Second Round Of Selection Research Articles

Two-Round Selection-Based Bit Flipping Decoding Algorithm for LDPC Codes

This paper presents a novel iterative reliability-based bit flipping (BF) algorithm for decoding low-density parity-check codes. The new decoder is a single BF algorithm called two-round selection -based bit flipping. It introduces the idea of a two-round selection of the flipped bit, based successively on hard and soft received channel values. In the first stage, a set of unreliable bits is identified, and then a second selection is used, to pick out among them the bit to flip. In the second round of selection, the initial belief about received signals, contributes efficiently to selecting the best candidate bit. We demonstrate through simulations over the binary-input additive white Gaussian noise channel and the Rayleigh fading channel that the proposed algorithm exhibits better decoding performance when compared with some well-known soft decision BF algorithms. A complexity analysis of the proposal and a comparison to other BF decoders are also presented.

Oral booster vaccine antigen-Expression of full-length native SARS-CoV-2 spike protein in lettuce chloroplasts.

Oral booster vaccine antigen-Expression of full-length native SARS-CoV-2 spike protein in lettuce chloroplasts.

Forecasting Credit Risk of SMEs in Supply Chain Finance Using Bayesian Optimization and XGBoost

Supply chain finance plays a crucial role as a financing channel for small‐ and medium‐sized enterprises (SMEs). However, issues such as financial problems and credit defaults have led to disruptions in this channel. To address credit risk control in SME financing within the field of supply chain finance, this paper focuses on a sample of 506 equipment manufacturing companies listed on the SME board of the Shenzhen Stock Exchange from 2016 to 2020. Taking into consideration, the overall risks faced by these enterprises, the study establishes seven first‐level indicators and identifies 84 candidate second‐level indicators. Partial correlation and variance analysis are then used for the first round of indicator screening, followed by the use of a BP neural network for the second round of selection. As a result, a system of 26 indicators for supply chain financial risk is constructed. The XGBoost model is employed to evaluate the constructed risk index system, while SVM and random forest models are used as comparison models. Bayesian optimization is utilized for parameter tuning of the three models. Empirical results demonstrate that the BO‐XGBoost model reduces prediction errors in comparison to the control models. Furthermore, statistical tests reveal that the predicted values of the BO‐XGBoost model significantly differ from those of the other control models. Compared to other individual models, the BO‐XGBoost model exhibits increased accuracy in credit risk prediction and a significant discriminative effect. These findings highlight the effectiveness of constructing an efficient risk indicator system and utilizing Bayesian optimization for parameter tuning in XGBoost to better differentiate between risky and normal enterprises, thereby minimizing default losses. The research results underscore the advantages of employing Bayesian optimization in XGBoost, which can be applied in credit default prediction for SMEs and serves as a valuable tool in financial risk management and control.

Fenpropathrin resistance in Asian citrus psyllid, Diaphorina citri Kuwayama: risk assessment and changes in expression of CYP and GST genes associated with resistance

Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), populations exhibit varying levels of resistance to several insecticide classes, including pyrethroids. Artificial laboratory selection was conducted to investigate mechanisms underlying fenpropathrin resistance. After ten generations of selection, the LC50 for fenpropathrin increased from 0.18 to 11.60 ng/μL, and the associated resistance ratio increased by 96.67-fold. The realized heritability of resistance (h2 ) to fenpropathrin changed from 0.17 to 0.44 during the first and second round of selection, respectively. We did not find high cross-resistance to imidacloprid (RR = 3.58) or dimethoate (RR = 1.77) in the fenpropathrin-selected strain, but found cross-resistance to bifenthrin (RR = 28.21). RT-qPCR analysis showed that expression of CYP6A2-1 was significantly increased in the selected population relative to the laboratory susceptible population. Our data indicate that D. citri has the capacity to develop a high level of fenpropathrin resistance following persistent selection and that cytochrome P450 (CYP) and glutathione S-transferase (GST) metabolic detoxification may be contributing mechanisms. Rotation programs that include neonicotinoids and organophosphates should manage resistance to fenpropathrin for D. citri.

Inheritance of virulence inFusarium circinatum, the cause of pitch canker in pines

Wildtype strains ofFusarium circinatum, the causal agent of pitch canker, were crossed to obtain an F1generation. Progeny of this cross were tested for virulence by inoculatingPinus radiataseedlings, and were found to induce a wide range of lesion lengths. Two strains from the F1generation that induced long lesions (= high virulence) were used as parents to produce an F2generation, followed by a second round of selection for high virulence to obtain an F3generation. Mean lesion lengths were not significantly different between the three generations (P ≥ 0.196). A parallel set of crosses was performed to select for low virulence by using progeny in the F1and F2generations that induced short lesions as parents for F2and F3generations, respectively. In this case, both rounds of selection resulted in a significant reduction in mean lesion length, from 33.8 ± 0.8 mm in the F1generation, to 19.7 ± 0.7 and 12.9 ± 0.7 mm in the F2and F3generations, respectively. Thus it is apparent thatF. circinatumretains the genetic capacity for avirulence to pines, which could reflect a lack of strong selection for virulence in nature. Progeny of a cross between high and low virulence parents manifested nearly continuous variation in lesion lengths, consistent with virulence being a quantitatively inherited trait. Based on this cross, broad‐sense heritability (H2) was determined to be 0.74, which suggests that virulence is under strong genetic control.

MTR-08MODELING TMZ RESISTANCE IN PATIENT-DERIVED BRAIN TUMOR- INITIATING CELLS

Intrinsic and required resistance hampers the inability for Temozolomide to give prolonged therapeutic response in glioblastoma. To this end we developed an in vivo model to investigate the mechanism behind TMZ resistance. Using TMZ-sensitive patient-derived brain tumor-initiating cells (BTIC; BT73 and BT206), intracranial tumors were established in immunocompromised mice. Once visible, animals were treated with TMZ [50 mg/kg/day (one cycle) followed by 5 cycles of 10 mg/kg/day (cycle = 5d on, 2d off)] and followed for recurrence. Tumor cells were isolated and re-implanted into animals for a second round of selection [TMZ 50 mg/kg/day (one cycle); 30 mg/kg/day (two cycles)]. BTICs established from these animals were termed BT73R and BT206R and confirmed for their acquired resistance to TMZ both in vitro and in vivo. Using cytokine profiling arrays and global gene expression arrays, we identified candidate genes/factors related to the development of TMZ resistance. Assessment of tumor interstitial fluid from BT73R and BT206R uncovered changes in several cytokines/chemokines including an increase in CX3CL1, IL16 and IL23 and a decrease in MDC, IFNa2, IP-10 and CCL2. In addition, we observed a greater than two-fold change in 131 genes found in common between the TMZ-resistant xenografts including 30 genes related to tumor adhesive, invasive and drug resistance that were selected for further validation. To date, of the common genes, 11 were confirmed including 7 up-regulated genes (IGFBP5, EFEMP1, TCEAL7, SLFN11, FAM129A, PCDH10 and DCX) and 4 down-regulated genes (DACH1, ICAM1, MLKL and NFTAC2). In addition, MGMT protein was detected in BT73R but not BT206R. The data presented here suggests that acquired resistance to TMZ in the BTICs may not be mediated through a single mechanism, but rather via multiple genes/pathways warranting further evaluation of these models in the hope of identifying potential therapeutic markers or targets for acquired TMZ resistance.

Additive Genetic Effects for Resistance to Foliar Powdery Mildew in Strawberry Revealed through Divergent Selection

Powdery mildew (PM) of strawberry (Fragaria sp.) is a ubiquitous, wind-spread disease caused by the obligate parasite Podosphaera aphanis. To control PM, multiple fungicide applications are necessary each season, and none of the major cultivars in Florida have high levels of resistance. Therefore, the objectives of this study were to observe the response to selection and to estimate genetic parameters for PM and related traits in the University of Florida breeding population. In 2010, clonally replicated individuals from seven biparental crosses arising from 11 parents were included in a field trial in which clonally replicated seedlings were evaluated visually for percent coverage of PM mycelium using a modified Horsfall-Barratt scale of 0 to 6. Broad- (H2) and narrow-sense (h2) heritabilities for PM score were (mean ± se) 0.50 ± 0.08 and 0.40 ± 0.39, respectively, for the base population. After the second round of selection in the resistant population, no additive variance was detected, indicating that alleles for PM resistance had become fixed. In contrast, after two rounds of divergent selection in the susceptible population, there remained considerable additive variance (h2 = 0.42 ± 0.65). Moderate to high heritability estimates and a clear response to selection indicate that resistance to PM is genetically controlled through mostly additive effects. Selection of parents based on field trials with natural inoculum should result in good progress toward more resistant cultivars. The consistently moderate to strong genotypic and genetic correlations among PM and canopy density (CD) indicate that selection for PM resistance will result in reduced CD. Therefore, CD must be monitored over successive rounds of selection for low levels of PM to prevent CD falling below the commercially acceptable range.

Agrobacterium tumefaciens-mediated genetic transformation of Salix matsudana Koidz. using mature seeds

An Agrobacterium tumefaciens-mediated transformation method was developed for Salix matsudana Koidz. using mature seeds as starting material. Multiple shoots were induced directly from embryonic shoot apices of germinating seeds. Although thidiazuron, 6-benzylaminopurine and zeatin induced multiple shoot induction with high frequency, zeatin (4.5 μM) was more effective for elongation of shoots and roots. The binary vector pCAMBIA1303, which contained neomycin phosphotransferase as a selectable marker gene and β-glucuronidase as a reporter gene, was used for transformation. Factors affecting transformation efficiency were examined for optimization of the procedure. Up to 35 of 180 seeds regenerated kanamycin-resistant shoots under optimal transformation conditions as follows: seeds were precultured for 4 days, apices of embryonic shoots were removed and infected with A. tumefaciens strain LBA4404 grown to a cell density equivalent (OD600) of 0.6, and then the infected explants were cultivated at 21 °C for 4 days. Storage of seeds at -20 °C for as long as 3 years had no significant effect on the induction of kanamycin-resistant shoots. Using this method, transgenic plants were obtained within ∼5 months with a transformation frequency of 7.2%. Analysis by polymerase chain reaction (PCR) showed that 36.4-93.8% of plants from all 13 tested kanamycin-resistant lines were PCR positive. Several 'escapes' were eliminated by a second round of selection. PCR, Southern blot and reverse transcriptase-PCR analyses of selected transgenic individuals 2 years after cutting propagation confirmed the successful generation of stable transformants. Our method, which minimizes the duration of axenic culture, may provide an alternative procedure for transformation of other recalcitrant Salix species.

Optimization of Factors Influencing Microinjection Method for Agrobacterium tumefaciens-Mediated Transformation of Tomato

A simple and efficient protocol for Agrobacterium-mediated genetic transformation of tomato was developed using combination of non-tissue culture and micropropagation systems. Initially, ESAM region of 1-day-old germinated tomato seeds were microinjected for one to five times with Agrobacterium inoculums (OD(600) = 0.2-1.0). The germinated seeds were cocultivated in the MS medium fortified with (0-200mM) acetosyringone and minimal concentrations of (0-20mg L(-1)) kanamycin, and the antibiotic concentration was doubled during the second round of selection. Bacterial concentration of OD(600) = 0.6 served as an optimal concentration for infection and the transformation efficiency was significantly higher of about 46.28%. In another set of experiment, an improved and stable regeneration system was adapted for the explants from the selection medium. Four-day-old double cotyledonary nodal explants were excised from the microinjected seedlings and cultured onto the MS medium supplemented with 1.5mg L(-1) thidiazuron, 1.5mg L(-1) indole-3-butyric acid, 30mg L(-1) kanamycin, and 0-1.5mg L(-1) adenine sulphate. Maximum of 9 out of 13 micropropagated shoots were shown positive to GUS assay. By this technique, the transformation efficiency was increased from 46.28 to 65.90%. Thus, this paper reports the successful protocol for the mass production of transformants using microinjection and micropropagation techniques.

Efficient selection of Gal-knockout pig cells for somatic cell nuclear transfer

BackgroundThe process of selecting transgenic cells has been one of the bottlenecks in the generation of transgenic animals by somatic cell nuclear transfer (SCNT). In particular, selection for the Gal double-knockout (Gal-DKO) genotype has been time consuming and inefficient. The objective of this work was to generate a highly efficient system to select Gal-DKO cells to be used in SCNT without affecting the efficiency in production of Gal-null pigs. Materials and methodsFetal liver–derived cells deficient in Gal-expression were initially selected by fluorescence-activated cell sorting (FACS) using IB4 conjugated to a fluorescent dye. Cells recovered by FACS were cultured and expanded, followed by a second round of selection using streptavidin magnetic beads and IB4 lectin biotin. ResultsRecovery efficiency of target cells was 0.04% for the first selection using FACS and 0.3% for the second round by magnetic beads. Full reprogramming was obtained on selected Gal-DKO cells after FACS and magnetic beads selection, when used for SCNT to produce the Gal-null piglets. Cells obtained from magnetic beads developed 48 colonies; the Gal-null genotype was found in 44 of them (91.7%). Three of these colonies were used to generate piglets by SCNT. From three recipients receiving embryos, two became pregnant and produced 17 piglets, all of them DKO. ConclusionsSequential selection of Gal-DKO cells by FACS/magnetic beads is a highly efficient system to generate null cells. Selected cells were successfully used to generate healthy double-knockout piglets by SCNT.

“Pure Hoosier”: Fieldwork, Food Guides, and Cultural Distinction

"Pure Hoosier":Fieldwork, Food Guides, and Cultural Distinction1 Danille Elise Christensen (bio) Joanne Raetz Stuttgen Cafe Indiana: A Guide to Indiana's Down-Home Cafes. Madison: University of Wisconsin Press, 2007. 288 pages. Joanne Raetz Stuttgen and Jolene Ketzenberger Cafe Indiana Cookbook. Madison: University of Wisconsin Press, 2010. 224 pages. The cover of Cafe Indiana, Joanne Raetz Stuttgen's 2007 guide to "Indiana's down-home cafes," features a photo of chrome stools bathed in early morning light while upended mugs await customers at the counter. The guide's companion publication, a cookbook, bears a cover dominated by a single serving of coconut cream pie—"the most popular pie in Hoosier cafes," according to Stuttgen (2010, 114). These images succinctly suggest the goals and foci of the linked volumes. Cafe Indiana is an impressive ethnographic endeavor: between 2004 and 2007, Stuttgen visited hundreds of Indiana cafes, and the counter was her preferred place to observe, interact with, and evaluate the people and food she happened upon. Cafe Indiana Cookbook, published three years later with co-author and food columnist Jolene Ketzenberger, incorporates some of the anecdotes and observations from the 2007 guide, but its focus is on teaching readers to prepare a discrete body of recipes. Both books are overtly evaluative: the first claims authority to distinguish authenticity and quality of experience, pointing readers to cafes that have met specific criteria. The second is more [End Page 97] subtle in its brokering: as in any collection, the cookbook directs its audience to read its contents as representative, but the cull pile—the stack of recipes not chosen for inclusion—is nowhere to be found. The two books are helpfully read together, then, since Cafe Indiana gives a sense of what Cafe Indiana Cookbook elides or enhances. As these accessible paperbacks call attention to good food, good stories, and good company, they also foster thinking about the nuts and bolts of fieldwork, modes of self-representation and heritage construction, and the consequences of bringing people and places to broader public notice. Cafe Indiana (2007) is modeled on Cafe Wisconsin (1993), a guide Stuttgen published while pursuing a PhD in Folklore from Indiana University. Updated and republished in 2004 by Terrace Books (the trade imprint of the University of Wisconsin Press), Cafe Wisconsin includes a preface that establishes Stuttgen's interest in traditional vernacular culture (i.e., those "things that maintain a familiar sameness despite change over time and across regions" [1993, 9]). The preface also reviews previous roadfood literature, outlines the author's procedures for selecting field sites and her criteria for recommending particular eateries, and previews the layout of the book, which is organized by intrastate regions. Cafe Indiana includes a version of this essay that is revised in its Hoosier-state particulars but consistent in focus and methods: for both projects, Stuttgen looked for cafes located in the original business districts of towns with fewer than 10,000 residents. Ideal cafes for her purposes were owner operated, demonstrated some level of historical continuity, and catered to a significantly local (and primarily daytime) clientele. In both Wisconsin and Indiana she began with 400 or so possible restaurants, but only about half the list made the next cut. In this second round of selection, Stuttgen favored cafes with primarily "home-cooked" food, clean yet idiosyncratic décor, and friendly staff and patrons; she stayed away from the "trendy," the mass-produced, and the aloof (i.e., staff or patrons who seemed begrudging or inarticulate; 2007, xiv, xvii; cf. Stuttgen 1993, 11-13). While in 1993 it was enough to say that she'd "concentrated entirely on traditional cafes hiding in small towns off the beaten path" (11), by the time Stuttgen explored Indiana eateries she needed to distinguish her work from several recently published guides to that state's 'Main Street' diners (e.g., Duffy [2001] 2006; Trogdon 2001). Consequently, in Cafe Indiana she emphasizes her "deep focus on Hoosier food traditions and the varied roles cafes fill in the lives of individuals [End Page 98] and communities" (2007, xiv). Indeed, Stuttgen attends to the taste, texture, and preparation of food served, but she also comments on the décor...

Selective Breeding for Susceptibility to Myopia Reveals a Gene–Environment Interaction

Purpose. To test whether the interanimal variability in susceptibility to visually induced myopia is genetically determined. Methods. Monocular deprivation of sharp vision (DSV) was induced in outbred White Leghorn chicks aged 4 days. After 4 days' DSV, myopia susceptibility was quantified by the relative changes in axial length and refraction. Chicks in the extreme tails of the distribution of susceptibility to DSV were kept and paired for breeding (high- and low-susceptibility lines). A second round of selection was then performed. The third generation of chicks, derived from the selected parents, was assessed after either monocular DSV (4 or 10 days) or lens wear. Results. After two rounds of selective breeding, the chicks from the high-susceptibility line developed approximately twice as much myopia in response to 4 days' DSV as did those from the low-susceptibility line (P < 0.001). All ocular component dimensions differed significantly (P < 0.001) between the two selected lines, both before treatment and in the responses of the treated eye. When DSV was conducted for 10 days, the relative changes in axial length and refractive error were still significantly different between the high and low lines (P < 0.001). The chicks bred for high or low susceptibility to DSV also showed significantly different responses to minus lens wear, but not to plus lens wear. Additive genetic effects explained ∼50% of the interanimal variability in response to DSV. Conclusions. Genes and environment interact to shape refractive development in chicks.

Amplification of Adenine Phosphoribosyltransferase Suppresses the Conditionally Lethal Growth and Virulence Phenotype of Leishmania donovani Mutants Lacking Both Hypoxanthine-guanine and Xanthine Phosphoribosyltransferases

Leishmania donovani cannot synthesize purines de novo and obligatorily scavenge purines from the host. Previously, we described a conditional lethal Deltahgprt/Deltaxprt mutant of L. donovani (Boitz, J. M., and Ullman, B. (2006) J. Biol. Chem. 281, 16084-16089) that establishes that L. donovani salvages purines primarily through hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and xanthine phosphoribosyltransferase (XPRT). Unlike wild type L. donovani, the Deltahgprt/Deltaxprt knock-out cannot grow on 6-oxypurines and displays an absolute requirement for adenine or adenosine and 2'-deoxycoformycin, an inhibitor of parasite adenine aminohydrolase activity. Here, we demonstrate that the ability of Deltahgprt/Deltaxprt parasites to infect mice was profoundly compromised. Surprisingly, mutant parasites that survived the initial passage through mice partially regained their virulence properties, exhibiting a >10-fold increase in parasite burden in a subsequent mouse infection. To dissect the mechanism by which Deltahgprt/Deltaxprt parasites persisted in vivo, suppressor strains that had regained their capacity to grow under restrictive conditions were cloned from cultured Deltahgprt/Deltaxprt parasites. The ability of these suppressor clones to grow in and metabolize 6-oxypurines could be ascribed to a marked amplification and overexpression of the adenine phosphoribosyltransferase (APRT) gene. Moreover, transfection of Deltahgprt/Deltaxprt cells with an APRT episome recapitulated the suppressor phenotype in vitro and enabled growth on 6-oxypurines. Biochemical studies further showed that hypoxanthine, unexpectedly, was an inefficient substrate for APRT, evidence that could account for the ability of the suppressors to metabolize hypoxanthine. Subsequent analysis implied that APRT amplification was also a potential contributory mechanism by which Deltahgprt/Deltaxprt parasites displayed persistence and increased virulence in mice.

Role of Transmembrane Domain 4 in Ligand Permeation by Crithidia fasciculata Equilibrative Nucleoside Transporter 2 (CfNT2)

Equilibrative nucleoside transporters play essential roles in nutrient uptake, cardiovascular and renal function, and purine analog drug chemotherapies. Limited structural information is available for this family of transporters; however, residues in transmembrane domains 1, 2, 4, and 5 appear to be important for ligand and inhibitor binding. In order to identify regions of the transporter that are important for ligand specificity, a genetic selection for mutants of the inosine-guanosine-specific Crithidia fasciculata nucleoside transporter 2 (CfNT2) that had gained the ability to transport adenosine was carried out in the yeast Saccharomyces cerevisiae. Nearly all positive clones from the genetic selection carried mutations at lysine 155 in transmembrane domain 4, highlighting lysine 155 as a pivotal residue governing the ligand specificity of CfNT2. Mutation of lysine 155 to asparagine conferred affinity for adenosine on the mutant transporter at the expense of inosine and guanosine affinity due to weakened contacts to the purine ring of the ligand. Following systematic cysteine-scanning mutagenesis, thiol-specific modification of several positions within transmembrane domain 4 was found to interfere with inosine transport capability, indicating that this helix lines the water-filled ligand translocation channel. Additionally, the pattern of modification of transmembrane domain 4 suggested that it may deviate from helicity in the vicinity of residue 155. Position 155 was also protected from modification in the presence of ligand, suggesting that lysine 155 is in or near the ligand binding site. Transmembrane domain 4 and particularly lysine 155 appear to play key roles in ligand discrimination and translocation by CfNT2.

Establishing a base population for a breeding program in aquaculture, from multiple subpopulations, differentiated by genetic drift: I. Effects of the number of subpopulations, heritability and mating strategies using optimum contribution selection

Guidelines for constructing base populations in fish were examined using a drift model. A large number of drift differentiated, wild subpopulations, with inbreeding Fwild (Fwild∈[0.05–0.20]), and a trait with heritability htot2 (htot2∈[0.10–0.30]), were assumed. A base population was generated by sampling 2N fish from each of nsub subpopulations (nsub∈[1–8]), and then mated to produce 160 or 240 families per generation. The matings were either at random, in a structured diallel-cross, or within subpopulations to produce generation 1. Optimal contribution selection on phenotype was applied from generation 1 to11, with a rate of inbreeding of 0.5% per generation. Genetic gain (Δg) improved with nsub, the improvement being largest when going from one to two subpopulations, with diminishing returns. Most of the potential improvement was obtained with nsub=4, and was well predicted by ΔgnsubΔg1≈(1−Fwild/nsub)(1−Fwild). Initial matings across subpopulations improved Δg from the second round of selection compared to within. Consequently the effect of the mating strategy were first observed 3 generation after decisions were made and persisted over subsequent generations, but with minimal effect on the accumulated additive genetic gain in generation 11. It was concluded that sampling from at least four subpopulations was beneficial and that mating across subpopulations should start as early as possible.

Establishment of liver metastasis model of human gallbladder cancer and isolation of the subpopulation with high metastatic potential

To establish an experimental liver metastatic model of gallbladder cancer and isolate the subpopulation with high metastatic potential from the model, which may serve as a reliable tool in research on liver metastasis of gallbladder cancer in vivo and in vitro. Human gallbladder cancer cells of the line GBC-SD were cultured. Ten nude athymic BALB/c mice, aged 4 approximately 5 weeks, underwent inoculation of suspension of GBC-SD cells into the spleens and then their spleens were resected. Three weeks later laparotomy was performed to observe if liver metastasis occurred. Once liver metastasis was discovered, the mice were killed and the livers were taken out to undergo microscopy, tumor cells were isolated from the metastatic foci and cultured in vitro, and then inoculated into other 10 mice in the same way as their parents cells for the second round of selection. The similar steps were repeated, altogether for three rounds of selection and a total 30 mice were inoculated in 3 rounds so as to find subpopulation with high metastatic potential. Another 10 mice underwent subcutaneous inoculation, 90 days later the mice were killed to observe if pulmonary metastasis occurred. PCR was used to detect the 3 microsatellite sequences D14S68, D18S69, and D20S199 in the DNA samples of the gallbladder cancer cells of the parent cell line GBC-SD, the isolated cells of the subpopulation with high metastatic potential, and the liver of experimental animal. 90% of the mice inoculated subcutaneously with the GBC-SD cells developed subcutaneous tumors, however, no mouse in this group died and no pulmonary metastasis was found. The liver metastatic rate was 65% in the 10 mice undergoing intrasplenic inoculation. Thus a metastatic model of human gallbladder cancer in nude mice was established. The liver metastatic tumors were uniformly distributed throughout the liver parenchyma with predominance to the periphery. Despite multiple sites of involvement, the left lobes were most commonly affected in all experimental animals. Histological examination of the metastatic lesion demonstrated adenocarcinoma. Gross hepatic metastasis was detected 10, 7, and 5 weeks after the inoculation respectively in the first, second, and third round selection respectively with an incidence rate of metastases of 60%, 70%, and 90% respectively. From the third round metastatic model a subpopulation with high metastatic potential was isolated and designated as GBC-SD/M, which exhibited the similar histological characteristics as its parent cell line GBC-SD under inverted light microscopy. The three amplified bands at the sites 14S68, D18S69, and D20S199, amplified with the three pairs of specific primers for the three homo-specific microsatellites, were detected in the GBC-SD cells and GBC-SD/M cells, but not in the liver of tumor-bearing animal. A liver metastasis model of human gallbladder cancer has been established, a reliable and efficient tool for study on the metastasis of gallbladder cancer to liver in vivo. Isolated from hepatic metastasis, the line of GBC-SD/M is a subpopulation with high metastatic potential, retaining the histological properties and identification of genetic background of its parent cell line GBC-SD.

Construction and characteristics of a transformed lepidopteran cell clone expressing baculovirus p35

A transformed cell line was constructed fromMythimna separata cells Ms7311 by lipofection method. TMs7311 cells were generated using a double selection technique involving a selection in the antibiotic Zeocin, followed by a second round of selection by exhibiting cell characterization. A cell clone expressing p35 was obtained with high level of AcMNPV and recombinant proteins. Compared with wild type Ms7311 cells, the cell clone showed increased resistance to Actinamycin D-induced apoptosis and a profound resistance to nutrient development (PBS). When the cell clone was infected with recombinant baculoviruses expressing secreted alkaline phosphatase (SEAP) and β-galactosidase, expression of the recombinant proteins from TMs7311 cells exceeded that from parental Ms7311 cells. Production of budded virus and occlusion body was significantly higher than that from parental cells Ms7311.

Acquisition of rifabutin resistance by a rifampicin resistant mutant of Mycobacterium tuberculosis involves an unusual spectrum of mutations and elevated frequency

BackgroundMutations in a small region of the rpoB gene are responsible for most rifamycin resistance in Mycobacterium tuberculosis. In this study we have sequentially generated resistant strains to first rifampicin and then rifabutin. Portions of the rpoB gene were sequenced from 131 randomly selected mutants. Second round selection resulted in a changed frequency of specific mutations.MethodsMycobacterium tuberculosis (strain Mtb72) rifamycin resistant mutants were selected in vitro with either rifampicin or rifabutin. One mutant R190 (rpoB S522L) selected with rifampicin had a rifampicin MIC of 32 μg/ml but remained sensitive to rifabutin (MIC<0.8 μg/ml). This mutant was subjected to a second round of selection with rifabutin.ResultsAll 105 first round resistant mutants derived from the parent strain (Mtb72) screened acquired mutations within the 81 bp rpoB hotspot. When the rifampicin resistant but rifabutin sensitive S522L mutant was subjected to a second round of selection, single additional rpoB mutations were identified in 24 (92%) of 26 second round mutants studied, but 14 (54%) of these strains contained mutations outside the 81 bp hotspot (codons 144, 146, 148, 505). Additionally, spontaneous rifabutin resistant mutants were produced at >10 times the frequency by the S522L mutant than the parent strain.ConclusionFirst round selection of mutation S522L with rifampicin increased the frequency and changed the spectrum of mutations identified after selection with rifabutin.

Evaluation of metastatic potential in prostate carcinoma: An in vivo model

Prostate cancer is frequently associated with bone metastases, which are in fact the main cause of morbidity and mortality for this tumor. To better investigate this process, animal models of bone and bone marrow metastases need to be developed. However, experimental prostate cancer bone metastases are difficult to be obtained in vivo, and some typical clinical patterns remain irreproducible. In this study, we injected PC3 prostate cancer cells into the left cardiac ventricle of nude mice, thus reproducing the basic biological phenomenon of tumor cell spreading in the arterious blood stream, and compared the outcome with direct injection of cells in the bone marrow cavity of the tibia. Mice were monitored by X-ray analysis. After 40 days, 100% of intratibially-injected and 64% of heart-injected mice revealed osteolytic lesions. The heart injection was then used to select PC3 cell subpopulations by serial inoculation and recovery from bone metastasis. One of the resulting cell populations, obtained by a second round of selection and denominated PCb2, showed a more invasive phenotype compared to parental PC3, both in vitro and in vivo. Although PCb2 cells had a growth rate comparable to that of PC3 cells, they generated a higher number of bone lesions in nude mice and crossed more easily the Matrigel barrier in vitro in the presence of several chemoattractants. This phenomenon was partially due to an increased capacity to adhere to laminin and to release MMP-2 at higher level relative to the original PC3 cells. This study demonstrates that heart injection of prostate cancer cells in nude mice may represent a good experimental model to investigate the pathophysiology of bone and bone marrow metastases in vivo.

Stable genetic transformation of Vigna mungo L. Hepper via Agrobacterium tumefaciens.

Vigna mungo is one of the large-seeded grain legumes that has not yet been transformed. We report here for the first time the production of morphologically normal and fertile transgenic plants from cotyledonary-node explants inoculated with Agrobacterium tumefaciens carrying binary vector pCAMBIA2301, the latter of which contains a neomycin phosphotransferase ( nptII) gene and a beta-glucuronidase (GUS) gene ( uidA) interrupted with an intron. The transformed green shoots, selected and rooted on medium containing kanamycin, tested positive for nptII and uidA genes by polymerase chain reaction (PCR) analysis. These shoots were established in soil and grown to maturity to collect the seeds. Mechanical wounding of the explants prior to inoculation with Agrobacterium, time lag in regeneration due to removal of the cotyledons from explants and a second round of selection at the rooting stage were found to be critical for transformation. Analysis of T(0) plants showed the expression and integration of uidA into the plant genome. GUS activity in leaves, roots, flowers, anthers and pollen grains was detected by histochemical assay. PCR analysis of T(1) progeny revealed a Mendelian transgene inheritance pattern. The transformation frequency was 1%, and 6-8 weeks were required for the generation of transgenics.