Replication of M13 single-stranded DNA bearing a sitespecific ethenocytosine lesion by Escherichia coil cell extracts

Previous investigation on the mutagenic effects of 3,N4-Ethenocytosine (εC), a nonpairing DNA lesion,revealed the existence of a novel SOS-independent inducible mutagenic mechanism in E. coli termed UVM for UV modulation of mutagenesis. To investigate whether UVM is mediated by an alteration of DNA replication, we have set up an in vitro replication system ill which phage M13 viral single-stranded DNA bearing a single site-specific (εC) residue is replicated by soluble protein extracts from E. coli cells. Replication products were analyzed by agarose gel electrophoresis and the frequency of translesion synthesis was determined by restriction endonuclease analyses. Our data indicate that DNA replication is strongly inhibited by εC, but that translesion DNA synthesis does occur in about 14% of the replicated DNA molecules. These results are very similar to those observed previously in vivo, and suggest that this experimental system may be suitable for evaluating alterations in DNA replication in UVM-induced cells.

Biotransformation of nitro-polycyclic aromatic compounds by vegetable and fruit cell extracts

Extracts from various vegetables and fruits were investigated for their abilities to reduce nitro-polycyclic aromatic hydrocarbons （NPAHs）. The extracts from grape and onion exhibited an interesting selectivity, yielding corresponding hydroxylamines or amines as major products under mild conditions of 30 ℃ and pH 7.0. Grape extracts reduced the 4-nitro-l,8-naphthalic anhydride with the highest conversion rate （〉99%） and the highest ratio of hydro- xylamine to amine （95：5）. In contrast, the onion extracts reduced 4-nitro-l,8-naphthalic anhydride with a conversion rate of 94% and a ratio of hydroxylamine to amine of 8：92. The thiol-reducing agent, 13-mercaptoethanol, and metal cations, Ca2＋ and Mg2＋, greatly increased the reductive efficiency. This work provides an alternative strategy for biotransformation of nitro-polycyclic compounds.

Dual-calibration coefficient:a more accurate protocol for simultaneous determination of superoxide and hydrogen peroxide in human Hep G2 cell extracts

We present a more accurate method for the quantification of superoxide anion （02-） and hydrogen peroxide （H202） simulta- neously in human HepG2 cell extracts. After the xanthine/xanthine oxidase system was added into cell extract which was devoid of O2＊- and H202, steady-state and in-situ produced O2＊- and H202 by xanthine/xanthine oxidase system was labeled by fluorescent probes and subsequently separated by microchip electrophoresis. Based on this method, two differential equations with the calibration coefficients were established for O2 and H202, respectively. Using the established dual-calibration coefficients, we obtained the calibrated concentrations of 02＊ and H202 that produced in human HepG2 cells, which were lower （0.66±0.03 and 0.82±0.04 lamol/L for 02＊-and H202, respectively） than that （0.85±0.03 and 0.96±0.03 gmol/L for O. and H202, respectively） obtained from statutory working curve. The proposed dual-calibration coefficient protocol takes into account both the complex matrix effect of the biological system and real time decaying of O2 ＊- and H202, providing a method with higher accuracy.

Progress in hole-transporting materials for perovskite solar cells

In recent years the photovoltaic community has witnessed the unprecedented development of perovskite solar cells（PSCs） as they have taken the lead in emergent photovoltaic technologies. The power conversion efficiency of this new class of solar cells has been increased to a point where they are beginning to compete with more established technologies. Although PSCs have evolved a variety of structures, the use of hole-transporting materials（HTMs） remains indispensable. Here, an overview of the various types of available HTMs is presented. This includes organic and inorganic HTMs and is presented alongside recent progress in associated aspects of PSCs, including device architectures and fabrication techniques to produce high-quality perovskite films. The structure, electrochemistry, and physical properties of a variety of HTMs are discussed, highlighting considerations for those designing new HTMs. Finally, an outlook is presented to provide more concrete direction for the development and optimization of HTMs for highefficiency PSCs.

Effects of Spinach Powder Fat-Soluble Extract on Proliferation of Human Gastric Adenocarcinoma Cells

Four kinds of assays were used to study the effect of a fat-soluble extract of spinach powder (SPFE) on the proliferation of human gastric adenocareinoma cell line (SGC-7901) in vitro.These studies included: (Ⅰ) cell growth assay, (Ⅱ) colony forming assay, (Ⅲ) MTT colorimetric assay, and (Ⅳ) 3H-TdR incorporation assay. The concentrations of SPFE expressed as the level of β-carotene in the medium were 2×10-8, 2×10-7 and 2×10-6 mol/L β-carotene in assays (Ⅰ)～(Ⅲ), but 4×10- 8, 4×10-7 and 4×10-6 mol/L β-caretene in assay (Ⅳ) respectively. The results indicated that SPFE inhibited the prolifendion and colony forming ability of SGC-7901 cells. And in MTT assay, SPFE inhibited the viability of SGC7901 cells, but no inhibitory effect of SPFE was observed on the viability of lymphocytes in peripheral blood of healthy people. Finally, in the 3H-TdR incorporation test, both SPFE and β-carotene showed significant inhibitory effects on DNA synthesis in SGC-7901 cells, but SPFE was more effective than β-carotene.

Effect of infusion pressure during cataract surgery on ganglion cells measured using isolated-check visual evoked potential

AIM： To detect the relationship between infusion pressure and postoperative ganglion cells function.METHODS： This prospective observational cohort study included sixty-one eyes that underwent uncomplicated cataract surgery. Patients were divided into two groups according to infusion time（IT） recorded using surgery equipment [Group A： IT〉IT_（mean）（27 eyes）; Group B： IT

Automatic cell object extraction of red tide algae in microscopic images

Extracting the cell objects of red tide algae is the most important step in the construction of an automatic microscopic image recognition system for harmful algal blooms.This paper describes a set of composite methods for the automatic segmentation of cells of red tide algae from microscopic images.Depending on the existence of setae,we classify the common marine red tide algae into non-setae algae species and Chaetoceros,and design segmentation strategies for these two categories according to their morphological characteristics.In view of the varied forms and fuzzy edges of non-setae algae,we propose a new multi-scale detection algorithm for algal cell regions based on border-correlation,and further combine this with morphological operations and an improved GrabCut algorithm to segment single-cell and multicell objects.In this process,similarity detection is introduced to eliminate the pseudo cellular regions.For Chaetoceros,owing to the weak grayscale information of their setae and the low contrast between the setae and background,we propose a cell extraction method based on a gray surface orientation angle model.This method constructs a gray surface vector model,and executes the gray mapping of the orientation angles.The obtained gray values are then reconstructed and linearly stretched.Finally,appropriate morphological processing is conducted to preserve the orientation information and tiny features of the setae.Experimental results demonstrate that the proposed methods can effectively remove noise and accurately extract both categories of algae cell objects possessing a complete shape,regular contour,and clear edge.Compared with other advanced segmentation techniques,our methods are more robust when considering images with different appearances and achieve more satisfactory segmentation effects.

Bone Marrow Mononuclear Cell Extraction. Does the Operator Performance Affect Processing Efficacy？

Different cell populations from bone marrow are used in various clinical trials for cardiac diseases during last decade. Four clinical studies are on going in our institution and enrol patients with cardiac diseases, coronary disease and type 2 diabetes, patients with osteoarthritis. Density gradient is used to separate bone marrow mononuclear cells. Cell processing looses are significant. To find out critical control points we analysed processing process and differences in cell yields between operators performing cell extraction. Bone marrow mononuclear cells were isolated using Ficoll density gradient centrifugation. Cells were counted using flow cytometry for mononuclear cell total counts, CD34＋ population count and viability analysis. The patients who underwent bone marrow aspiration followed by cell isolation received cell suspension for transplantation. Two cells processing for separate patients were performed at once. Same standard operation procedures were applied. Processing looses between operators performing cell extraction were analysed. Bone marrow samples from eight patients were processed. Mononuclear cells were extracted. Operator performances were compared. Operator A average bone marrow mononuclear cell yield in starting material was 9,97 ± 9,98 %, CD34＋ population yield 75,46±79,67%. Operator B average bone marrow mononuclear cell yield in starting material was 24,68 ± 14,8 %, CD34＋ population yield 70,42 ±44.84%. Operator A average cell viability in starting material was 45,24 ± 9,55%, after cell processing 42,96 ± 23,66 %. Operator B average cell viability in starting material was 49,85 ± 5,48%, after cell processing 69,52 ± 6,65 %.

PV Solar Yield Applicable Simulator Based on Free Irradiance Data Source:Applied Comprehensive Tool for Solar Engineers

Accurate PV system simulators are implemented with expensive software platforms using paid irradiance data.The main purpose of this paper is to develop and validate a PV system simulator,beginning with a solar cell parameter extraction model,then test and validate long-termIrradiance data using free online source(Typical Meteorological Year TMY in(PVGIS)European website),and finally building full solar generator simulator to run in working real conditions.Comparing results with Accurate Paid PV simulators(which use theMuneer model)showed good accuracy of the proposed simulator.Work flow starts with the Irradiance model’s data processing,then solar cell 5 parameters model data processing(to extract cell parameters),and finally full system simulator.MATLAB coding programs in real working conditions are used for simulation.Results of solar cell parameter extraction show 99.6%to 99.99%matching with data sheet and cell performance under standard test conditions.Systemmodel simulation output shows 8%less yearly generated energy compared to the PVGIS 2022 long-term simulation(hourly basis(one-year time)).This is due to incident energy variations(between the years 2016 and 2022)of 4.02%.The novelty of the algorithm is the methodology,as it tests irradiance data on an hourly basis and validates results for a whole continuous year.Also,the 5-parameter solar cell model is used to be validated in long term analysis,not only STC conditions and could be applied on any PV solar cell.The algorithm and block diagramused are scalable,modular,and interchangeable with similarmodels to be tested.This simulator could test severalmethods andmodels in solar pv technology.

Biosynthesis of Callose and Cellulose by Detergent Extracts of Tobacco Cell Membranes and Quantification of the Polymers Synthesized in vitro

The conditions that favor the in vitro synthesis of cellulose from tobacco BY-2 cell extracts were determined. The procedure leading to the highest yield of cellulose consisted of incubating digitonin extracts of membranes from 11-day-old tobacco BY-2 cells in the presence of 1 mM UDP-glucose, 8 mM Ca^2＋ and 8 mM Mg^2＋. Under these conditions, up to nearly 40% of the polysaccharides synthesized in vitro corresponded to cellulose, the other polymer synthesized being callose. Transmission electron microscopy analysis revealed the occurrence of two types of structures in the synthetic reactions. The first type consisted of small aggregates with a diameter between 3 and 5 nm that associated to form fibrillar strings of a maximum length of 400 nm. These structures were sensitive to the acetic/nitric acid treatment of Updegraff and corresponded to callose. The second type of structures was resistant to the Updegraff reagent and corresponded to straight cellulose microfibrils of 2-3 nm in diameter and 200 nm to up to 5 μm in length. In vitro reactions performed on electron microscopy grids indicated that the minimal rate of microfibril elongation in vitro is 120 nm/min. Measurements of retardance by liquid crystal polarization microscopy as a function of time showed that small groups of microfibrils increased in retardance by up to 0.047 nm/min per pixel, confirming the formation of organized structures.

The Anti-Sickling Properties of Medicinal Plants, Insights in Botanical Medicine*

SCD is one of the most prevailing homogeneous inherited haemoglobinopathies causing a plethora of various clinical complications to the patients. The high mortality and morbidity severely concern the Western community, where numerous clinical trials and research for a cure are in process. In order to alleviate patients from the severe symptoms of the disease, avoiding the side effects, Botanical Medicine exhibits concrete evidence, as a gold candidate, to be the salvation to the problem. The Preferred Reporting Items for Systematic Review (PRISMA) protocol has been used to achieve extensive research on the topic, focusing on the identification and evaluation of the phytochemical properties of common medicinal plants. Meta-analysis has also been implemented on the results of published literature. Forest plots have been plotted, comparing and evaluating the results’ validity and significance. The meta-analysis results have undoubtedly demonstrated the importance and significance of the medicinal plants and their properties against various clinical complications, focusing on the pathogenicity of SCD. Surprisingly, their effectiveness to suppress haemoglobin polymerisation and increase the Fe2+/Fe3+ ratio in patients, enhanced the normal morphological erythrocytes’ appearance by suppressing the sickle shape of drepanocytes. Research made on the epidemiology of SCD associates the disease with the geographical frequency of malaria infection. Based on the natural selection theory of Charles Darwin, nature aids in the population’s survival by the endemicity of various medicinal plants in areas with increased SCD patients. Limitations to the medicinal plants’ consumptions and further therapeutic options have been discussed.

Impacts of low level aflatoxin in feed and the use of modified yeast cell wall extract on growth and health of nursery pigs

This study was to investigate the effect of corn naturally contaminated with aflatoxins(AF)under the regulatory level on the growth performance and health of nursery pigs,and the efficiency of yeast cell wall based feed additive(YC)mainly composed ofβ-glucans and mannan oligosaccharide(MOS)(Integral A+,Alltech,Lexington,KY)in prevention of mycotoxicosis.Pigs(60 barrows and 60 gilts at 6.02 ± 0.83kg BW)were randomly allotted to 4 treatments in a randomized complete block design based on a 2×2 factorial arrangement with 10 pens(5 barrow and 5 gilt pens)per treatment and 3 pigs per pen.Pigs were fed experimental diets for 5 wk.First factor was AF(0 or 20 mg/kg in feed)and the second factor was YC(0 or 2 g/kg in feed).Feed intake and body weight were measured weekly,and blood samples were used to measure blood cell counts,immunoglobulin G(Ig G),tumor necrosis factor-a(TNF-a),oxidative damage status,and serological evaluation related to liver health.Aflatoxin decreased(P<0.05)the number of platelet count(247.4 to 193.5×10~3/μL),and it also tended to increase the level of albumin(P=0.055,3.46 to 3.63 g/d L),albumin:globulin ratio(P=0.050,2.09 to 2.37),and Ca(P=0.080,10.79 to 10.97 mg/d L).Yeast cell wall based feed additive increased(P < 0.05)ADG(493 to 524 g/d),and ADFI(796 to 846 g/d)of pigs whereas G:F was not affected,and it also tended to increase(P=0.055)albumin level(3.46 to 3.63 g/d L).Interactions(P < 0.05)on hemoglobin,hematocrit,and platelet count indicated that YC further increased their levels when pigs were eating AF contaminated feed.Interactions(P < 0.05)on urea nitrogen and blood urea N to creatinine ratio indicated that YC further decreased their levels when feed were contaminated with AF.In conclusion,low level of 20 mg AF/kg under the regulatory level had minor effects on hematology without affecting growth performance,however the supplementation of 2 g/kg YC as a source ofβ-glucans and MOS in feed can improve feed intake and therefore the growth of pigs.

Effects of yeast cell wall on growth performance, immune responses and intestinal short chain fatty acid concentrations of broilers in an experimental necrotic enteritis model

Subclinical necrotic enteritis(NE) causes devastating economic losses in the broiler chicken industry,especially in birds raised free of in-feed antibiotics. Prebiotics are potential alternatives to in-feed antibiotics. Yeast cell wall extract(YCW) derived from Saccharomyces cerevisiae is a prebiotic with known immune modulating effects. This study examined the effects of YCW and antibiotics(AB) during subclinical NE on broiler growth performance, intestinal lesions, humoral immune response and gut microflora metabolites. The study employed a 2 × 3 factorial arrangement of treatments. Factors were:NE challenge(yes or no) and feed additive(control, AB, or YCW). Each treatment was replicated in 8 floor pens with 15 birds per pen. Challenged birds had higher feed conversion ratio(FCR) than unchallenged birds on d 35(P < 0.05). Dietary inclusion of AB decreased FCR regardless of challenge(P < 0.05) on d 24 and 35. Inclusion of YCW reduced serum interleukin-1(IL-1) concentration in NE challenged birds(P < 0.01) and increased immunoglobulin(Ig) G(P < 0.05) and Ig M(P < 0.05) levels compared to other dietary treatments regardless of challenge. Yeast cell wall extract increased formic acid concentration in cecal contents during challenge and increased butyric acid concentration in unchallenged birds on d 16.This study indicates YCW suppressed inflammatory response, promoted generation of immunoglobulin and increased short chain fatty acid production suggesting potential benefits to bird health.

Acaricidal activities of whole cell suspension,cell-free supernatant,and crude cell extract of Xenorhabdus stokiae against mushroom mite (Luciaphorus sp.)

Xenorhabdus bacterium has been used as a biological control agent against Luciaphorus sp., a mushroom mite endemic in Thailand. To develop an effective formulation ofXenorhabdus stokiae, treatments using different parts of X. stokiae isolate PB09 culture, including whole cell suspension, cell-free supernatant, and crude cell extract, were performed. The results show that different parts ofX. stokiae isolate PB09 culture could induce variable effects on mite mortality and fecundity. Application with cell-free supernatant of X. stokiae culture resulted in both the highest mite mortality rate [（89.00＋3.60）%] and the lowest mite fecundity [（41.33_＋23.69） eggs/gravid female]. Whole cell suspen- sion of X. stokiae isolate PB09 culture was found to be slightly less effective than its cell-free supernatant, suggesting that X. stokiae was more likely to release its metabolites with acaricidal activities to the surrounding culture media. Crude cell extract of X. stokiae was not effective against mites. Cell-free supematant of X. stokiae isolate PB09 was the most effective biological control agent and it could be conveniently used in future formulations instead of live bacteria.

Methods to reduce variability in E. Coli-based cell-free protein expression experiments

Cell-free protein synthesis(CFPS)is an established biotechnology tool that has shown great utility in many applications such as prototyping proteins,building genetic circuits,designing biosensors,and expressing cytotoxic proteins.Although CFPS has been widely deployed,the many,varied methods presented in the literature can be challenging for new users to adopt.From our experience and others who newly enter the field,one of the most frustrating aspects of applying CFPS as a laboratory can be the large levels of variability that are present within experimental replicates.Herein we provide a retrospective summary of CFPS methods that reduce variability significantly.These methods include optimized extract preparation,fully solubilizing the master mix components,and careful mixing of the reaction.These have reduced our coefficient of variation from 97.3%to 1.2%.Moreover,these methods allow complete novices(e.g.semester rotation undergraduate students)to provide data that is comparable to experienced users,thus allowing broader participation in this exciting research area.

Determination of the Bioaccessibility of Cadmium in Golden Thread by Physiologically Based Extraction Test Digestion Using the in vitro/Caco2 Cell Model and Subsequent Risk Assessment

Background:The ingestion of golden thread contaminated with heavy metals through the food chain leads to detrimental effects to human health.During digestion,not all of the heavy metals could be released to the gastrointestinal tract and readily to be absorbed by human body.Thus,bioaccessibility is an important issue in health risk assessments.Aims and Objectives:The aims and objectives of this study were to investigate the bioaccessibility of Cd in golden thread and assess the associated health risks based on the exposure to bioaccessible Cd.Materials and Methods:Inductively coupled plasma mass spectrometry(ICP-MS)has been applied to determine the Cd content in golden thread.Physiologically based extraction test(PBET)digestion was performed in the in vitro/Caco2 cell model to investigate the bioaccessibility of Cd in golden thread.Furthermore,the target hazard quotient(THQ)was used to assess the risks of the total and the bioaccessible content of Cd in golden thread.Results:The results revealed that the total Cd content in six batches of golden thread ranged from 3.203 to 5.723 mg/kg.After uptake by Caco2 cells,the bioaccessibility of Cd ranged from 42.36%to 59.73%.The results of the risk assessment indicated that prior to uptake by Caco2 cells,the THQ values of Cd for all batches of golden thread were greater than 1.However,after uptake by Caco2 cells,the THQ values of Cd in all samples were less than 1,thus suggesting that the risks were at a safe level.Conclusion:This study was the first to perform health risk assessment with bioaccessible heavy metals present in traditional Chinese medicine by PBET digestion using an in vitro/Caco2 cell model,thus enabling us to obtain more accurate and objective results while allowing us to avoid unnecessary government intervention and to establish more reasonable limit standards for heavy metals.

Effect of Early Intervention with Extract of Huannao Yicong Decoction(还脑益聪方) on the Pathologic Picture of Hippocampus and Neurocyte Apoptosis in APP Transgenic Mice Model of Dementia

Objective：To observe the effect of early intervention using extract of Huannao Yicong Decoction （还脑益聪方,HYD） on the pathological picture of hippocampus,neurocyte apoptosis,and associated regulatory genes inβ-amyloid precursor protein（APP） transgenic mice model of dementia.Methods：Sixty APP695^（V7171） transgenic mice were randomly divided into four groups of 15.The model group was treated with distilled water, the positive control group was treated with donepezil（0.65 mg/kg）,and the two HYD groups were treated with high dose（2.8 g/kg） and low dose（1.4 g/kg） HYD,respectively.All testing drugs were administered through gastrogavage by dissolving in equal volume of distilled water,once a day for six successive months.In addition, a normal control group with 15 healthy C57BL/6J mice of the same age and genetic background was set up with distilled water treatment.The pathologic picture of brain tissue was observed by microscopy with HE stain;the amount of apoptosis cells in the hippocampal CA1 area was detected by TUNEL;and expressions of associated genes,Bcl-2,and Bax were determined by immunohistochemical method.Results：Pathologic pictures of hippocampus showed that in the model group,cells messily arranged,neurons markedly decreased, and the surrounding tissue of some cells was loosened with edema,necrosis,and widened gap with glia cells proliferation.Compared with those in the normal group,the amount of apoptosis cells in the CA1 area was increased,Bcl-2 expression decreased,and Bax expression increased significantly,with markedly reduced Bcl-2/Bax ratio in the model group.Compared to the model group,the pathological changes were significantly milder in the HYD-treated groups,showing rather regularly arranged cells,significantly increased neurons, only few denatured necrotic cells with milder edema,less proliferation of glia cells,and obviously reduced cell apoptosis in CA1 area（P0.05 or P0.01）.Besides,Bcl-2 expression was up-regulated and Bax expression down-regulated,and Bcl-2/Bax ratio significantly increased in the two HYD groups（P0.05 or P0.01）. Conclusion：Early intervention with HYD could improve the abnormal pathologic picture of hippocampus and regulate the expressions of associated genes to suppress cell apoptosis,which might be its mechanism of action in alleviating cognitive functional disorder.