Combinatorial Interaction Testing (CIT) Benchmarks

<p>There are five subject sets used in our experiments. The details are summarized below: [Syn-2] contains 14 pairwise (2-way) synthetic models without constraints. These are shown in the leftmost column of Table I.  These models are benchmarks that have been used both to compare mathematical constructions as well as search based techniques [2], [10], [11], [18], [32]. We take these from Table 7 from the paper by Garvin et al.  [2]. [Syn-3] contains 15 3-way synthetic models without constraints.  These are shown in the second column of Table I. These models are benchmarks that have been used for mathematical constructions and search [10], [33], [34]. We take these from Table 7 from the paper by Garvin et al. [2]. [Syn-C2] contains 30 2-way synthetic models with constraints (see Table I, rightmost two columns). These models were designed to simulate configurations with constraints in real-world programs, generated by Cohen et al. [35] and adopted in follow-up research by Garvin et al. [2], [25]. [Real-1] contains real-world models from a recent benchmark created by Segall et al. [21], shown in Table II. There are 20 CIT problems in this subject set, generated by or for IBM customers. The 20 problems cover a wide range of applications, including telecommunications, healthcare, storage and banking systems. [Real-2] contains 6 real-world constrained subjects shown in Table II, which have been widely studied in the literature [2], [25], [30], [35], [36]. The TCAS model was first presented by Kuhn et al. [36]. TCAS is a traffic collision avoidance system from the ‘Siemens’ suite [37]. The rest of the models in this subject set were introduced by Cohen et al. [30], [35]. SPIN-S and SPIN-V are two components for model simulation and model verification. GCC is a well known compiler system from the GNU Project. Apache is a web server application and Bugzilla is a web-based bug tracking system.</p

Table_1_Transcriptional Analysis of the Effects of Gambogic Acid and Neogambogic Acid on Methicillin-Resistant Staphylococcus aureus.docx

Methicillin-resistant Staphylococcus aureus (MRSA) infection is a major threat to human health, as this bacterium has developed resistance to a variety of conventional antibiotics. This is especially true of MRSA biofilms, which not only exhibit enhanced pathogenicity but also are resistant to most antibiotics. In this work, we demonstrated that two natural products with antitumor activity, namely, gambogic acid (GA) and neogambogic acid (NGA), have significant inhibitory activity toward MRSA. GA and NGA can not only effectively inhibit planktonic MRSA strains in vivo and in vitro, but also have strong inhibitory effects on MRSA biofilms formation. By transcriptome sequencing, Q-RT-PCR and PRM, we found that GA and NGA could reduce the expression of S. aureus virulence factors by inhibiting the saeRS two-component, thus achieving inhibition of MRSA. We found that GA and NGA had anti-MRSA activity in vivo and in vitro and identified saeRS to be the target, indicating that saeRS inhibitors may be used to treat biofilm-related infections.</p

Numbers of detected peaks and distinct loci.

<p>The stacked bar plots show the number of peaks detected for each trait using X-QTL selections in each cross. The first parent listed in each cross was MATα and the second parent was MAT<b>a</b>. The grey dots indicate the number of distinct loci detected in a condition after peak grouping.</p

CDK5RAP2 colocalizes with dynein.

<p>(A) MDA-MB-231 cells expressing GFP-CDK5RAP2 were stained with anti-DIC and fluorescent secondary antibodies. Arrowheads point to overlapping CDK5RAP2 and DIC signals, which are shown magnified in the insets. Scale bar, 10 μm. (B) Transport of GFP-CDK5RAP2 in living cells. The dynamics of CDK5RAP2 was monitored using MDA-MB-231 cells that stably expressed GFP-CDK5RAP2 at levels similar to that of the endogenous protein. Here a time series is shown for GFP-CDK5RAP2, with time points indicated at the upper left corner. Arrowheads point to a mobile CDK5RAP2-containing particle (again magnified in insets). Images shown here are representatives from at least three independent experiments.</p

Genome-wide plots of detected loci.

<p>(A) Loci detected for each cross and trait, with green indicating loci selected in the direction of the MATα parent and red indicating loci selected in the direction of the MATa parent. For each trait, the crosses are vertically ordered as follows: BYxRM, BYxYJM, BYxYPS, RMxYJM, RMxYPS, YJMxYPS. (B) The number of traits affected by loci within each 50-kb window. The grey dotted line shows the threshold for significance, while the black dotted line highlights the bins in which only one trait was affected.</p

Construction of the FRET Pairs for the Visualization of Mitochondria Membrane Potential in Dual Emission Colors

Mitochondria membrane potential (MMP) play significant roles during metabolism, signaling, and other important bioevents. Visualization of MMP levels is essential for many biological researches. However, fluorescent probes for monitoring MMP levels in dual emission colors are still deficient, which greatly limited the development of relative research areas. In this work, a pair of fluorescent probes have been designed and synthesized to monitor the MMP levels in dual emission colors based on Forster resonance energy transfer (FRET) mechanism. The FRET donor (FixD) is constructed by linking a benzyl chloride group to a fluorophore with bright-green emission. The FixD could target mitochondria and be immobilized in mitochondria by linking to the thiol group of mitochondrial proteins. The FRET acceptor (LA) is designed with green absorption and deep-red emission. In live cells with high MMP levels, FixD and LA both target mitochondria, and deep-red (DR) emission could be detected with the excitation of 405 nm. Particularly, the spectral shift of fluorescence upon the decrease of MMP is up to 110 nm, which is greatly favorable for the clear observation of MMP levels. With the decrease of MMP, LA would be released from mitochondria while FixD would still be immobilized in mitochondria, and decreased DR emission and increased green fluorescence could be detected due to the absence of FRET. In this manner, the MMP levels could be monitored in dual emission colors

CDK5RAP2 binds to DLC8.

<p>(A) Extracts of HEK293T cells transfected with FLAG-CDK5RAP2 (706–1241) or the empty vector were used in immunoprecipitation assays with anti-FLAG. Immunoprecipitated proteins stained on SDS-PAGE gels were digested with trypsin, extracted, and analyzed by mass spectrometry. Arrows show the bands corresponding to DLC8 and the CDK5RAP2 fragment. (B) Mapping the DLC8-binding site in CDK5RAP2. Myc-DLC8 and various CDK5RAP2 fragments (FLAG tagged) were co-transfected into HEK293T cells. After immunoprecipitation with the anti-FLAG antibody, captured samples and cell extracts were probed with anti-FLAG and anti-Myc antibodies. (C) Direct binding of DLC8 to CDK5RAP2. Recombinant CDK5RAP2 (706–925) and DLC8 were subjected to an in vitro binding assay: after pulling-down GST-DLC8 with GSH-beads, anti-His₆ immunoblotting was performed to detect His₆-CDK5RAP2 (706–925). (D) Alignment of the DLC8-binding motif in human, mouse and chicken CDK5RAP2. (E) Anti-GFP immunoprecipitation was performed using HEK293T cells co-expressing wild-type or mutant GFP-CDK5RAP2 with Myc-DLC8 and the immunoprecipitates were probed on blots with anti-Myc and anti-GFP antibodies. Ctrl, GFP vector; WT, CDK5RAP2 wild-type; Mu, CDK5RAP2 (Q874A/T875A). Blots shown here are representatives of three separate experiments.</p

Encapsulation of Tetraphenylethylene Derivative in Liposome Vesicles as Promising Aggregation-Induced Electrochemiluminescence Emitter for Detection of Human Epidermal Growth Factor Receptor 2

In this work, a sensitive signal-on electrochemiluminescence biosensor using liposome-encapsuled 1,1,2,2-tetra(4-carboxylphenyl)ethylene (TPE) as a promising aggregation-induced electrochemiluminescence (AIECL) emitter for detection of biomarkers was developed. Aggregation-induced enhancement occurs internally through the spatial confinement effect and intramolecular self-encapsulation of encapsulating TPE and triethylamine (TEA) molecules in liposome cavities. Peptide sequence WTGW­CLNP­EEST­WGFC­TGSF (WF-20) was used to replace the antibody for reducing the steric hindrance of the sensing surface while taking into account the affinity. The proposed sensing strategies showed satisfactory properties for detection of human epidermal growth factor receptor 2 (HER2) ranging from 0.01 to 500 ng/mL with a limit of detection of 6.65 pg/mL. The results confirmed that encapsulation of luminescent molecules in the vesicle structure for triggering the AIECL phenomenon is a promising method to prepare a signal label for a trace detection biomarker

CDK5RAP2 associates with dynein.

<p>(A) Extracts of HEK293T cells expressing FLAG-CDK5RAP2 were incubated with taxol-polymerized microtubules in the absence or presence of AMP-PNP; control samples lacked polymerized microtubules. After incubation, microtubules were spun down through a sucrose cushion and the pellets were immunoblotted with an anti-FLAG antibody for CDK5RAP2; α-tubulin in these pellets was quantified by staining with anti-α-tubulin antibody (n = 3, <i>p</i><0.001). (B) DIC was immunoprecipitated from HEK293T extracts using an anti-DIC antibody, normal mouse IgG served as a control. The immunoprecipitates (“IP” here and in other figures) and cell extracts were probed for DIC and CDK5RAP2 by Western blotting (“WB” here and in other figures). (C) Mapping the DIC-binding region in CDK5RAP2. Anti-DIC immunoprecipitation was performed on HEK293T ectopically expressing CDK5RAP2 fragments. Immunoprecipitates and cell extracts were analyzed for DIC and for the CDK5RAP2 fragments with anti-FLAG. A control immunoprecipitation (Ctrl) was performed using normal mouse IgG on lysates of 706–1893-expressing cells.</p