove is wide and shallow. Thus, fully modified 4′-thioDNA takes on the A-form characteristic of RNA, both locally and globally. The only structure reported for 4′-thioDNA showed that partially modified 4′-thioDNA that contained some 2′-deoxy-4′-thionucleosides took on the B-form in the crystalline form. We have determined the structure of 4′-thioDNA in solution for the first time, and demonstrated unexpected differences between the two structures. The origin of the formation of the A-form is discussed. The remarkable biochemical properties reported for fully modified 4′-thioDNA, including nuclease-resistance, are rationalized in the light of the elucidated structure

Unexpected A-form formation of 4’-thioDNA in

solution, revealed by NMR, and the implications as to the mechanism of nuclease resistanc

Increased Urinary 3-Mercaptolactate Excretion and Enhanced Passive Systemic Anaphylaxis in Mice Lacking Mercaptopyruvate Sulfurtransferase, a Model of Mercaptolactate-Cysteine Disulfiduria

Mercaptopyruvate sulfurtransferase (Mpst) and its homolog thiosulfate sulfurtransferase (Tst = rhodanese) detoxify cyanide to thiocyanate. Mpst is attracting attention as one of the four endogenous hydrogen sulfide (H2S)/reactive sulfur species (RSS)-producing enzymes, along with cystathionine β-synthase (Cbs), cystathionine γ-lyase (Cth), and cysteinyl-tRNA synthetase 2 (Cars2). MPST deficiency was found in 1960s among rare hereditary mercaptolactate-cysteine disulfiduria patients. Mpst-knockout (KO) mice with enhanced liver Tst expression were recently generated as its model; however, the physiological roles/significances of Mpst remain largely unknown. Here we generated three independent germ lines of Mpst-KO mice by CRISPR/Cas9 technology, all of which maintained normal hepatic Tst expression/activity. Mpst/Cth-double knockout (DKO) mice were generated via crossbreeding with our previously generated Cth-KO mice. Mpst-KO mice were born at the expected frequency and developed normally like Cth-KO mice, but displayed increased urinary 3-mercaptolactate excretion and enhanced passive systemic anaphylactic responses when compared to wild-type or Cth-KO mice. Mpst/Cth-DKO mice were also born at the expected frequency and developed normally, but excreted slightly more 3-mercaptolactate in urine compared to Mpst-KO or Cth-KO mice. Our Mpst-KO, Cth-KO, and Mpst/Cth-DKO mice, unlike semi-lethal Cbs-KO mice and lethal Cars2-KO mice, are useful tools for analyzing the unknown physiological roles of endogenous H2S/RSS production

Effect of P1 concentration on NV centers created by electron beam irradiation

1. IntroductionNegatively charged nitrogen-vacancy (NV-) center in diamond is expected as quantum sensor to measure small changes in physical quantities, such as magnetic and electric field, temperature, strain, etc. The quantum sensors with high sensitivity require diamond containing high concentration of NV centers. Electron irradiation is a good method to create high concentration of NV centers. The high energy electrons knock carbon atoms out of the diamond lattice, creating vacancies. Annealing an irradiated diamond causes all the vacancies to become mobile and subsequently trapped by substituted nitrogen (P1 centers), forming NV centers. It is known that the process of NV center creation depends on the initial P1 concentrations, and the irradiation fluence dependence at low initial P1 concentrations below 1 ppm is still uncleared. In this study,we irradiate samples with different initial P1 concentrations, especially low concentration, and estimate the fluence dependency of the charge state and the amount of created NV centers.2. ExperimentsThe diamonds synthesized by High Pressure and High Temperature (HPHT) and Chemical Vapor Deposition (CVD) were used. The initial concentration of P1 was in the range from 40 ppb to 100 ppm. 2 MeV electrons were irradiated with a fluence up to 2E18 cm-2 at room temperature. Then, the samples were annealed at1000℃ for 2 hours to create NV center. The concentration of P1 was measured by Electron Spin Resonance (ESR) using JES-X330 (JEOL). The ratio between NV0 and NV- was estimated from photoluminescence (PL) spectrum measurement obtained using LabRAM HR Evolution (HORIBA). All these measurements wereperformed at room temperature.3. Results & DiscussionIn the case of type Ib HPHT diamond containing initial P1 concentration of ~100 ppm, the most of NV centers are thought to be NV-, and PL measurement revealed that only NV- was detected when the fluence of 2E18 cm-2. P1 center acts as electron donor, and provide electron to NV0. In contrast to high P1 concentration,an appreciable fraction of NV center is present as NV0 for samples with low initial P1 concentration (~ 10 ppm). We examined the relationship between the concentration of P1 and the fluence, using HPHT diamonds with initial P1 concentration of ~0.8 ppm. The concentration of P1 decreased to ~0.3 ppm when the fluence of 1.5E17 cm-2. This result suggests that P1 centers of ~0.5 ppm change to either NV center or positively charged substitutional nitrogen. PL spectrum when the fluence of 1.5×1017 cm-2, shows that both NV0 and NVarecreated. The ratio of NV- to NV0 decreased as irradiation fluence, to be 0.70 after irradiated with 1.0×1017 cm-2 and 0.53 after irradiated with 1.5E17 cm-2. It is noted that NV0 was formed even though P1 center which can act as electron donor to NV0 center remains. Similar results were observed for CVD diamonds with initial P1 concentration of ~40 ppb. The adequate fluence to maximize NV- concentration for diamonds containing low P1 concentration will be discussed by examining the fluence dependency of the charge state and total amount of NV center.New Diamond and Nano Carbons 2020/202

Overexpression of LIM and SH3 Protein 1 leading to accelerated G2/M phase transition contributes to enhanced tumourigenesis in oral cancer.

BACKGROUND: LIM and SH3 protein 1 (LASP-1) is a specific focal adhesion protein involved in several malignant tumors. However, its role in oral squamous cell carcinoma (OSCC) is unknown. The aim of this study was to characterize the role and molecular status/mechanism of LASP-1 in OSCC. METHODS: We evaluated LASP-1 mRNA and protein expressions in OSCC-derived cell lines and primary OSCCs. Using an shRNA system, we analyzed the effect of LASP-1 on the biology and function of the OSCC cell lines, HSC-3 and Ca9-22. The cells also were subcutaneously injected to evaluate tumor growth in vivo. Data were analyzed by the Fisher's exact test or the Mann-Whitney U test. Bonferroni correction was used for multiple testing. RESULTS: Significant up-regulation of LASP-1 was detected in OSCC-derived cell lines (n = 7, P<0.007) and primary OSCCs (n = 50, P<0.001) compared to normal controls. LASP-1 knockdown cells significantly inhibited cellular proliferation compared with shMock-transfected cells (P<0.025) by arresting cell-cycle progression at the G2 phase. We observed dramatic reduction in the growth of shLASP-1 OSCC xenografts compared with shMock xenografts in vivo. CONCLUSION: Our results suggested that overexpression of LASP-1 is linked closely to oral tumourigenicity and further provide novel evidence that LASP-1 plays an essential role in tumor cellular growth by mediating G2/M transition

Kinesin Family member 4A: A Potential Predictor for Progression of Human Oral Cancer

<div><p>Background</p><p>Kinesin family member 4A (KIF4A), a microtubule-based motor protein, was implicated in regulation of chromosomal structure and kinetochore microtubule dynamics. Considering the functions of KIF4A, we assumed that KIF4A is involved in progression of oral squamous cell carcinomas (OSCCs) via activation of the spindle assembly checkpoint (SAC). However, little is known about the relevance of KIF4A in the behavior of OSCC. We investigated the KIF4A expression status and its functional mechanisms in OSCC.</p> <p>Methods</p><p>The KIF4A expression levels in seven OSCC-derived cells were analyzed by quantitative reverse transcriptase-polymerase chain reaction and immunoblotting analyses. Using a KIF4A knockdown model, we assessed the expression of (SAC)-related molecules (BUB1, MAD2, CDC20, and cyclin B1), cell-cycle, and cellular proliferation. In addition to <i>in</i><i>vitro</i> data, the clinical correlation between the KIF4A expression levels in primary OSCCs (n = 106 patients) and the clinicopathologic status by immunohistochemistry (IHC) also were evaluated. </p> <p>Results</p><p>KIF4A mRNA and protein were up-regulated significantly (<i>P</i> < 0.05) in seven OSCC-derived cells compared with human normal oral keratinocytes. In the KIF4A knockdown cells, SAC activation was observed via increased BUB1 expression on the kinetochores, appropriate kinetochore localization of MAD2, down-regulation of CDC20, up-regulation of cyclin B1, and cell-cycle arrested at G2/M phase. The results showed that cellular proliferation of KIF4A knockdown cells decreased significantly (<i>P</i> < 0.05) compared with control cells. IHC showed that KIF4A expression in primary OSCCs was significantly (<i>P</i> < 0.05) greater than in the normal oral counterparts and that KIF4A-positive OSCCs were correlated closely (<i>P</i> < 0.05) with tumoral size. </p> <p>Conclusions</p><p>Our results proposed for the first time that KIF4A controls cellular proliferation via SAC activation. Therefore, KIF4A might be a key regulator for tumoral progression in OSCCs.</p> </div

Evaluation of KIF4A protein expression in primary OSCCs.

<p>Representative IHC results for KIF4A protein in normal oral tissue (<b>A</b>) and primary OSCC (<b>B</b>). <b>A</b>, <b>B</b>: Original magnification, ×400. Scale bars, 10 μm. Strong KIF4A immunoreactivity was detected in primary OSCCs; normal oral tissues show almost negative immunostaining. <b>C</b>: The state of KIF4A protein expression in primary OSCCs (n=106) and the normal counterparts. The KIF4A IHC scores were calculated as follows: IHC score = 0× (number of unstained cells in the field) +1× (number of weakly stained cells in the field) +2× (number of moderately stained cells in the field) +3× (number of intensely stained cells in the field). The KIF4A IHC scores for normal oral tissues and OSCCs ranged from 26.5 to 103 (median, 66.5) and 62.3 to 188 (median, 131), respectively. KIF4A protein expression levels in OSCCs are significantly (<i>P</i> < 0.05, Mann-Whitney’s U test) higher than in normal oral tissues.</p

Expression of KIF4A in shKIF4A cells.

<p>(<b>A</b>) qRT-PCR shows that KIF4A mRNA expression in the shKIF4A cells (HSC-3-and Ca9-22-derived transfectants; 2 clones each) is significantly lower than in the shMock cells (<i>P</i> < 0.05, Mann-Whitney U test). (<b>B</b>) Immunoblotting analysis shows that the KIF4A protein levels in shKIF4A-transfected cells (HSC-3- and Ca9-22-derived transfectants; 2 clones each) also have decreased markedly compared with that in the shMock cells (<i>P</i> < 0.05, Mann-Whitney U test).</p