Escherichia coli (E. coli) O157:H7.An aptamer based biosensor (aptasensor) was developed and evaluated for rapid colorimetric detection of E. coli O157:H7 and aptamer at the interface of the vesicle lead to blue-red transition of PDA which was readily visible to the naked eyes and could be quantified by colorimetric responses (CR). Confocal laser scanning microscope (CLSM) and transmission electron microscopy (TEM) was used to confirm the specific interactions between the truncated aptamer and E. coli O157:H7. The aptasensor could detect cellular concentrations in a range of 104∼ 108 colony-forming units (CFU)/ml within 2 hours and its specificity was 100% for detection of E. coli O157:H7. Compared with the standard culture method, the correspondent rate was 98.5% for the detection of E. coli O157:H7 on 203 clinical fecal specimens with our aptasensor.The aptasensor was assembled by modifying the truncated lipopolysaccharides (LPS)-binding aptamer on the surface of nanoscale polydiacetylene (PDA) vesicle using peptide bonding between the carboxyl group of the vesicle and the amine group of the aptamer. Molecular recognition between The new aptasensor represents a significant advancement in detection capabilities based on the combination of nucleic acid aptamer with PDA vesicle, and offers a specific and convenient screening method for the detection of pathogenic bacteria. This technic could also be applied in areas from clinical analysis to biological terrorism defense, especially in low-resource settings. Escherichia Coli (E. coli) have been widely spread and grown as public health problems. Most of what we know about pathogenic E. coli comes from the outbreak studies of E. coli O157:H7 infection, which was first identified as a pathogen in 1982 E. coli O157:H7 infections accounted for 32 outbreaks between 2003 and 2008 in United States E. coli O157:H7 is a specific serotype of E. coli, which can cause watery diarrhea, hemorrhagic colitis, hemolytic-uremia syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP), especially in young children and the elderly. The conventional assays to detect E. coli O157:H7 are specimen culture and colony counting which are sensitive and selective enough. But, these methods are labor intensive, time consuming (requiring 5 to 7 days) and professional operation limited. Many types of E. coli O157:H7 rapid detection tests have been developed during the last decades, involving nucleic acid-based polymerase chain reaction (PCR) technology In recent years, outbreaks of foodborne diseases associated with pathogenic E. coli O157:H7, including microarray biosensor the antibody disadvantages associated with their production, stability and crossreaction have prompted us to seek alternatives of molecular recognition component. Other biosensors, which are based on oligonucleotide Various kinds of biosensors have been also developed to detect the Due to their unique optical properties, polydiacetylene (PDA) and its derivatives have been widely exploited for biosensing applications in vitro selection referred to as systematic evolution of ligands by exponential enrichment (SELEX) Nucleic acid aptamers are relatively short single-stranded DNA or RNA oligonucleotides that have been engineered through repeated rounds of E. coli O157:H7. 10, 12-pentacosadiynoic acid (PCDA), one of the well-known diacetylene monomers, was used to prepare PDA vesicles. It consists of hydrophillic head group and hydrophobic tail, can be easily self-assembled into vesicles in solution. The free carboxyl of nanoscale PDA vesicle was activated with N-hydroxysuccinimide (NHS) and 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and formed reactive intermediate. The aptasensor was obtained by modifying the truncated 5′-amino-aptamer on the surface of nanoscale PDA vesicle using peptide bonding between the carboxyl group of the vesicle and the amine group of the aptamer (E. coli O157:H7 and aptamer at the interface of the vesicle lead to blue-red transition of PDA vesicle which was readily visible to the naked eyes and could be quantified by colorimetric responses (CR).In this paper, an aptamer based biosensor (aptasensor) was developed and evaluated for rapid colorimetric detection of  aptamer . MoleculUnpolymerized vesicles were made by using PCDA, DMPC/PCDA or LPS/DMPC/PCDA in the dark room. Then the PDA vesicles were polymerized via 1, 4-addition reaction to form alternating polymer chains under UV light irratidiation at 254 nm. The resulting polyers appeared deeply blue which is directly visible to the unaided eye and can be characterized by the UV-vis spectrum showing an absorption peak at 640 nm .E. coli O157:H7, we prepared fluorescein isothiocyanate (FITC) labeled aptamers before and after truncated. Positive controls were investigated with FITC labeled E17F-72 (or E18R-72) instead of FITC labeled E17F-37 (or E18R-42). Further, Salmonella typhimurium was used instead of E. coli O157:H7 in the experiment as negative controls. E. coli O157:H7 after incubating of FITC labeled E17F-37, E18R-42, E17F-72and E18R-72 separately. Green fluorescence was detected on E. coli O157:H7 with all four kinds of FITC labeled aptamers. It was well known that the recognition and binding between the aptamers and target molecules were depended on their structures, like the interactions between antigen and antibody, the results also might indicate that the aptamers before and after truncated had the same loops structures and similar functions. Moreover, no green fluorescence was detected on Salmonella typhimurium with FITC labeled E17F-37, E18R-42 /ml) at 37°C temperature under stirring, the color of aptasensors turned from deeply blue to red, which could be easily seen by naked eyes. In contrast to pure PCDA vesicles  and pure PCDA vesicles, and drew the CR%-time curve. As shown in E. coli O157:H7.The aptasensors appeared deeply blue and showed an absorption maximum at 640 nm. After being incubated with To quantify the sensitivity of the aptasensors, we challenged with an increasing concentration of bacteria and generated a calibration curvet. As shown in 4 ∼ 108CFU/ml. E. coli O157:H7 was Y = 6.0163X-10.5468 (R2 = 0.99087), the detect limitation was 104 CFU/ml (three times the standard deviation of blank). The E. coli O157:H7: the linear equation was Y = 6.1055X-13.8503 (R2 = 0.98982) and the detect limitation was 104 CFU/ml too, These detection limits were 104-fold lower than previously reported PDA-based approach 2-fold lower Also, a good linear relationship was shown when the concentrations of bacteria was in the range of 10E. coli O157:H7, the aptasensors were challenged with other types of bacteria including E. coli , Salmonella typhimurium, Salmonella typhi, Salmonella paratyphi A, Salmonella paratyphi B, Shigella sonnei, Shigella flexneri, Proteus vulgaris, Enterobacter aerogenes, Citrobacter freundii, Staphylococcus aureus and Monilia albican.The recognition of aptamer to target molecule was highly specific which could identify the target accurately from similar molecules with subtle difference. In order to confirm that the color shift was specifically caused by E. coli O157:H7 was diluted to the concentration of 107CFU/ml while other bacteria were diluted to the concentration of 108CFU/ml by deionized water, and then be incubated with aptasensors for 2 hours. The values of CR%s were calculated as previously mentioned. As shown in E. coli O157:H7 could be recognized by the aptasensors, which means our biosensors could only specially detect the E. coli O157:H7. Importantly, this selectivity can be visualized with the naked eye.E. coli O157:H7 were red, whereas all others (10-fold higher than E. coli O157:H7) have remained blue. This clearly illustrated that the aptasensors was only specific to the E. coli O157:H7 and did not respond to the wide range of background bacteria even to other E. coli.Indeed, only the aptasensors containing E. coli O157:H7 in artificially contaminated fecal samples and It is feasible to separate E. coli O157:H7 as pretreatment process. However, the pretreatment process inevitably decreased the concentration of E. coli O157:H7 and the CR% of group A were less than those of E18R-42/PCDA vesicles with E. coli O157:H7 (108 CFU/ml). Furthermore, in order to quantitatively compare the difference responses of E18R-42/PCDA vesicles to the artificially contaminated and uncontaminated fecal samples, a Student’s t-test was conducted. Based on The 100 fecal samples artificially contaminated were exposed to E18R-42/PCDA vesicles and pure PCDA vesicles for 2 hours. As shown in n = 203) were tested for detecting E. coli O157:H7 by standard culture method and our E18R-42/PCDA vesicles. Samples were deemed to be aptasensor – positive if CR%s were equal or greater than 10%. All samples resulted in 98.5% (200 to 203) agreement of E. coli O157:H7 detection between standard culture method and our E18R-42/PCDA vesicles. The agreement ratewas the proportion of all samples diagnosed correctly by E18R-42/PCDA vesicles. It was computed from the proportion diagnosed correctly by E18R-42/PCDA vesicles of the fourfold table given in p>0.05) for the detection of E. coli O157:H7 with the standard culture method and our E18R-42/PCDA vesicles. It was thus clear that our aptasensor was feasible and reliable for primary screening E. coli O157:H7 in clinical fecal samples.In addition, the clinical fecal specimens , Salmonella typhimurium (CMCC50115), Salmonella typhi (CMCC50071), Salmonella paratyphi A(CMCC50433), Salmonella paratyphi B (CMCC50004), Shigella sonnei (ATCC25931), Shigella flexneri (CMCC51571), Proteus vulgaris (CMCC49072), Enterobacter aerogenes (ATCC 13048), Citrobacter freundii (ATCC8090), Staphylococcus aureus (ATCC25923) and Monilia albican (ATCC14053) were obtained from Wenzhou Medical College.PCDA was purchased from GFS chemicals. . 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC), NHS and 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, hydrochloride (EDC·HCl) were purchased from Sigma-Aldrich. . DNA oligonucleotides modified with amine group or FITC at the 5′ end were obtained from Bioneer Co. Ltd. . All solvents used in this study were of analytical grade and were prepared by using deionized water of resistivity not less than 18.2 MΩ cm.PURELAB Classic was purchased from ELGA Lab Water . Scientz-IID ultrasonic homogenizer and SCIENTZ03-II UV crosslinker were purchased from Ningbo Scientz Biotechnology Co.Ltd. . CLSM Fluoview FV1000 was purchased from Olympus . TEM H-7500 was purchased from Hitachi . Varioskan Flash spectral scan multimode plate reader was purchased from Thermo Fisher Scientific .PCDA monomers were dissolved in chloroform and the solvent was removed by purging with nitrogen to generate a thin lipid film on the glass surface, and continued to be dried by vacuum for 2 hours. After adding the deionized water (18.2 MΩ cm) to make the 1 mM lipids, the lipids were sonicated at 72°C for 15 min until it turned clarity. The lipids were cooled in the dark bottle at 4°C overnight. They were polymerized by using UV light irradiation at 254 nm until turning blue, and then the vesicle solution was obtained. The quality of vesicle solution was monitored with Varioskan Flash spectral scan multimode plate reader and TEM. Most of the procedures for preparing LPS/PCDA/DMPC vesicle or PCDA/DMPC vesicle were the same as pure PCDA vesicle’s, excepting for replacing the PCDA monomer by a mixture containing PCDA monomer, DMPC and LPS (molar ratios as 6∶2: 0.1), or a mixture containing PCDA monomer and DMPC (molar ratios as 6∶2) to a total lipid concentration of 1 mM.The aptamers were conjugated to the pure PCDA vesicles by using the carbodiimide method in the presence of the EDC and the NHS . Briefly−1, 10−2, 10−3, 10−4, 10−5, 10−6, 10−7 and 10−8) of the bacterial suspension were prepared in saline (0.9%). A 0.1 ml of each dilution was spread plated on to Luria Broth agar plate and the plates were incubated overnight at 37°C for 24 h. Finally, we got the E. coli O157:H7 samples of different concentrations from 103 to 108 CFU/ml via suspending bacteria with different volume deionized water.Bacteria were cultured in the Luria Broth at 37°C overnight, and then washed 3 times with deionized water. Then appropriate 10-fold serial dilutions . The mixtures were redispersed finally in the same buffer (10 µl) and placed onto a glass slide. After complete drying, the slides covered with coverslips were observed under CLSM equipped with an excitation wavelength of 488 nm and an emission wavelength of 520 nm.As shown in And then, the E17F-37 and the E18R-42 were modified with the amide groups at the 5′ region and were used to prepare for the aptamer/PCDA vesicles. We performed the affinity tests to verify the binding sites of the E17F-37 and the E18R-42 by mixing the aptamer/PCDA vesicles to the LPS/DMPC/PCDA vesicles and the DMPC/PCDA vesicles. The DMPC/PCDA vesicles were used as the negative control. The results were observed by transmission electron microscopy (TEM).The absorbance was obtained by using the Varioskan Flash spectral scan multimode plate reader from 228 nm to 700 nm at room temperature.E. coli O157: H7 (103 to 108 CFU/ml) at 37°C for 2 hours. Absorbance measurements were performed at different time points using absorption spectroscopy from 400 nm to 700 nm. The aptamers of E17F-37 and E18R-42 automatically folded to capture the E. coli O157:H7, which would change the structure of PCDA backbone, and then induced the color turning blue to red. To quantify the degree of color change, we used the following formula 0– PBf)/PB0]×100%. Where the PB  =  A640 nm/(A640 nm + A540 nm); the PB0 was in the absence of E. coli O157:H7, while the PBf was in the presense of E. coli O157:H7. The higher CR% represents more efficient transition from the blue color to the red color.The data of A640 and A540 was collected before and after adding the O157:H7 from at least three independent experiments; the CR% values were then calculated.Sensitivity test was performed as follows: the aptamer/PCDA vesicles were incubated with various concentrations of 8 CFU/ml but E. coli O157:H7 at the concentration of 107 CFU/ml. The bacteria were as follows: E. coli , Salmonella typhimurium, Salmonella typhi, Salmonella paratyphi A, Salmonella paratyphi B, Shigella sonnei, Shigella flexneri, Proteus vulgaris, Enterobacter aerogenes, Citrobacter freundii, Staphylococcus aureus, Monilia albican and E. coli O157: H7.Specificity test was performed as follows: the aptasensors were used to detect other different bacteria at the concentration of 10n = 203), that were used for detecting E. coli O157:H7 by standard culture method and our E18R-42/PCDA vesicles, were from patients from the First Affiliated Hospital of Wenzhou Medical College. For artificially contaminated fecal samples, 1 ml of 108 CFU/ml E. coli O157:H7 was added to each 1 g of fecal samples (n = 100) that were culture negative for E. coli O157:H7. For uncontaminated fecal samples, 1 ml of 10 mM sterile PBS buffer (pH 7.2) was added to each 1 g of fecal samples (n = 100) that were culture negative for E. coli O157:H7. All three kinds of samples need to be pretreated simply before application of E18R-42/PCDA vesicles. 1 g of fecal samples was homogenized in 8 ml of 10 mM sterile PBS buffer (pH 7.2) and was centrifuged at 500 rpm for 10 min. Then the supernatant was filtered through a 0.45 µm filtration device. Agitate the filter membrane carefully with 8 ml of sterile water in order to resuspend any bacteria that were caught in the filter membrane. Finally, bacteria were centrifuged at 12000 rpm for 10 min and resuspended in 1 ml of sterile water.The clinical fecal specimens  pure PCDA vesicles. (B) aptamer/PCDA vesicles. (C) DMPC/PCDA vesicles. (D) LPS/DMPC/PCDA vesicles. Scale bar is 100 nm.(TIF)Click here for additional data file.Figure S2E.coli O157:H7. (B) FITC labeled E18R-42 with E.coli O157:H7. (C) FITC labeled E17F-37 with Salmonella typhimurium. (D) FITC labeled E17F-72 with E.coli O157:H7. (E) FITC labeled E18R-72 with E.coli O157:H7. (F) FITC labeled E18R-42 with Salmonella typhimurium.CLSM images of affinity tests. (A) FITC labeled E17F-37 with (TIF)Click here for additional data file.


Insulin-like growth factor (IGF) signaling through human insulin receptor isoform A (IR-A) contributes to tumorigenesis and intrinsic resistance to anti-IGF1R therapy. In the present study, we (a) developed quantitative TaqMan real time-PCR-based assays (qRT-PCR) to measure human insulin receptor isoforms with high specificity, (b) evaluated isoform expression levels in molecularly-defined breast cancer subtypes, and (c) identified the IR-A:IR-B mRNA ratio as a potential biomarker guiding patient stratification for anti-IGF therapies.mRNA expression levels of IR-A and IR-B were measured in 42 primary breast cancers and 19 matched adjacent normal tissues with TaqMan qRT-PCR assays. The results were further confirmed in 165 breast cancers. The tumor samples were profiled using whole genome microarrays and subsequently subtyped using the PAM50 breast cancer gene signature. The relationship between the IR-A:IR-B ratio and cancer subtype, as well as markers of proliferation were characterized.The mRNA expression levels of IR-A in the breast tumors were similar to those observed in the adjacent normal tissues, while the mRNA levels of IR-B were significantly decreased in tumors. The IR-A:IR-B ratio was significantly higher in luminal B breast cancer than in luminal A. Strong concordance between the IR-A:IR-B ratio and the composite Oncotype DX proliferation score was observed for stratifying the latter two breast cancer subtypes.The reduction in IR-B expression is the key to the altered IR-A:IR-B ratio observed in breast cancer. The IR-A:IR-B ratio may have biomarker utility in guiding a patient stratification strategy for an anti-IGF therapeutic. The mature human insulin receptor (IR) has two isoforms, insulin receptor isoform A (IR-A) and insulin receptor isoform B (IR-B). Both isoforms result from alternative splicing of the same primary transcript. IR-A differs from IR-B by the exclusion of exon 11, which encodes a 12 amino acid fragment (residues 717–728) of the insulin receptor α-subunit The fine regulation of the IR-A:IR-B expression ratio may be rendered more complex by coexpression of the cognate IGF1R, which may form hybrid receptors with IRs. Several studies have demonstrated the existence of IR-A/IGF1R hybrid receptors. These hybrid receptors are heterodimers comprised of an αβ chain from the IR and an αβ chain from the IGF1R, both in normal As a key signaling component of the IGF pathway, IGF1R is the target of several investigational agents in clinical development. These agents were developed to specifically inhibit IGF1R while sparing IR-A due to concerns that co-targeting IGF1R and IR would result in unacceptable toxicity As therapeutic strategies for co-targeting IGF1R and IR-A move forward in clinical development, evaluating IR-A levels in clinical tumor samples becomes critical for identifying the most appropriate population to receive IGF-targeted therapy. To date, the most common method to specifically measure human IR-A and IR-B expression has been described by Frasca and colleagues Forty-two grade I to III infiltrating breast ductal carcinomas were purchased from ILSbio . Nineteen of the 42 breast tumor tissue samples had matched normal adjacent breast tissue (NAT) samples. In the case of breast cancer mastectomy, normal tissue from the opposite breast was taken and serves as the normal adjacent tissue. The ages of patients ranged from 31 to 88 years. All the breast cancer samples were estrogen receptor (ER) and progesterone receptor (PR) positive and HER2 negative according to immunohistochemistry. All samples were freshly frozen and collected before initiation of any treatment. Tumor samples were macrodissected to remove normal tissue, and tumor purity in all samples was greater than 85%. Normal samples were macrodissected to remove non-glandular tissue.Four breast cancer tissue cDNA arrays  were purchased from OriGene Technologies . The arrays contain cDNAs from 15 normal breast tissues  and 165 unique breast adenocarcinoma tissues. The tumor stage ranged from stage I to IV and the tissues were comprised of 50–90% tumor.Total RNA was extracted from snap-frozen tissue specimens using the ZR RNA MicroPrep kit . RNA purity and concentration were determined spectrophotometrically (260/280>1.9). RNA quality was assessed on an Agilent 2100 Bioanalyzer using the RNA 6000 Nano LabChip®.Full-length mRNA transcript sequences for the IR-A (NM_001079817) and IR-B (NM_000208) isoforms were retrieved from the NCBI Reference Sequences database. For the IR-A assay design, we targeted the exon 10/12 junction region for the gene specific probe; the exon 10 coding region for the forward primer pairs; and the exon 12 coding regions for the reverse primer pairs. For the IR-B assay design, we targeted the exon 11 interior coding region for the gene specific probe; exon 11/12 junction for the forward primer pairs; and exon 12 for the reverse primer pairs. All primer/probe designs were imported into the Primer Express  software tool to ensure optimal design for utilization in the TaqMan Gene Expression assay procedure. All probes were designed to incorporate a minor groove binding moiety (MGB), and were labeled with a fluorescent dye (FAM) for detection and a non-fluorescent quencher. Primers and probes were custom ordered from Applied Biosystems. Sequences for all primer/probe combinations are as follows:5′-TCCCCAGGCCATCT -3′;IR-A: The sequence of the probe is 5′-TGAGGATTACCTGCACAACG -3′;The sequence of the forward primer is 5′- ACCGTCACATTCCCAACATC -3′.The sequence of the reverse primer is CCGAGGACCCTAGGC -3′;IR-B: The sequence of the probe is 5′ 5′- CGTCCCCAGAAAAACCTCTTC -3′;The sequence of the forward primer is 5′-GGACCTGCGTTTCCGAGAT -3′.The sequence of reverse primer is Commercially available cDNA clones that contain the full-length cDNA clones of IR-A (cloned in pCMV6-XL4) and IR-B (cloned in pCMV6-XL5) were purchased from OriGene Technologies, Inc . The sequence verification of each IR isoform clone was conducted in-house at MedImmune. The empty plasmids pCMV6-XL4 and pCMV6-XL5 were used as negative control DNA for IR-A and IR-B assays, respectively.Standard TaqMan qRT-PCR Gene Expression assays were conducted in a 384-well format for all primer/probe and template combinations. Reactions consisted of 5 µL of TaqMan Universal Master Mix (Applied Biosystems), 0.5 µL of 10x Gene Expression Assay Mix, and 4.5 µL of varying copy numbers of either the IR-B or IR-A cDNA clone, for a final volume of 10 µL per well. Each primer/probe and template combination was repeated at least 3 times. All assay plates were run on an Applied Biosystems 7900HT detection system using standard settings (cycling program included 10 min incubation at 95°C followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min). Data values  were extracted from each assay with the SDS v2.0 software tool (Applied Biosystems).TaqMan Gene Expression assays were purchased from Applied Biosystems. The assays include: INSR (Assay ID: Hs00961554_m1); ESR1 (Assay ID: Hs00174860_m1); PGR (Assay ID: Hs01556707_m1); ERBB2 ; tumor proliferation genes : BIRC5 , AURKA , CCNB1 (Assay ID: Hs00259126_m1), MKI67 (Assay ID: Hs01032443_m1), MYBL2 (Assay ID: Hs00942543_m1); and reference genes: ACTB (Hs99999903_m1), GUSB (AssyID:Hs99999908_m1), GAPDH (Assay ID: Hs99999905_m1), RPLP0 (Assay ID: Hs99999902_m1), TFRC (Assay ID: Hs99999911_m1).The BioMark™ Dynamic Array (Fluidigm Corporation) microfluidics system allows for high throughput real-time PCR , producing high quality data with low variability and a tight correlation with conventional RT-PCR. Single stranded cDNA was generated from total RNA using the SuperScript® III First-Strand Synthesis SuperMix . cDNA samples were pre-amplified using TaqMan Pre-Amp Master Mix, according to the manufacturer's instructions. Reactions contained 5 µL of cDNA, 10 µL Pre-Amp Master Mix and 5 µL of 0.2X gene expression assay mix  for a final volume of 20 µL. Reactions were cycled with the recommended program for 14 cycles and then diluted 1∶5 with TE buffer. Pre-amplified cDNA was either utilized immediately or stored at −20°C until processed.To prepare samples for loading into 48×48 dynamic array chips (Fluidigm), the reaction mix contained 2.5 µL 2X Universal Master Mix (Applied Biosystems), 0.25 µL Sample Loading Buffer (Fluidigm Corporation), and 2.25 µL pre-amplified cDNA. To prepare the primer/probes, the reaction mix contained 2.5 µL 20X TaqMan Gene Expression Assay and 2.5 µL Assay Loading Buffer (Fluidigm Corporation). Prior to loading the samples and assay reagents into the inlets, the chip was primed in the IFC Controller. Five µL of sample prepared as described was loaded into each sample inlet of the dynamic array chip, and 5 µL of 10X gene expression assay mix was loaded into each detector inlet. The chip was placed on the IFC Controller for loading and mixing. Upon completion of the IFC priming step, the chip was loaded on the BioMark™ Real-Time PCR System for thermal cycling (10 min at 95°C followed by 40 cycles of 95°C for 15 sec and 1 min at 60°C). The number of replicates and the composition of the samples varied depending on the particular experiment, but were never less than triplicate. Average Ct values were used to determine sensitivity and specificity of the designed probes. The average Ct values of all available reference gene assays within a sample were utilized for ΔCt calculation.TM Premier RNA Amplification Kit . The concentration and purity of the cRNA product were determined spectrophotometrically. Fifteen micrograms of each biotin-labeled cRNA was fragmented for hybridization on Affymetrix Human Genome U133 Plus 2.0 GeneChip® arrays. All GeneChip® washing, staining, and scanning procedures were performed with Affymetrix standard equipment. Data capture and initial array quality assessments were performed with the GeneChip Operating Software (GCOS) tool. Stratagene's  ArrayAssist® Lite software was used to calculate probe-level summaries (GC-RMA and MAS5) from the array CEL files.Generation of biotin-labeled amplified cRNA from 75 ng of total RNA was accomplished with the MessageAmpA subset of ER+/PR+ and HER2- primary breast tumors (n = 40) and a subset of matched normal adjacent breast tissue samples (n = 15) were profiled on Affymetrix Human Genome U133 Plus 2.0 GeneChip® arrays. Two of the primary breast samples and four of the normal adjacent breast tissue samples analyzed on the Fluidigm platform were not processed on GeneChip due to insufficient RNA quantity. Breast cancer molecular subtype classification, with regards to luminal-A and luminal-B subtype, was conducted utilizing our whole genome array data.P-value <1.0×10−12  (n = 459 probes) were used for an unsupervised hierarchical clustering analysis. Several additional probeset selection thresholds were utilized and resulted in similar clustering patterns (data not shown).Two methods for determining putative sample classification were implemented. The first classification method utilized a published PAM50-gene shrunken centroid classifier 7 copies of DNA template). The DNA was serially diluted to 10−4 pg (approximately 10 copies DNA template). Each sample was tested in duplicate. The slope of the standard template dilution curve was determined by plotting cycle-threshold (Ct) values as a function of the log DNA copy number. The results are shown in r2 value) were ≥0.999 (P≤0.0001). Linearities were maintained in the DNA concentration ranges described above in both assays, demonstrating a wide dynamic range and yielding accurate Ct values. The results indicate that both the IR-A and IR-B assays have the appropriate sensitivity, and are able to detect the corresponding isoform to approximately 35 copies of DNA.Probe sensitivity was tested by performing IR-A or IR-B TaqMan qRT-PCR assays starting with 100 pg template stock solutions of IR-A or IR-B (approximately 107 copies of IR-B DNA). Likewise, the IR-B assay does not amplify the IR-A DNA template in the range tested. In contrast, we found the commercial Applied Biosystems assays  had strong cross-interaction and could not measure each isoform accurately (data not shown).The specificity of the assays was also assessed by testing the IR-A assay in the presence of the IR-B DNA template or, alternatively, the IR-B assay in presence of the IR-A DNA template. The details of the testing results are shown in The assay efficiency was assessed by the slopes of the standard dilution curves for both assays . The sloThese TaqMan-based assays have been successfully implemented on the BioMark™ Dynamic Array (Fluidigm), facilitating their use in a higher throughput system and demonstrating their performance across multiple platforms.P<0.0001). No significant differences were observed in the mRNA levels of IR-A in breast cancer when compared with normal breast tissue (P = 0.450).In order to assess the IR-A and IR-B mRNA expression in breast cancer, 42 primary breast tissue samples that are ER+, PR+ and HER2-, and 19 matched normal adjacent breast tissues were evaluated. Random hexamer primed cDNAs were pre-amplified and assayed for expression levels of total IR, IR-A, and IR-B transcripts by TaqMan qRT-PCR (Fluidigm). Samples were normalized to the average of five housekeeping genes as described in the methods section. The results are shown in (−ΔCt) calculation. The results are shown in P<0.0001). The results suggest that the significantly decreased IR-B levels in tumor contribute to an overall increase in the proportion of IR-A in tumor samples compared to normal tissue.We calculated the proportion of IR-A relative to total insulin receptor composition (IR-A + IR-B) in matched tumor and normal pairs as determined by a 2P<0.0001)  values were calculated for all samples utilizing the within-sample reference gene panel (average Ct) for normalization purposes. The mean IR-A:IR-B ΔCt ±95% CI were -0.20±0.27 and 1.81±0.31 in the normal (n = 19) and primary tumors (n = 42), respectively. A two-tailed Welch's t-test analysis identified a significant difference between normal and tumor samples in relation to observed IR-A:IR-B ΔCt (<0.0001) . The resP<0.0001).To further validate the above results, we assessed mRNA expression ratios of IR-A and IR-B in an additional breast cancer tissue panel. PCR arrays containing cDNAs from 15 normal breast tissues (obtained from 10 independent donors) and 165 unique breast adenocarcinoma tissues were used. Equal amounts of cDNA were pre-amplified and assayed for expression levels of IR-A, IR-B, and ER by TaqMan qRT-PCR (Fluidigm). The ΔCt differential (IR-B ΔCt–IR-A ΔCt) was calculated for all samples utilizing the within-sample reference gene panel  for normalization purposes. The results are shown in AURKA, BIRC5, CCNB1, MKI67 and MYBL2 are well characterized genes involved in breast cancer proliferation Using mRNA expression profiles, ER+ breast cancers can be further classified by hierarchical cluster analysis into luminal-A and luminal-B subtypes P<0.001). The results indicate that IR-A:IR-B ratios are significantly increased in luminal-B cancers relative to luminal-A cancers, with the greatest differential observed between luminal-B and normal breast tissue. Additionally, we found that these luminal-B samples exhibited a significant increase in expression of a proliferation gene panel (data not shown). Increased proportion of IR-A in luminal-B patients may play an important role in inducing the increased proliferation observed in this study and others To address this question we conducted whole genome array analysis on 40 ER+/PR+ and HER2- negative breast tumors and 15 normal breast samples. We initially utilized a published PAM50-gene shrunken centroid classifier P<0.001).In addition to the shrunken centroid classifier, we identified an independent panel of differentially expressed transcripts from our GeneChip data using a two-sample Welch's t-test and unsupervised hierarchical clustering analysis. Samples were divided into two groups  based on pathology assessments performed prior to conducting the statistical analysis. Sub-populations identified were classified as normal, luminal-A, or luminal-B as a function of transcript panel composition for the three primary clusters identified. Differentiation of luminal-A and luminal-B sub-types was primarily determined by over-expression of proliferation and cell cycle associated genes  in the putative luminal-B tumors and over-expression of extracellular matrix associated genes  in the putative luminal-A tumors. IR-A:IR-B ΔCt differentials in the normal, luminal-A and luminal-B were then compared. The results are shown in Overall, an 80% concordance between the two classification methods implemented was observed, based on our unbiased assessments. Unfortunately, we are naïve to the true sub-type classification of the tumor samples so it is difficult to resolve differences in classification between the two methodologies. However, the overall concordance between the results from comparisons of the IR-A:IR-B ΔCt differential with both the published PAM50 classifier and an independent gene selection and clustering method further supports our observation that the IR-A:IR-B ratio is significantly increased in the luminal-B subtype of breast cancer.We have described specific TaqMan qRT-PCR-based assays that accurately measure the mRNA levels of human IR isoforms IR-A and IR-B. In the absence of specific reagents to quantitatively distinguish the two isoforms, previous research utilized an RT-PCR/gel quantification protocol to distinguish IR-A and IR-B and measure their relative amounts in normal and cancer tissues Over-expression of hybrid IGF1R and IR-A receptors has been reported in breast cancer The functional consequences of an altered IR-A:IR-B ratio on cancer progression may also be mediated, in part, by changes in receptor dimerization patterns and the subsequent effect on cell signaling pathways. An increase in the relative abundance of IR-A might significantly increase formation of IGF1R and IR-A hybrid receptors, IR-A/IR-B heterodimers, and IR-A homodimers in breast cancer tissues. These changes in dimerization patterns could result in an increased number of binding sites for IGFs, leading to increases in mitogenic signaling in breast cancer cells Gene expression profiling studies have consistently revealed biologically distinct breast cancer subtypes with different prognoses and treatment responses Previous work has shown that protein levels of the total insulin receptor are elevated in breast cancer tissue compared to normal breast tissue In summary, the specific TaqMan qRT-PCR-based assays that we developed can easily be incorporated into routine clinical practice and measure the relative abundance of IR-A and IR-B mRNAs in human tissues with high sensitivity and specificity. Studies based on these assays may provide value in evaluating the therapeutic benefit of targeting both IGF1R and IR-A pathways by inhibiting the growth factors IGF1 and IGF2, as well as potentially identifying patients more likely to respond to any anti-IGF therapy under clinical development. The results reported here provide a promising foundation to support these future efforts.Table S1Specificity of IR-A and IR-B assays. Assay specificity was tested by performing IR-A, IR-B, and IR qRT-PCR assays with a serial dilution of approximately 107 to 10 copies of plasmid DNA of IR-A and IR-B. Cross reactions of either IR-A assay to IR-B DNA or IR-B assay to IR-A DNA were not observed.(PDF)Click here for additional data file.


Mycoplasma pneumoniae is an important cause of respiratory tract infection and is increasingly being associated with other diseases such as asthma and extra-pulmonary complications. Considerable cross-reactivity is known to exist between the whole cell antigens used in the commercial serological testing assays. Identification of specific antigens is important to eliminate the risk of cross-reactions among different related organisms. Adherence of M. pneumoniae to human epithelial cells is mediated through a well defined apical organelle to which a number of proteins such as P1, P30, P116 and HMW1-3 have been localized, and are being investigated for adhesion, gliding and immunodiagnostic purposes.(N-27), corresponding to the N-terminal region of M. pneumoniae P116 gene was cloned and expressed. A C-terminal fragment P1(C-40), of P1 protein of M. pneumoniae was also expressed. Three IgM ELISA assays based on P116(N-27), P1(C-40) and (P116 (N-27) + P1(C-40)) proteins were optimized and a detailed analysis comparing the reactivity of these proteins with a commercial kit was carried out. Comparative statistical analysis of these assays was performed with the SPSS version 15.0.A 609 bp fragment P116(N-27) protein was well recognized by the patient sera and was immunogenic in rabbit. P1(C-40) of M. pneumoniae was also immunogenic in rabbit. In comparison to the reference kit, which is reported to be 100% sensitive and 75% specific, ELISA assay based on purified P116(N-27), P1(C-40) and (P116(N-27) + P1(C-40)) proteins showed 90.3%, 87.1% and 96.8% sensitivity and 87.0%, 87.1% and 90.3% specificity respectively. The p value for all the three assays was found to be < 0.001, and there was a good correlation and association between them.The expressed P116M. pneumoniae infection. The IgM ELISA assays based on the recombinant proteins seem to be suitable for the use in serodiagnosis of acute M. pneumoniae infections. The use of short recombinant fragments of P116 and P1 proteins as specific antigens may eliminate the risk of cross-reactions and help to develop a specific and sensitive immunodiagnostic assay for M. pneumoniae detection.This study shows that an N-terminal fragment of P116 protein holds a promise for serodiagnosis of  M. pneumoniae is among the most common causes of community-acquired respiratory tract infection [M. pneumoniae causes upper and lower respiratory illness in all age groups and accounts for 3.3-40% of community-acquired pneumonia (CAP) cases worldwide with an attack rates ranging from 25-71% among closed populations. Approximately 25% of individuals infected with M. pneumoniae develop extra-pulmonary complications [nfection . M. pneuM. pneumoniae infection is frequently seen in the patients with respiratory illnesses [M. pneumoniae has been reported in acute exacerbation of bronchial asthma and chronic obstructive pulmonary disease (COPD) [M. pneumoniae needs to be considered in the differential diagnosis of various respiratory and non-respiratory infections as the pathogen respond well to antibiotics, such as tetracyclines, macrolides and quinolones [llnesses . Recentle (COPD) -5, acutee (COPD) ,7, polyae (COPD) , stroke e (COPD) , Guillaie (COPD) , coronare (COPD) ,12 and ie (COPD) . Therefoinolones .M. pneumoniae possess an apical organelle that mediates adherence to the host epithelium. The cytadherence is a complex multifactorial process requiring a group of proteins such as P1(170 kDa), P30(30 kDa), P116(116 kDa), HMW1-3 and proteins A, B and C. These proteins cooperate structurally and functionally for adherence as well as for the gliding mobility [M. pneumoniae was identified as a membrane protein in triton-X-114 soluble fraction of the pathogen and its gene was identified. P116 protein is encoded in an operon, consisting of a 3093 bp ORF encoding a protein of a predicted molecular mass, 116 kDa and a 408 bp ORF that is predicted to code a 16 kDa protein (5'-16 kDa ORF-116 kDa ORF-3'). Northern blot analysis and RT-PCR amplification of the intergenic region established that the ORFs encoding the 16 kDa and 116 kDa proteins are transcribed as a single mRNA. Swenstrup and co-workers showed that the P116 protein is expressed on the surface of M. pneumoniae and a polyclonal antibody Pab(rP116) inhibited the adherence of M. pneumoniae to Hep-2 cells [M. pneumoniae P116 in cytadherence, like the other two adhesins P1 [M. pneumoniae for their immunodiagnostic potential. In the present study, we expressed and purified an N-terminal fragment (203aa) of P116 protein and showed that the protein is well recognized by sera of M. pneumoniae infected Indian patients. We next compared the immunoreactivity of recombinant P116 and P1 proteins with that of a commercially available ELISA kit - the Serion ELISA classic IgM. Our results show that a short N-terminal fragment (203aa) of P116 protein holds a promise for serodiagnosis of M. pneumoniae infection.Mycoplasmas are cell wall deficient, the smallest known self-replicating organisms. They possess a small genome (0.58-2.20 Mbp) . M. pneuuch as P10 kDa, P3mobility . In addi-2 cells . These resins P1  and P30 esins P1 . A couplesins P1 . Our groThe study was carried out with the approval of the Institute's human & animal ethics committee (Ref. 51/7.1.2004/AIIMS). Blood Samples were collected over a period of two and a half years i.e Sep.05 through March 07 from 62 patients between 1 m to 70 yrs of age, admitted in the wards and intensive care units of AIIMS hospital. Each patient's medical history was recorded in a specific format and written informed consent was obtained.M. pneumoniae antigens, was used as screening test as well as reference test for comparison. This kit was selected, because of its availability and its use in various studies [Commercially available Serion ELISA classic IgM  kit, which uses mixture of undefined  studies ,26,27.M. pneumoniae standard strain FH (NCTC 10119), was grown in Edward Hayflick medium containing PPLO basal broth with supplements including glucose and phenol red (0.0002%) as indicator in glass tubes. The culture was grown at 37°C, aerobically until the colour of the medium changed (red-orange). Mycoplasma cells were harvested at this stage by centrifugation at 15,000 rpm for 15 min, washed twice with PBS (pH 7.2) and were stored at -70°C until use. To confirm that the colour change of culture medium is due to M. pneumoniae growth, the broth culture was plated on PPLO agar plates and the plates were incubated at 37°C in a CO2 incubator. The plates were examined microscopically once in five days with X10 magnification. The suspected colonies were stained with Diene's stain and checked under light microscopy. Further confirmation of the colonies was carried out by growth inhibition assay using M. pneumoniae polyclonal antisera (NCTC).M. pneumoniae P116 gene that codes for a protein of 203 amino acids, primers were designed using published sequence (accession no. Z71425) [M. pneumoniae genomic DNA was extracted as per the method of Stauffer et al [To clone 609 bp fragment of  Z71425) . M. pneuer et al . Followi2 and 0.1% gelatin), 200 μm dNTP's, 20 pmol of each primer, 1U Taq Polymerase  and template DNA (50 ng) in a GeneAMP PCR system 9700 . The PCR conditions were-initial denaturation at 94°C for 5 min followed by 30 cycles of amplification  with final extension at 72°C for 5 min. The PCR product was analysed by 1% agarose gel electrophoresis in 0.5% Tris-borate EDTA buffer and purified by gel extraction kit .The F1 was positioned at 786 nt whereas R1 at 1375 nt within the gene coding for P116 protein. PCR amplification was performed in a reaction volume of 50 μl containing 1× PCR buffer agar plates containing 100 μg/ml of ampicillin, 20 mg of X-gal and 200 mg/ml of IPTG . The selected white colonies were further analyzed for the presence of P116 gene fragment. Recombinant plasmids were extracted using Miniprep plasmid extraction kit (Qiagen).The purified P116 gene fragment was ligated into pGEMT-easy vector and the ligation mixture was used to transform DH5-α Bam HI and Pst I restriction sites. The ligated plasmid DNA was used to transform competent M-15 cells. Transformants were selected on ampicillin (100 μg/ml) and kanamycin (25 μg/ml) plate. M-15 cells containing the recombinant plasmids were cultivated in 5 ml of LB broth and the protein expression was induced by the addition of 1 mM IPTG final concentration, followed by 3 hrs of shaking at 37°C. Bacterial pellet was subjected to SDS-PAGE and western blotting using anti-His antibody to analyze the expression of recombinant protein.For the expression and purification of P116 protein fragment, the 609 bp fragment was cloned in pQE-30 vector with (N-27)) recombinant protein, induced and un-induced E. coli pellets from 1 ml of grown cultures were resuspended in 100 μl of SDS sample buffer  and boiled for 5 min. The proteins were resolved on 10% SDS-PAGE gel and stained with Coomassie brilliant blue R-250. For immunoblotting, after separating proteins on gel, the resolved proteins were transferred onto a nitrocellulose membrane (Sigma-Aldrich) in a transblot apparatus . The membrane was blocked in blocking buffer (5% skimmed milk in PBS-T) for 2 h. The blots were washed and incubated with anti-His primary antibody  or anti-M. pneumoniae antibody  or sera from M. pneumoniae infected patients (1:50) for 1 h. Later the blots after washing, were incubated with secondary antibody -H2O2 (Sigma-Aldrich).To analyse the expression of the (P116E. coli cells and the protein was found to be expressed in inclusion bodies. For the preparation of inclusion bodies E. coli. cells were disrupted by sonication in buffer ((0.05 M Tris (pH 8.0), and 0.3 M NaCl) with 1-min pulses at 1-min intervals 10 times using mini probe . The soluble and insoluble fractions were separated by centrifugation at 14,000 rpm at 4°C for 30 min and were analyzed by SDS-PAGE. To purify the protein from the inclusion bodies, E. coli pellet from 200 ml of culture was suspended in 1/50 original volume of lysis buffer (0.05 M Tris (pH 8.0), 0.3 M NaCl and 8 M Urea). The lysate was loaded onto a Ni-NTA column (Qiagen) and the bound protein was eluted under denaturing conditions in a buffer containing 0.05 M Tris (pH 8.0), 0.3 M NaCl, 0.3 M imidazole and 8 M Urea. The denatured protein was refolded by dialysis using buffers containing 0.05 M Tris (pH 8.0), 0.3 M NaCl and decreasing concentrations of Urea, 6M-0.5M. The refolded protein was stored in buffer containing 0.05 M Tris (pH 8.0), 0.15 M NaCl, 0.5 M Urea and 5% glycerol. Purified recombinant P116 protein fragment (P116(N-27)) was analysed for its reactivity with positive anti-M. pneumoniae human sera in western blot assay.Sub-cellular localization studies were carried out to analyse the expression of P116 protein fragment in Expression and purification of P1 protein fragment was carried out by a protocol described by Chaudhry et al .(N-27) or P1(C-40) protein emulsified in 300 μl Complete Freund adjuvant  intramuscularly (i.m.). Rabbits were subsequently boosted with 300 μl (250 μg) of the same protein in 300 μl Incomplete Freund adjuvant , through the same route on the 21st and 42nd days. Control rabbits were injected with complete and incomplete Freund's adjuvant in normal saline according to the immunization schedule. Blood samples were obtained by ear vein puncture on 0th, 14th, 21st, 28th, 35th, 49th and 56th days. IgM and IgG antibody responses against the two purified recombinant proteins were analysed by ELISA and end point titres were determined. In order to confirm the specificity of the antisera western blotting with whole M. pneumoniae as antigen was also performed.The protein concentration of both the antigens preparation was determined by Bradford assay. To check the immunogenicity of the two recombinant proteins-P116 and P1, four White New Zealand rabbits (two tests and two controls) were selected. Each one of the two test rabbits was immunized with 300 μl (250 μg) of purified recombinant P116M. pneumoniae IgM antibody (which is an indicator of the acute infection), using Serion ELISA kit. Among the sixty two sera samples tested, thirty one samples were found positive for the anti-M. pneumoniae IgM antibodies. We next compared the antibody response seen with Serion kit with the response seen with recombinant P116(N-27), P1(C-40) or P116(N-27) + P1(C-40) together for IgM antibodies. The experiment was done in duplicate. Briefly, 100 ng of P116(N-27) or P1(C-40) was added to each well of 96-well microtiter plates. In case of P116(N-27) + P1(C-40) assay, P116(N-27) and P1(C-40) antigens were mixed in 1:1 ratio. The plates were incubated overnight at 4°C and further for one hour at 37°C next day. The plates were washed with PBS-T and blocked with 5% BSA in PBS for 2 h. The plates were subsequently washed twice with PBS-T, once with PBS and incubated with 1:50 diluted patient sera at 37°C for 1 h. The positive control  and negative control  were included in each ELISA assay and they were also diluted to the same extent. The wells were washed and incubated with HRP-conjugated goat anti-human IgM (Sigma-Aldrich) diluted 1:3000 in PBS-T, for 1 h. The enzyme reaction was developed by the addition of the substrate ortho-phenylenediamine (Sigma) (1 mg/ml) diluted in phosphate-citrate buffer (pH 5.0) containing 0.03% (v/v) hydrogen peroxide. The enzymatic reaction was stopped with 100 μl of 2N H2SO4 and absorbance was read at 490nm with ELISA (Bio-Tek Microplate) reader.Sera obtained from sixty two patients suffering from respiratory tract infections were analysed for the presence of anti-Comparative statistical analysis of ELISA assays for P116 and P1 proteins was done taking Serion IgM kit (100% sensitive and 75% specific as per the manufacturer's claim) as reference test. Analysis was performed with the SPSS version 15.0. The parameters of in house ELISA assays were calculated by using Epitable module of Epiinfo (6.04D) software. Cohen's Kappa test was used to find the agreement between two different modalities. The chi-square test of proportions was applied to compare two proportions with the p value < 0.05 considered statistically significant. Cut-off values were calculated by taking median values of the controls.M. pneumoniae antibody (data not shown).A 609 bp fragment of P116 gene (786nt-1394nt) coding for a protein of 203 amino acids was successfully amplified by PCR using the designed primers, and was cloned into pGEM-T easy vector. Figure (N-27) protein was purified under denaturing conditions on a Ni-NTA column  was recognized by both the sera strongly. This reactivity was specific as sera from healthy humans (age matched persons without any history of the respiratory tract infections) failed to recognize any such protein  protein with eleven sera from different patients infected with M. pneumoniae and six uninfected patients sera (negative controls), for IgM antibody. All the eleven sera showed reactivity with the P116(N-27) protein, while sera from uninfected patients failed to recognize the P116(N-27). These results suggested that the N-terminal region (27 kDa) is one of the immunodominant region of the P116 protein.Immunoblot analysis was carried out using rabbit anti-(N-27) and P1(C-40) are immunogenic in nature, we formulated these recombinant proteins with CFA and injected them in rabbits. High antibody response was generated against the corresponding protein. The time course of the immune response for each of the recombinant proteins  and P1(C-40) were > 2,56,000 for both the proteins. Western blotting with whole M. pneumoniae as antigen confirmed the specificity of the antisera raised as they recognized single specific bands for each antigen without any cross-reactivity  and P1(C-40) proteins along with Serion  kit was carried out to determine immunodiagnostic potential of these recombinant antigens in immunodiagnosis of M. pneumoniae infections. A total of 62 sera were analysed for their reactivity to the recombinant proteins individually and together in ELISA assays. In the beginning, these sera were also tested with the commercial Serion kit and thirty one sera were found positive for IgM antibodies to M. pneumoniae. Although most patient sera shown to be positive for M. pneumoniae antibodies by Serion kit were also positive for their reactivity towards P116(N-27) and P1(C-40) proteins, however few differences were also observed. Of the 31 positive sera selected based on the reactivity with commercial kit, 28, 27 and 30 sera reacted with P116(N-27) and P1(C-40), and P116(N-27) + P1(C-40) ELISA assay respectively  protein or P116(N-27) and P1(C-40) combined  protein. Western blot could not be performed for one serum sample due to its inadequate volume. Among the seropositive group, 3 sera were negative by P116(N-27) based ELISA. Of these 3, one was also negative by P1(C-40) based assay. However, all these 3 sera were positive in P116(N-27) + P1(C-40) based ELISA.Interestingly, four out of 31 seronegative patients as determined by Serion kit showed reactivity with the P116d Figure . Western(N-27), P1(C-40), P116(N-27) + P1(C-40) ELISA and assay with Serion kit (the reference test), was done (Table (N-27), 87.1% and 87.1% for P1(C-40), and 96.8% and 90.3% for (P116(N-27) + P1(C-40)) respectively. The p value for all the three assays was found to be < 0.001, which is highly significant and there was a good correlation and association between them. There was an observed agreement of 77.4% and 74% between P116(N-27), P1(C-40) and commercial ELISA respectively and is the highest (87%) between the P116(N-27) + P1(C-40) combined ELISA and commercial ELISA.A comparative analysis of the P116M. pneumoniae infections, as it is often difficult to differentiate different etiological agents by clinical symptoms. Even though a number of clinical tests such as PCR have been developed and applied to identify M. pneumoniae infections, serology is probably the most frequently used method to diagnose M. pneumoniae infections as PCR based tests are expensive and require specialized equipment [M. pneumoniae. An ELISA employing purified M. pneumoniae surface proteins would be having inherent advantage of specificity. One of the major difficulties of developing serological test for the detection of M. pneumoniae is the difficulty to express its proteins in heterologous expression system as the Mycoplasmas use the UGA opal codon to incorporate tryptophan rather than as a stop codon as in the universal genetic code [M. pneumoniae 116-kDa virulence factor (P116). We have been systematically analysing the serodiagnostic potential of various surface adhesion antigens of M. pneumoniae and previously showed that a C-terminal fragment of P1 and P30 protein hold potential to be used for the diagnosis of M. pneumoniae infections [Laboratory investigations are important in the diagnosis of quipment . Serologquipment  as the ctic code , leadingfections ,19.E. coli expression system with an N-terminal 6His-tag. The expressed protein was ~27 kDa in size and was purified on a Ni-NTA column under denaturation conditions. The yield of the protein was ~5 mg/l. A simple refolding protocol was developed to get the protein in soluble form. Even though the protein was expressed in inclusion bodies, a significant yield of the protein after refolding (~2 mg/l) could be obtained. The refolded soluble protein was recognized by anti-M. pneumoniae antibodies, by most of the M. pneumoniae infected patient sera and also by experimentally infected rabbit sera by immunoblot analysis. Our results were in agreement with a previous lone study by Duffy et al who expressed GST fusion protein fragments of P116 protein and showed that a 53 kDa P116 protein fragment (pGEX-3X-MP661) encompassing 9aa-474aa was recognized by most of the patient sera in ELISA [(N-27) was also found to be immunogenic in rabbit. The gene sequence of the P116(N-27) fragment was found similar to the sequence of P116 gene of M. pneumoniae strains FH and M-129 given in the database. The nucleotide sequence of the gene encoding the 116 kDa protein, and consequently also the epitopes of the protein, is highly conserved between M. pneumoniae M-129 and FH [M. pneumoniae groups. To know about the serodiagnostic potential of P116(N-27) IgM ELISA, sixty two Indian patients suffering with M. pneumoniae infections were analyzed. All these patients sera were also analyzed by the commercially available Serion  kit. In comparison to the reference kit, which is reported to be 100% sensitive and 75% specific, ELISA assay based on purified P116(N-27), P1(C-40) and (P116(N-27) + P1(C-40)) proteins showed 90.3%, 87.1% and 96.8% sensitivity and 87.0%, 87.1% and 90.3% specificity respectively. The p value for all the three assays was found to be < 0.001, which is highly significant and there was a good correlation and association between them. Our results also demonstrated that an ELISA assay based on two recombinant proteins was more sensitive and specific. During the course of this study, a report by Drasbek et al described the utility of P116 and P1 recombinant fragments for correct diagnosis of atypical pneumonia caused by M. pneumoniae [M. pneumoniae infection is to avoid possible cross-reactions with antibodies to M. genitalium [(N-27) protein of M. pneumoniae with M. genitalium. Thus, our study using the short recombinant fragment of P116 and P1 proteins together is an important step towards developing M. pneumoniae specific and sensitive diagnostic assay. Importantly, since present study was conducted in a geographically distinct location than the study by Drasbek et al, the study provides an important confirmation of the global applicability of the findings of Drasbek et al. Together these limited trials also indicate that recombinant P116 and P1 proteins can be useful as serodiagnostic agents.In furtherance to these studies, we expressed an N-terminal fragment of P116 protein  using anin ELISA . The P119 and FH , which aeumoniae . The difnitalium . To overnitalium . We coulM. pneumoniae. The report further describes the immunodiagnostic potential of P116 and P1 proteins individually or combined by ELISA on parallel patient's serum samples. Since both protein fragments are also immunogenic, it will be worth-while also to look for vaccine potential of these recombinant proteins. Surveillance or reporting system for M. pneumoniae infections eg. positive serological results for M. pneumoniae based on rapid and specific diagnostic tests, would be useful for physicians in understanding the regional epidemiology of M. pneumoniae infections, and also decide the empirical antibiotic treatment to be used in these cases.The present study describes a simplified approach for the large-scale production of an immunodominant fragment of P116 protein of The authors declare that they have no competing interests.M. pneumoniae lysate. All authors read and approved the final manuscript.This work is a part of Ph.D. thesis of IT, under supervision of RC, Professor, Deptt. of Microbiology, All India Institute of Medical Sciences, New Delhi, India. RC and PM participated in the study design and coordination. RC provided the reagents and other facilities. PM designed the primers and provided technical guidance. Both of them edited the manuscript. BK performed western blotting with The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/10/350/prepub


Henipavirus, are recently emerged, highly pathogenic and often lethal zoonotic agents against which there are no approved therapeutics. Two surface glycoproteins, the attachment (G) and fusion (F), mediate host cell entry. The crystal structures of the Hendra G glycoprotein alone and in complex with the ephrin-B2 receptor reveal that henipavirus uses Tryptophan 122 on ephrin-B2/B3 as a “latch” to facilitate the G-receptor association. Structural-based mutagenesis of residues in the Hendra G glycoprotein at the receptor binding interface document their importance for viral attachments and entry, and suggest that the stability of the Hendra-G-ephrin attachment complex does not strongly correlate with the efficiency of viral entry. In addition, our data indicates that conformational rearrangements of the G glycoprotein head domain upon receptor binding may be the trigger leading to the activation of the viral F fusion glycoprotein during virus infection.Hendra virus and Nipah virus, comprising the genus  Pteropodidae) The henipaviruses are recently emerged highly pathogenic paramyxovirus zoonoses and include Hendra virus (HeV) and several distinct isolates of Nipah virus (NiV) Paramyxovirinae, the henipavirus attachment glycoprotein does not hemagglutinate and neither binds to sialic acid, nor retains neuraminidase activity, and instead binds cell surface protein receptors 0 which form trimeric oligomers that are then proteolytically processed into the disulphide-linked subunits F1 and F2For many enveloped viruses, entry is mediated by viral fusion glycoproteins that contain two distinct activities: receptor attachment and membrane fusion. The fusion activity is triggered either by receptor binding or exposure to an acidic environment following endocytosis The pre-fusion trimeric F glycoprotein is proposed to be in a “metastable” conformation and associated with its oligomeric attachment glycoprotein partner. A current, and widely accepted, model of paramyxovirus fusion suggests that upon receptor binding, the F glycoprotein is activated, presumably involving direct contacts between the attachment and fusion glycoproteins, and inserts its fusion peptide into the host cell membrane. The activation process facilitates a series of conformational changes in F and the glycoprotein transitions into its post-fusion, six-helix-bundle conformation concomitant with the merging of the viral membrane envelope and the host cell plasma membrane Materials and Methods. The structure was determined at 2.2 Å resolution (Table S1) using molecular replacement with NiV-G (PDB ID 3D11) as a search model. Similar to other paramyxovirus attachment proteins, the HeV-G's head domain folds as a six-blade (B1–B6) β-propeller (The HeV-G globular head domain (174–602) was expressed using the baculovirus/insect-cell system as described in the ropeller Figure 12, HeV: 768 Å2), as well as morbilliviruses (MeV: 1087 Å2 in average), than in other paramyxovirus family members  While gel-filtration and analytical ultracentrifugation indicate that the HeV-G head domain is monomeric in solution, it packs as a dimer within one asymmetric unit in the crystals Figure 12, slightly smaller than the 1354 Å2 buried in the NiV-G/ephrin-B3 complex and the 1393 Å2 - in NiV-G/ephrin-B2 complex. In the complex, ephrin-B2 attaches to the upper face of the HeV-G β-propeller  used in our structural studies here was expressed in stably-transfected human HEK293 cells. Thus, its glycosylation pattern is more physiologically relevant than in the previously published structures, which used ephrin expressed in either yeast, bacteria or in the presence of glycosylation inhibitor. In addition to the previously observed glycosylation site N36 Ephrin-B2 undergoes very minor conformational changes upon binding to HeV-G and its structure in the HeV-G/ephrin-B2 complex can be superimposed on the unbound ephrin-B2 structure with the root mean square deviation (RMSD) between equivalent Cα positions of approximately 0.5 Å It has been suggested that HeV-G has a lower than NiV-G affinity for its receptors and our data here provides a structural basis for this observation. The structures reported here indicate that the HeV-G head domain dimer is dissociated upon ephrin binding. Interestingly, most of the ephrin-binding residues of HeV-G are in loop regions, with the exception of these forming the F117-binding pocket, which are located on the B6S2 and B6S3 β-strands Figure 5Virus-receptor association and dissociation is a dynamic process, which is usually difficult to study using conventional crystallographic methods; however, our crystals provide some very useful snapshots of this process. Specifically, we observed two rotameric forms of W122 in ephrin-B2 among the four HeV-G/ephrin-B2 complexes in the asymmetric unit  firmly into their pockets, stabilizing the whole central hydrophobic region of the ephrin-G interface. Thus, the structural data supports a “lock, key and latch” model for the association between the G glycoprotein and its receptors with the W122 residue of ephrin-B2 serving as the “latch”  HeV-G, targeting various residues which make up the pockets that accommodate the ephrin-B2 G-H loop residues F117, P119, L121 and W122, or the ephrin-B3 G-H loop residues Y120, P122, L124 and W125. These HeV-G mutations included V401A, N402A, Q490A, W504A, E505A, G506A, Q559A, Y581A, and I588A. In addition, E501A and E533A mutations were also generated, which targeted the HeV-G-ephrin salt bridges at the periphery of the HeV-G-ephrin interface.We first examined whether the G-glycoprotein mutations effected the HeV-G/ephrin association as measured by co-immunoprecipitation. The series of G glycoprotein mutants were expressed in ephrin receptor negative cells either alone or together with their F glycoprotein partner. Cellular lysates were prepared and a series of precipitation assays were carried out using ephrin-B2, ephrin-B3 or G-specific polyclonal antibodies as a control Figure 7Materials and MethodsTo explore the correlation between HeV-G/ephrin-B2/B3 binding results with the fusion promotion activity of HeV-G, we next examined the impact of the HeV-G mutations on the efficiency of virus entry. Wild-type or the mutant HeV-G constructs were expressed together with HeV-F to generate a series of lentivirus-based, reporter gene encoding pseudovirions as described in the To further validate these observations, the same series of HeV pseudovirions where used to infect ephrin-B3 expressing cells, and here the effects on virus entry were more profound. Furthermore, in addition to the same G residues showing inhibition upon mutation to alanine, three other amino acid substitutions  also completely or significantly abrogated virus entry in ephrin-B3 expressing cells . These data indicate that the ephrin receptor binding and its fusion triggering activities on HeV-G can be uncoupled. One possible explanation to account for these observations in the context of the fusion models discussed previously is that these HeV-G mutants no longer associate with its partner F glycoprotein. To examine these mutations in the context of the fusion models, the E501A and I588A HeV-G mutants, as well as the mutant Q559A that appeared to enhance virus entry on ephrin-B2 and ephrin-B3 expressing cells, were tested for their ability to bind HeV-F using a HeV-G/F co-precipitation assay Thus, most of the mutations, which disrupted HeV entry in ephrin-B2 expressing cells in the context of a pseudotyped virus particle , were not doing so because the mutant G glycoprotein was poorly incorporated into the particles, nor did they have a defect in their ability to bind the ephrin-B2 receptor. The minor difference in the behavior of the W504A substitution in HeV-G, which destabilizes the HeV-G/ephrin-B2 complex, from that of the equivalent mutation in NiV-G, which does not seem to affect the NiV-G/ephrin-B2 binding The fact that various mutations at the HeV-G/ephrin interface appear to have little impact on binding while clearly affecting viral entry, suggest that they are likely preventing conformational changes or triggering steps linking ephrin binding with F glycoprotein activation. Indeed the conformational relay from the receptor-binding pocket of the HeV-G protein to its homodimerization interface Figure 5The structure-based models discussed above also shed light on the dynamic process of the virus-receptor attachment stage, as well as on the mechanism of the G glycoprotein initiated fusion-promotion step that facilitates F activation. The structures provide a clear basis to design further experiments for evaluation of models of henipavirus attachment and entry into host cells and to examine certain key steps in the paramyxovirus entry process in general. Further, the series of structure-based point mutations made in the HeV-G glycoprotein underscore the importance of the individual residues forming the ephrin binding interface, and indicate that both the crystallographically-observed hydrophobic and electrostatic interactions are crucial not only for cell attachment, but also for the subsequent membrane fusion and viral entry events.In addition, our structure-based mutagenesis experiments reveal three important and unique features of the HeV entry process: First, that single substitutions of interface resides affect more strongly the HeV-G binding and entry to ephrin-B3-expressing cells as compared to ephrin-B2-expressing cells, suggesting that the HeV-G/ephrin-B2 attachment is more robust and resistant to minor structural perturbations; Second, that that the stability of the HeV-G/ephrin association does not strongly correlate with the efficiency of viral entry, suggesting that, particularly for ephrin-B2-expressing cells, viral attachment is not the rate limiting step in the viral entry process; And third, that it is possible to alter the HeV-G/ephrin interface in a way that does not affect the overall stability and/or affinity of the association, but that does affect the efficiency of viral entry, in the context of henipavirus pseudovirions, supporting a model where subtle structural changes at the HeV-G/ephrin interface are relayed at a distance to trigger large conformational rearrangements in the HeV-G-HeV-F complex architecture, initiating membrane fusion.Based on the alignment between the HeV G and NiV G glycoproteins, we designed a construct containing residues 171–602, as well as several shorter constructs containing further N-terminal truncations. All HeV-G constructs were subcloned into the pMA152 vector baculovirus expression vector (provided by Alexander Antipenko) with a C-terminal Fc tag and expressed as previously described for NiV-G 2SO4. The HeV-G/ephrin-B2 complex was generated and crystallized as described in The Fc tag was removed from HeV-G and ephrin-B2 by thrombin cleavage, followed by further purification on a SD200 gel-filtration column (GE Lifesciences) and concentration to 15 mg/ml. Unbound HeV-G was crystallized in vapor diffusion sitting drops against a reservoir containing 20% PEG2000MME and 130 mM (NH4)Crystals were cryo-protected in mother-liquor with 25% glycerol added. X-ray diffraction data were collected at the Northeastern Collaborative Access Team, Advance Photon Source beamline 24ID-C. The diffraction images were integrated and scaled with DENZO/SCALEPACK A series of selected alanine substitution point mutations were made in full length HeV-G via site-directed mutagenesis using the Quick-Change II Site-directed Mutagenesis Kit . The template for the reactions consisted of a codon optimized full length HeV-G cloned in the pCAGGs expression vector 2 flasks. Cell lysates were prepared using lysis buffer (100 mM Tris-HCl (pH 8.0), 100 mM NaCl, and 1% Triton X-100) and clarified by centrifugation. For co-precipitations of HeV-G with receptors, cell lysates were incubated with 2 µg human ephrin-B3/FC or mouse ephrin-B2/FC  followed by precipitation with Protein-G Sepharose (Amersham). As a control and for comparison of expression, equal amounts of lysates were co-immunoprecipitated with 1 µl of HeV-G specific rabbit polyclonal antisera at 4°C for one hr. Samples were washed twice with lysis buffer followed by one wash with 100 mM Tris-HCl (pH 8.0), 100 mM NaCl, 0.1% sodium deoxycholate, and 0.1% SDS (DOC wash buffer). Samples were boiled in sample buffer with 2-mercaptoethanol, analyzed by SDS-PAGE and visualized by Western blotting under reducing conditions with mouse polyclonal HeV-G-specific antisera at 1∶25,000.Sub-confluent HeLa-USU cells were transfected for 48 h with the various alanine mutation-containing Gs or wild-type G either alone or in the presence of full length F, using the Fugene-6 transfection reagent . Cells were transfected with 3 µg total DNA per T-25 cm5 cells/well) and infection experiments were performed in triplicate. After infecting for 2.5-3 hr, cells were washed and incubated for additional 72 hr before lysis with 0.5% TritonX-100 in PBS. A 50 µl aliquot of the resulting lysate was assayed for luciferase activity using luciferase substrate . To quantify the incorporation of the HeV-F and -G glycoproteins into pseudotyped HIV-1 particles, equal amounts of sucrose cushion purified virus particles based on p24 content were lysed in buffer containing 100 mM Tris-HCl (pH 8.0), 100 mM NaCl, 2% Triton X-100 and protease inhibitors at 4°C for 30 min. Samples were boiled in SDS-PAGE sample buffer with 2-mercaptoethanol and separated on a 4–12% Bis-Tris gradient gels (Invitrogen), transferred to nitrocellulose, and probed with a cross-reactive polyclonal rabbit antiserum to recombinant HeV soluble G at a concentration of 1∶25,000 or a rabbit polyclonal F1 specific antiserum at a concentration of 1∶25,000, and HRP-conjugated goat anti-rabbit  as previously described Luciferase reporter gene-encoding HIV-1-based pseudovirus stocks were prepared by transfecting 293T cells with plasmids encoding the luciferase virus backbone pNL4-3-Luc-E-R+ S-peptide tagged HeV-F and un-tagged HeV-G encoding plasmids were co-transfected into sub-confluent HeLa-USU cells. HeV-G only and HeV-F only transfections were carried out in parallel as controls. At 48 hrs post transfection, cells were lysed in buffer containing 100 mM Tris-HCl (pH 8.0), 100 mM NaCl, 2% Triton X-100 and protease inhibitors at 4°C for 30 min, clarified by centrifugation, and divided into two equal portions. One half of each cell lysate was used to precipitate the HeV-F glycoprotein and the other half for precipitation of HeV-G. For total HeV-G present for precipitation, 1 µl of rabbit polyclonal anti-G antiserum was added followed by addition of 50 µl of a 20% slurry of Protein G-Sepharose 4B beads. For HeV-F precipitation, 30 µl of a 50% slurry S-protein agarose beads  was added to a separate and equal amount of lysate. In both cases, the complexed products and beads were washed twice in lysis buffer, and then boiled in sample loading buffer and the precipitated proteins were analyzed by SDS-PAGE and Western blotting under reducing conditions with either F or G specific mouse mAbs. The images of the precipitated F and G glycoproteins were quantified by densitometry using ImageQuantTL Software .Figure S1Related to ;Crystal packing helps to trap the “up” rotamer of W122. (A) Ephrin-B2 with the “down” W122 conformation contacts the adjacent HeV-G in crystal packing. (B) Ephrin-B2 with the “up” W122 conformation contacts the adjacent HeV-G in crystal packing. (C) Superimposition of the two HeV-G/ephrin-B2 complexes with different conformations of residue W122 (labeled in sticks). The different packing modes in the crystal prevent the “up” and “down” W122 rotamers from switching their conformations.(TIF)Click here for additional data file.Table S1(DOC)Click here for additional data file.


The asymmetric unit consists of one hep ligand and a CuBr unit. The Cu2+ ion is thereby coordinated by the N atom and the deprotonated hydroxy O atom in a distorted square-planar geometry that is completed by another O atom. The latter acts as bridging ligand towards the second, symmetry-equivalent, Cu atom, thus generating a centrosymmetric dimeric unit, with the inversion centre halfway between the Cu atoms. These units are linked via C—H⋯Br and C—H⋯O hydrogen bonds, leading to the formation of a hydrogen-bonded one-dimensional-polymeric chain along a..The title compound, [Cu DOI: 10.1107/S1600536811043637/fi2115Isup2.hkl            Structure factors: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials: