Proper application of DNA dyes in agarose gel electrophoresis.

Various dyes are used to visualize DNA bands in agarose gel electrophoresis (AGE) by the methods of pre- or post-staining. The DNA dye user's guides generally state that the binding of the dye to DNA will affect DNA mobility in electrophoresis, thus recommending post-staining for accurate measurement of DNA size. However, many AGE performers prefer pre-staining procedures for reasons such as convenience, real-time observation of DNA bands, and/or the use of a minimal amount of dye. The detrimental effect of the dye on DNA mobility and the associated risk for inaccurate measurement of DNA size are often overlooked by AGE performers. Here we quantitatively determine the impact on DNA migration imposed by frequently used dyes, including GelRed, ethidium bromide (EB), and Gold View. It was observed that pre-staining with GelRed and EB significantly slowed down DNA migration to cause as much as 39.1% overestimation on the size of sample DNA, whereas Gold View had little effect. The slowdown of DNA migration increased with dye concentration until it plateaued when the dye concentration reached a saturated level. Thus, to take advantage of pre-staining, saturated levels of DNA dyes should always be applied for both DNA samples and DNA markers to ensure a fair comparison of DNA sizes. In addition, GelRed and EB display much higher sensitivity than Gold View in the detection of DNA bands in post-staining. The saturated concentrations, cost considerations, and other useful features of these frequently used dyes are summarized for the information of AGE performers.

Design, synthesis, and in vivo and in vitro biological screening of pseudolaric acid B derivatives as potential anti-tumor agents.

Pseudolaric Acid B (PAB), a natural product with remarkable anti-tumor activity, is a starting point for new anticancer therapeutics. We designed and synthesized 27 PAB derivatives and evaluated their anti-proliferative activities against four cancer cell lines: MCF-7, HCT-116, HepG2, and A549. Compared with unmodified PAB, the PAB derivatives showed stronger anti-proliferative activity. The ability of compound D3 (IC50 = 0.21 µM) to inhibit HCT-116 cells was approximately 5.3 times that of PAB (IC50 = 1.11 µM) and the antiproliferative action was unrelated to cytotoxicity (SI=20.38), indicating its superior safety profile (PAB; SI=0.95). Compound D3 effectively suppressed the EdU-positive rate and reduced colony formation, arrested HCT-116 cells in the S and G2/M phases and induced apoptosis. In vivo experiments further demonstrated low toxicity of compound D3 while suppressing tumor growth in mice. In summary, given its strong anti-proliferative effect and relative safety, further development of compound D3 is warranted.

Natural Products-Pyrazine Hybrids: A Review of Developments in Medicinal Chemistry.

Pyrazine is a six-membered heterocyclic ring containing nitrogen, and many of its derivatives are biologically active compounds. References have been downloaded through Web of Science, PubMed, Science Direct, and SciFinder Scholar. The structure, biological activity, and mechanism of natural product derivatives containing pyrazine fragments reported from 2000 to September 2023 were reviewed. Publications reporting only the chemistry of pyrazine derivatives are beyond the scope of this review and have not been included. The results of research work show that pyrazine-modified natural product derivatives have a wide range of biological activities, including anti-inflammatory, anticancer, antibacterial, antiparasitic, and antioxidant activities. Many of these derivatives exhibit stronger pharmacodynamic activity and less toxicity than their parent compounds. This review has a certain reference value for the development of heterocyclic compounds, especially pyrazine natural product derivatives.

In Vitro and In Vivo Anti-Cancer Activity of Lasiokaurin in a Triple-Negative Breast Cancer Model.

Due to its intricate heterogeneity, high invasiveness, and poor prognosis, triple-negative breast cancer (TNBC) stands out as the most formidable subtype of breast cancer. At present, chemotherapy remains the prevailing treatment modality for TNBC, primarily due to its lack of estrogen receptors (ERs), progesterone receptors (PRs), and human epidermal growth receptor 2 (HER2). However, clinical chemotherapy for TNBC is marked by its limited efficacy and a pronounced incidence of adverse effects. Consequently, there is a pressing need for novel drugs to treat TNBC. Given the rich repository of diverse natural compounds in traditional Chinese medicine, identifying potential anti-TNBC agents is a viable strategy. This study investigated lasiokaurin (LAS), a natural diterpenoid abundantly present in Isodon plants, revealing its significant anti-TNBC activity both in vitro and in vivo. Notably, LAS treatment induced cell cycle arrest, apoptosis, and DNA damage in TNBC cells, while concurrently inhibiting cell metastasis. In addition, LAS effectively inhibited the activation of the phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway and signal transducer and activator of transcription 3 (STAT3), thus establishing its potential for multitarget therapy against TNBC. Furthermore, LAS demonstrated its ability to reduce tumor growth in a xenograft mouse model without exerting detrimental effects on the body weight or vital organs, confirming its safe applicability for TNBC treatment. Overall, this study shows that LAS is a potent candidate for treating TNBC.

Infectivity and antigenicity of pseudoviruses with high-frequency mutations of SARS-CoV-2 identified in Portugal.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has had a major impact on global human health. During the spread of SARS-CoV-2, weakened host immunity and the use of vaccines with low efficacy may result in the development of more-virulent strains or strains with resistance to existing vaccines and antibodies. The prevalence of SARS-CoV-2 mutant strains differs between regions, and this variation may have an impact on the effectiveness of vaccines. In this study, an epidemiological investigation of SARS-CoV-2 in Portugal was performed, and the VSV-ΔG-G* pseudovirus system was used to construct 12 spike protein epidemic mutants, D614G, A222V+D614G, B.1.1.7, S477N+D614G, P1162R+D614G+A222V, D839Y+D614G, L176F+D614G, B.1.1.7+L216F, B.1.1.7+M740V, B.1.258, B.1.258+L1063F, and B.1.258+N751Y. The mutant pseudoviruses were used to infect four susceptible cell lines (Huh7, hACE2-293T-293T, Vero, and LLC-MK2) and 14 cell lines overexpressing ACE2 from different species. Mutant strains did not show increased infectivity or cross-species transmission. Neutralization activity against these pseudoviruses was evaluated using mouse serum and 11 monoclonal antibodies. The neutralizing activity of immunized mouse serum was not significantly reduced with the mutant strains, but the mutant strains from Portugal could evade nine of the 11 monoclonal antibodies tested. Neutralization resistance was mainly caused by the mutations S477N, N439K, and N501Y in the spike-receptor binding domain. These findings emphasize the importance of SARS-CoV-2 mutation tracking in different regions for epidemic prevention and control.

Cell death mechanisms induced by synergistic effects of halofuginone and artemisinin in colorectal cancer cells.

Our previous study found that the combination of halofuginone (HF) and artemisinin (ATS) synergistically arrest colorectal cancer (CRC) cells at the G1/G0 phase of the cell cycle; however, it remains unclear whether HF-ATS induces cell death. Here we report that HF-ATS synergistically induced caspase-dependent apoptosis in CRC cells. Specifically, both in vitro and in vivo experiments showed that HF or HF-ATS induces apoptosis via activation of caspase-9 and caspase-8 while only caspase-9 is involved in ATS-induced apoptosis. Furthermore, we found HF or HF-ATS induces autophagy; ATS can't induce autophagy until caspase-9 is blocked. Further analyzing the crosstalk between autophagic and caspase activation in CRC cells, we found autophagy is essential for activation of caspase-8, and ATS switches to activate capase-8 via induction of autophagy when caspase-9 is inhibited. When apoptosis is totally blocked, HF-ATS switches to induce autophagic cell death. This scenario was then confirmed in studies of chemoresistance CRC cells with defective apoptosis. Our results indicate that HF-ATS induces cell death via interaction between apoptosis and autophagy in CRC cells. These results highlight the value of continued investigation into the potential use of this combination in cancer therapy.

Identification of Spodoptera frugiperda importin alphas that facilitate the nuclear import of Autographa californica multiple nucleopolyhedrovirus DNA polymerase.

Proteins containing nuclear localization signals (NLSs) are actively transported into the nucleus via the classic importin-α/ß-mediated pathway, and NLSs are recognized by members of the importin-α family. Most studies of insect importin-αs have focused on Drosophila to date, little is known about the importin-α proteins in Lepidoptera insects. In this study, we identified four putative importin-α homologues, Spodoptera frugiperda importin-α1 (SfIMA1), SfIMA2, SfIMA4 and SfIMA7, from Sf9 cells. Immunofluorescence analysis showed that SfIMA2, SfIMA4 and SfIMA7 localized to the nucleus, while SfIMA1 distributed in cytoplasm. Additionally, SfIMA4 and SfIMA7 were also detected in the nuclear membrane of Sf9 cells. SfIMA1, SfIMA4 and SfIMA7, but not SfIMA2, were found to associate with the C terminus of AcMNPV DNA polymerase (DNApol) that harbours a typical monopartite NLS and a classic bipartite NLS. Further analysis of protein-protein interactions revealed that SfIMA1 specifically recognizes the bipartite NLS, while SfIMA4 and SfIMA7 bind to both monopartite and bipartite NLSs. Together, our results suggested that SfIMA1, SfIMA4 and SfIMA7 play important roles in the nuclear import of AcMNPV DNApol C terminus in Sf9 cells.

[A Novel Chemically Defined Medium Enhanced the Osteogenic Potential and Periodontal Bone Regeneration of Dental Papilla Cells].

OBEJECTIVE: To investigate the role of a novel chemically defined medium (CDM) in the regulation of dental papilla cells (DPCs) functional phenotype in vitro and periodontal bone regeneration in vivo. METHODS: DPCs were isolated and cultured in conventional medium (CM) or CDM. The surface makers, and the proliferation, migration and osteogenic differentiation abilities of DPCs were evaluated. In vivo, the DPCs that mixed with collagen gel were implanted into the model rats in the defect of periodontal to repair the periodontal tissue. Regeneration of the tissues was examined by microcomputed tomography and histological observation. RESULTS: DPCs in the CM group and CDM group showed similar surface markers. Compared to the CM group, the CDM significantly enhanced the proliferation, colony-forming efficiency and migration of DPCs in vitro. In addition, real time PCR showed that the expression levels of osteogenesis-related genes, Runx2, Alp and Opn. were significantly enhanced in DPCs in the CDM group. DPCs cells treated with CDM also exhibited higher alkaline phosphatase activity and stronger ability of formation of mineralized nodules in vitro. In vivo, DPCs from CDM group significantly enhanced the periodontal bone regeneration and the reconstruction of periodontal bone tissues in rat periodontal defect model. CONCLUSION: CDM is a suitable medium to culture DPCs for periodontal bone regeneration. This research provided a substitute for basic research and set the stage for future clinical application of stem cell transplantation.

[The Study of Three-Dimensional Synchronous Fluorescence Spectral Characteristics of Liuwei Dihuang Pills].

Three-dimensional synchronous fluorescence of each component of Liuwei dihuang pills (Prepared rehmannia root, cornel, yam, alisma, poria cocos, and cortex moutan) is measured by using FLS920P fluorescence spectrometer. Feature parameters were extracted. It can be found that each component is fluorescent material and the lines are all different. Furthermore, Three-dimensional synchronous fluorescence of Liuwei dihuang pills boiling with standard water and non-standard water are all measured and there are significant differences between them. It can be applied in distinguishing different formula of Chinese medicine decoction. Experimental and Theoretical conclusion show that: the three-dimensional fluorescence spectrometry method and the combination of synchronous fluorescence spectroscopy method can further improve the sensitivity and selectivity of fluorescence spectroscopy, have a distinct advantage in a complex multi-component mixture of fluorescence spectroscopic analysis. The study can provide a convenient and reliable method for establishing a complete fingerprint of Chinese traditional medicine. It also can help identifying the component and the quality of Chinese patent medicine.

MoRad6-mediated ubiquitination pathways are essential for development and pathogenicity in Magnaporthe oryzae.

The ubiquitin system modulates protein functions through targeting substrates for ubiquitination. Here, E2 conjugating enzyme MoRad6-related ubiquitination pathways are identified and analyzed in Magnaporthe oryzae, the causal agent of rice blast disease. Disruption of MoRad6 leads to severe defects in growth, sporulation, conidial germination, appressorium formation, and plant infection. To depict the functions of MoRad6, three putative ubiquitin ligases, MoRad18, MoBre1 and MoUbr1, are also characterized. Deletion of MoRad18 causes minor phenotypic changes, while MoBre1 is required for growth, conidiation and pathogenicity in M. oryzae. Defects in ΔMobre1 likely resulted from the reduction in di- and tri-methylation level of Histone 3 lysine 4 (H3K4). Notably, MoUbr1 is crucial for conidial adhesion and germination, possibly by degrading components of cAMP/PKA and mitogen-activated protein kinase (MAPK) Pmk1 signaling pathways via the N-end rule pathway. Germination failure of ΔMoubr1 conidia could be rescued by elevation of cAMP level or enhanced Pmk1 phosphorylation resulting from further deletion of MoIra1, the M. oryzae homolog of yeast Ira1/2. These reveal vital effects of cAMP/PKA and MAPK Pmk1 signaling on conidial germination in M. oryzae. Altogether, our results suggest that MoRad6-mediated ubiquitination pathways are essential for the infection-related development and pathogenicity of M. oryzae.

[Soft Measurement of the Purity of the Synthetic Edible Pigment Powder Using Fluorescence Spectroscopy Combined with SVM].

The feature compression algorithm which was reformed from the original Moment method was used for the pre-processing of the fluorescence spectral data, then combined the data and the Weighted Least Squares Support Vector Machine(WLS-SVM) algorithm to establish a robust regression model, which is used for forecasting the purity of edible pigment powder. In this paper, brilliant blue and ponceau 4R served as an example to discuss the method of forecasting effect of edible pigment powder purity. The emission fluorescence spectra of two edible pigment at the optimal excitation wavelength were measured by FLS920 fluorescence spectrometer. The compression and transformation of the fluorescence spectral data was acquired by the feature compression algorithm reformed from the Original Moment method. On the one hand the feature compression algorithm shortened the operation time, on the other hand it improved the prediction accuracy of the model. Then, the concentration prediction model was established after inputting the fluorescence spectral data pre-processed into the Weighted Least Squares Support Vector Machine. The model gave anastomotic predicted spectral data with the actual experiments of the brilliant blue and ponceau 4R, and the average coefficient of determination in the half peak width was 0.700 and 0.930 respectively. There was a good linear relationship between the predicted and the nominal concentration of the brilliant blue and ponceau 4R, and the correlation coefficients were 0.997 and 0.992 respectively. It can be concluded that, the predicted concentration of the brilliant blue and ponceau 4R powder were got the results of 61.0% and 72.3% respectively.

[The Discrimination of Chinese Strong Aroma Type Liquors with Three-Dimensional Fluorescence Spectroscopy Combined with Principal Component Analysis and Support Vector Machine].

In this paper, a method for discrimination of different bands liquor with strong aroma type based on three-dimensional fluorescence spectrum technology was developed. Firstly, the three-dimensional fluorescence spectra of seven different brands liquor were measured by the FLS920 fluorescence spectrometer which produced by Edinburgh in England. The spectral show that different bands liquors have similar fluorescence characteristics and it's difficult to distinguish them only with Fluorescent characteristic parameters. Because of this, the first-order and second-order partial derivatives respect to fluorescence emission wavelength on each of the excitation wavelength were carried out in this paper. Daubechies7 (db7) orthonormal wavelet with compact support was used to compress the spectral data. The forth approximate coefficients were finally chosen as the new data matrix. Then the new data matrix was analyzed by principal component analysis (PCA) and the principal components were extracted to be used as the inputs of support vector machine (SVM). The K-fold cross validation was applied to optimize the parameters c and y and the prediction model was constructed in the end. Fourteen samples were selected randomly from each brand that in total of ninety-eight samples were selected as the training set, and the rest forty-two samples were collected as the prediction set. The effect of three different spectral data after processing on the model is compared, original data, the first-order and second-order partial derivatives on the spectral data. The results show that the three-dimensional fluorescence spectra with the pretreatment of second-order partial derivatives coupled with PCA and SVM can make a good performance on the brands identification of strong aroma type liquors, the accuracy of the established model and prediction accuracy were 98.98% and 100%, respectively. This method has the advantage of easy operation, high speed, low cost and provides a good help in the detection and identification of Chinese liquor.

[Comparative Study on the Molecular Structures and Spectral Properties of Ponceau 4R and Amaranth].

The Edinburgh FLS920P steady-instantaneous fluorescence spectrometer was applied on the detection of the absorption and the emission spectra of ponceau 4R and amaranth, which are isomers to each other. After that, the spectral parameters of them were compared. Then, the density functional theory (DFT) and time-dependent density functional theory (TD-DFT) were used on the optimization of ponceau 4R and amaranth under the ground and excited state, respectively, in order to compare the differences in configurations of them under different states. On the base of the results above, the absorption and emission spectra of the two isomers were calculated with TD-DFT, and the polarized continuum model (PCM) was applied on the base of 6-311++G (d, p). The fluorescence mechanism, the relationships between the properties of fluorescence spectra and the molecular geometry were all analyzed. The results shows that, the structures of the two molecules are non-planar, these two naphthalene rings are not co-planar, respectively, and there's hydrogen bond in amaranth. When the two isomers were on the ground state, the planarity of the naphthalene ring which exists the hydrogen bond mentioned above in amaranth is better than the corresponding part of ponceau 4R. The two isomers are nearly co-planar when they're on the excited state. The molecular structures of ponceau 4R and amaranth optimized above are basically reasonable, for the quantum chemistry calculation spectral results are agree with the experiments. The planarity of the naphthalene rings on the right side in ponceau 4R is worse than that in amaranth, the ponceau 4R molecule experienced more vibration and rotation from the excited to the ground state, lost more energy, which lead to the reduction of energy for emitting fluorescent photons. So ponceau 4R has longer fluorescence emission wave- length than amaranth. In this paper, the molecular structure information of ponceau 4R and amaranth were obtained, and the differences of the spectral characteristics between them were found out. The results can provide references for the study of the relationship between the spectral properties and the configuration of isomers.

[Quantum Chemistry Calculation of Ponceau 4R Molecule Structure and Research on the Fluorescence Mechanism].

The molecule structures of Ponceau 4R in ground state and the excited state were optimized by employing the Gaussian 09W program package. In addition, the electronic structure and frontier orbital of the ground state, the emission wavelength of the excited state was also investigated. And then, the Edinburgh FLS920P fluorescence spectrometer was applied to the measurement of the fluorescence spectra of cochineal solution, and the emission spectra was obtained. The calculated emission wavelength had a good coincidence with the experiment data, which indicates that the optimized structures mentioned above are reasonable. The structures comparison between the ground state and the excited state was also performed to analyze the mechanism of fluorescence spectrum. It can be concluded that the molecule structure of excited state is nearly planar, so Ponceau 4R is thought to have strong fluorescent characteristics, the emission fluorescence is the result of transition from orbit 139 to orbit 137.

[Identification of Prepared Rehmannia Root in Different Regions by Using Derivative Synchronous Fluorescence Spectrum].

Three-dimensional synchronous fluorescence spectrum of prepared rehmannia root from different regions were measured and feature parameters were extracted. It can be found that the effective fluorescent compositions of prepared rehmannia root from different regions are similar. The relationships between the spectrum-effect were established. This study can provide reference for clinicaldosage. First order derivative and second order derivative of prepared rehmanniaroot from different regions can be got by programming respectively. Magnifing synchronous emission spectrum of the shoulder strap, the subtle differences of the spectrum can be distinguished and the method, derivative synchronous fluorescence spectrum, to identify different regions of prepared rehmannia root are acquired.

[Year Discrimination of Mild Aroma Chinese Liquors Using Three-Dimensional Fluorescence Spectroscopy Combined with Parallel Factor and Neural Network].

Three-dimensional fluorescence spectroscopy coupled with parallel factor analysis and neural network was applied to the year discrimination of mild aroma Chinese liquors. The excitation-emission fluorescence matrices (EEMs) of 120 samples with various years were measured by FLS920 fluorescence spectrometer. The trilinear decomposition of the data array was performed and the loading scores of and the excitation-emission profiles of four components were also obtained. The scores were employed as the inputs of the BP neural networks and the PARAFAC-BP identification model was constructed. 10 samples were collected from 10, 20 and 30 years of liquors respectively, and 30 samples were selected as the test sets. The remaining 90 samples were used as the training sets to build the training model. The year prediction of unknown samples was also carried out, and the prediction accuracy was 90%, 100% and 100%, respectively. Meanwhile, the discrimination analysis method and the multi-way partial least squares discriminant analysis were compared, namely PARAFAC-BP and NPLS-DA. The results indicated that parallel factor combined with the neural network (PARAFAC-BP) has higher prediction accuracy. The proposed method can effectively extract the spectral characteristics, and also reduce the dimension of the input variables of neural network. A good year discrimination result was finally achieved.

[Analysis for related factors of upper urinary tract deterioration in patients with spinal cord injury].

OBJECTIVE: To evaluate the related factors of upper urinary tract deterioration in spinal cord injured patients. METHODS: Medical records of spinal cord injured patients from Jan.2002 to Sep.2009 were retrospectively reviewed. All the patients were divided into the upper urinary tract deterioration group and non-deterioration group according to the diagnostic criteria. Indexes such as demographic characteristic (gender, age), spinal cord injury information (cause, level, completeness), statuses of urinary tract system (bladder management, urine routine, urine culture, ultrasound, serum creatinine, fever caused by urinary tract infection) and urodynamics information(bladder compliance, bladder stability, bladder sensation, detrusor sphincter dyssynergia, detrusor leak point pressure, maximum cystometric capacity, relative safe bladder capacity, maximum flow rate, maximum urethra closure pressure) were compared between the two groups.Then Logistic regression analysis were performed. RESULTS: There was significantly difference between the two groups in spinal cord injury level(χ(2) = 8.840, P = 0.031),bladder management(χ(2) = 11.362, P = 0.045), urinary rutine(χ(2) = 17.983, P = 0.000), fever caused by urinary tract infection(χ(2)= 64.472, P = 0.000), bladder compliance(χ(2) = 6.531, P = 0.011), bladder sensation(χ(2) = 11.505, P = 0.009), maximum cystometric capacity(t = 2.209, P = 0.043), and detrusor-sphincter dyssynergia(χ(2) = 4.247, P = 0.039). The multiple-factor non-conditional Logistic regression analysis showed that bladder management (OR = 1.114, P = 0.006), fever caused by urinary tract infection(OR = 1.018,P = 0.000), bladder compliance (OR = 1.588, P = 0.040) and detrusor-sphincter dyssynergia(OR = 1.023, P = 0.034) were the key factors of upper urinary tract deterioration in spinal cord injured patients. CONCLUSION: Urinary tract infection, lower bladder compliance, detrusor-sphincter dyssynergia and unreasonable bladder management are the risk factors of upper urinary tract deterioration in spinal cord injured patients.

Determination of components of mixed color solutions by fluorescence spectroscopy combined with calibration algorithms.

The three-dimensional spectra of mixed solutions of allure red, sunset yellow and brilliant blue were obtained. Then the three synthetic food colors were determined by parallel factor analysis (PARAFAC) and alternating trilinear decomposition (ATLD) algorithms, respectively. The component number of model is three by core-consistency diagnostic. The average recoveries of allure red, sunset yellow and brilliant blue obtained by PARAFAC were 98.75% +/- 8.9%, 97.22% +/- 2.9% and 99.00% +/- 2.9% and those by ATLD algorithm were 99.78% +/- 5.9%, 92.52% +/- 5.5% and 97.23% +/- 5.8%, respectively. Results show that both of the algorithms can be used in direct and rapid determination of multi-components of mixtures. From further comparison, the PARAFAC is more stable and advantageous.

[Quantitative analysis of two food colors using excitation-emission matrix spectra coupled with parallel factor algorithm].

In the present paper, British Edinburgh FLS920P Steady State and Time-Resolved Fluorescence Spectrometer was applied to measure three dimensional fluorescence spectra of 12 pigment solution samples and the parallel factor algorithm was combined with the excitation-emission matrix to find a way to detect the food colors. In the experiment, making use of CORCONDIA determination method to confirm that the number of the components is 3 in mixed solution, and then by using parallel factor analysis (PARAFAC) algorithms, get the average recoveries of carminum and Allura red were 99.3% +/- 5.0% and 102.2% +/- 5.6%, and the root mean square errors of prediction (RMSEP) were 0.054 and 0.205, respectively. The results show that the method can be applied to determine carminum and Allura red in the mixed solution simultaneously even in the presence of interfering amaranth, which was simple and convenient, rapid, etc, and provides references for synthetic food pigments detection.