bioorthogonal reaction, which suggests the bioorthogonal reaction is an effective strategy for signal amplification in biochemical assays. In bioorthogonal reaction system, it is easy to achieve multiple signal amplification through layer-by-layer bio-conjugation strategy. This result shows that the Tz/TCO cycloaddition reaction is better than biotin-streptavidin recognition for signal amplification. The ligands of click chemistry are small molecules which are suitable to modify NPs or biomolecules, and this bio-conjugation can be controlled to satisfy the need of signal amplification and readout. In contrast, in the avidin-biotin system, the large molecular size of avidin (6 nm, 67 kDa) could potentially mask adjacent biotin sites. In addition, biotin must associate within a deep cleft inside the avidin protein, which could physically or spatially constrain certain binding configurations. By contrast, Tz is a small molecule that can interact with TCO (also a small molecule) on the surface of the antibody without physical restriction on neighboring TCO sites. The small molecule-based bioorthogonal chemistry allows nanoparticles to pack more ligands densely onto the antibody scaffolds, yielding greater signal amplification. Thus, bioorthogonal reaction is a more effective bio-conjugation method for biochemical analysis, especially for detection of targets (CTC, biomarkers, pathogen, virus) in complex sample at low-concentrations.
17 Read more
In this study, we investigated the cell entry characteristics of dengue virus (DENV) type 2 strain S1 on mosquito, BHK-15, and BS-C-1 cells. The concentration of virus particles measured by biochemical assays was found to be substantially higher than the number of infectious particles determined by infectivity assays, leading to an infectious unit-to-particle ratio of approximately 1:2,600 to 1:72,000, depending on the specific assays used. In order to explain this high ratio, we investigated the receptor binding and membrane fusion characteristics of single DENV particles in living cells using real-time fluorescence microscopy. For this purpose, DENV was labeled with the lipophilic fluorescent probe DiD (1,1 ⴕ -dioctadecyl-3,3,3 ⴕ ,3 ⴕ -tetramethyl- indodicarbocyanine, 4-chlorobenzenesulfonate salt). The surface density of the DiD dye in the viral membrane was sufficiently high to largely quench the fluorescence intensity but still allowed clear detection of single virus particles. Fusion of the viral membrane with the cell membrane was evident as fluorescence dequenching. It was observed that DENV binds very inefficiently to the cells used, explaining at least in part the high infectious unit-to-particle ratio. The particles that did bind to the cells showed different types of transport behavior leading to membrane fusion in both the periphery and perinuclear regions of the cell. Membrane fusion was observed in 1 out of 6 bound virus particles, indicating that a substantial fraction of the virus has the capacity to fuse. DiD dequenching was completely inhibited by ammonium chloride, demonstrating that fusion occurs exclusively from within acidic endosomes.
10 Read more
Synergist studies using PBO and DEF on three strains of German cockroaches collected from Alabama revealed that, despite being from the same geographic origin, P450 monooxigenases and hydrolases are strongly involved in permethrin and deltamethrin resistance in one strain while playing a minor role in two other strains . Undertaking biochemical assays, Lee et al.  characterized possible insecticide resistance mechanisms in four Malaysian field-collected strains of German cockroach. Elevated esterase activity was detected in all four strains, while elevated GST levels were present in only two strains studied. Classic bioassays and biochemical assays performed on German cockroaches collected from residential areas of Tehran (Iran) showed that oxidases, esterases and GSTs are involved in pyrethroid resistance [3, 23].
In our previous genetic studies, we showed that Drosoph- ila CLASP is required for Abl function in motor axons [Lee et al., 2004]. Although this in vivo analysis was the first to link CLASP and Abl function in any context, genetic analy- sis alone cannot reveal whether the interaction between CLASP and Abl is direct or indirect. In addition, our genetic epistasis experiments did not conclusively show that CLASP is regulated downstream of the kinase. Moreover, although CLASP and Abl sequences are highly conserved across species, the question remained as to whether the functional relationship between CLASP and Abl is retained in vertebrate cells. Using biochemical assays with vertebrate cells and proteins, we now address these questions using the neuronal-enriched gene CLASP2. We find that Abl binds to and phosphorylates CLASP2 in response to extracellular signals such as serum or PDGF. In vitro experiments indi- cate that CLASP2 is a direct substrate of Abl. Biochemical experiments with purified proteins show that Abl can mod- ulate CLASP2 binding to MTs and actin. Finally, analysis of CLASP2 in cultured vertebrate neurons reveals that Abl regulates CLASP2 localization and its interaction with both MTs and actin in the growth cone. Together our findings suggest that a functional relationship between Abl and CLASP2 is conserved across species that this may coordi- nate actin and MT behavior.
16 Read more
at levels that directly paralleled their initiator activities in the biochemical assays described above: K356R and K359A supported replication at similar levels to wild- type E1 (lanes 10 and 16 compared to 1), the activities of K356A and K356Q were reduced (K356A to a greater extent than K356Q; lanes 4 and 13 compared to 1), and K356E was completely inactive (lane 7). With increasing concentration of expression construct, this trend was maintained and no replication was detected for K356E, even with the highest amount of expression vector tested (lanes 7–9). Similar results were obtained at earlier and later times (36 and 72 h post-transfection, data not shown). The in vitro replication activity of E1 and mutant proteins was tested in a mammalian cell-free protein extract supplemented with ori-plasmid and the precursors of DNA synthesis (34). Replication products labelled with a- 32 PdCTP were resolved in agarose gels and visualised by phosphor-imaging (Figure 6B). Increasing concentrations of wild-type E1 promoted increasing levels of DNA synthesis (lanes 2–5). K356R and K359A (lanes 14–17 and 22–25) supported DNA synthesis at similar levels to
The present study using biochemical assays pertaining to Blood Glucose Levels of different animal models reveals that the aqueous and alcoholic extract (in the dose 400 mg/kg body weight) of roots of Mangifera indica was found to have the moderate antidiabetic activity. However, longer duration studies of Mangifera indica and its isolated compounds on chronic models are necessary to develop a potent antidiabetic drug.
As indicated previously, the disease mutation catalogues in common use for in silico prediction tool training and benchmarking suffer from circularity through a lack of independence on multiple levels . To address this, we have identified that data relating to biochemical assays of protein function, without significant overlap with disease mutation catalogues, should be highly valuable for variant effect prediction tool assessment (and training). These re- flect validated effects on protein function while achieving independence. Since highly curated and accessible data- bases with these properties are not available, we have engi- neered three such datasets, based on (1) mining functional mutagenesis data from UniProt, (2) the deep mutational scanning (DMS) protocol applied to BRCA1 and (3) the as- sessment of TP53 mutants by transactivation assay.
This study is the first to characterize the in vitro nucleic acid chaperone activity of the HIV-2 NC protein and to compare it with HIV-1 NC. Biochemical assays with sub- strates derived from the HIV-1 and HIV-2 genomes were performed to compare the ability of both proteins to chaperone nucleic acid aggregation, annealing and strand exchange in duplex structures. Using a truncated NCp8 mutant, we found that the short, basic N-terminal domain is crucial for NCp8 activity. NAC activity of NCp8 and NCp7 in assays with HIV-1 TAR oligonucleotides was similar, but when longer HIV-1 substrates or particularly those derived from the HIV-2 genome were used, interest- ing differences between these two proteins were discov- ered. In contrast to NCp7, NCp8 weakly facilitates annealing of HIV-2 TAR RNA to complementary TAR (−) DNA. Moreover, NCp8 was unable to efficiently stimulate tRNA Lys3 annealing to its respective HIV-2 PBS motif. Our data suggest that the NAC activity of HIV-1 and HIV-2 NC proteins is not equivalent and NCp8 exhibits lower chaperone activity in vitro than NCp7.
13 Read more
Urine samples from children with human immunodeficiency virus (HIV) infection and healthy controls were examined for mycoplasmas by culture. Standard biochemical assays, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and PCR (16S and 16S-23S spacer rRNA region) were used for identification of isolates. Mycoplasmas were identified from 13 (87%) of 15 HIV-positive patients and 3 (20%) of 15 HIV-negative control patients. The frequency and type of mycoplasma varied with the severity of HIV infection. Mycoplasma penetrans, Mycoplasma pirum, Mycoplasma fermentans, and Mycoplasma genitalium were isolated from patients with severe immunodeficiency. Mycoplasma hominis and Ureaplasma urealyticum were isolated more frequently from children in the early stages of HIV infection and from HIV-negative patients. Mycoplasma penetrans was isolated from one (50%) of two patients in Centers for Disease Control and Prevention (CDC) group B and from five (55.5%) of nine pediatric patients with AIDS (CDC group C). This is the first report that indicates that “AIDS-associated” mycoplasmas are more common in HIV-infected children than in HIV-negative controls.
The study of viral gene expression based on biochemical in vitro systems and mini-genes containing only a portion of the viral genome does not always reliably mimic the complex set of overlapping and redundant regulatory elements present within the viral genome. Although hnRNPs of the A/B family and the SR protein SC35 regulate expression of the Tat1 mRNA as predicted in an in vitro system (15, 41), downregulation of the Rev2 and Nef2 mRNAs following overexpression of SF2 and SRp40 does not correlate well with data obtained with a viral mini-gene (4). Discrepancies between the results presented here and the ones obtained in controlled biochemical assays or with partial genomic sequences are likely due to (i) the com- binatorial effect of multiple and overlapping viral sequences that may interact with several cellular factors, (ii) pleiotropic effects on mRNA metabolism of hnRNPs and SR proteins, and (iii) secondary effects on cellular genes required for viral gene expression, replication, and infectivity.
12 Read more
most useful. While it is possible to use small inhibitory RNAs (siRNA) to specifically knock-down the levels of the endogenous protein prior to introducing an siRNA- resistant variant transgene, these studies are tedious and may lead to misinterpretations as complete elimination of the endogenous wild-type protein is unlikely. Thus, the cell lines most commonly used are human cancer cell lines that have suffered mutational inactivation of the endogenous MMR genes. Previous studies have iden- tified cancer cell lines that lack MSH2, MLH1 or MSH6 expression where MMR functions can be restored by re- introduction of the wild-type gene [47-50]. Cancer cell lines generally grow well in culture and are immortal which makes it easy to generate large numbers of cells for performing biochemical assays. However, the genetic background of these lines is uncertain and likely un- stable possibly masking the function of some VUS. Some studies have avoided the need to use MMR-null cancer cell lines by adding a protein tag to the variant transgene to distinguish it from endogenous wild-type protein [32,51,52]. Through use of fluorescent protein fusions, we previously were able to track expression and cellular localization of the variant proteins in NIH-3T3 primary mouse embryonic fibroblasts (MEFs) (discussed further below) . This approach is limited to those assays in which the tagged protein can be isolated from the en- dogenous wild-type protein. In addition, the presence of a large protein tag may influence the function of the MMR protein, which needs to be examined in carefully controlled experiments .
10 Read more
Another drawback of the biochemical assays was the absence of fully susceptible reference strains for each spe- cies tested. The An. minimus s.s. population collected in 2003 in the Hoa Binh province was used as reference strain based on the bioassay results (100% mortality against DDT, permethrin, alpha-cypermethrin and lambda-cyhalothrin), but compared to the other field populations, this An. minimus s.s. population had relative high levels of P450 monooxygenases. Significant lower levels of P450 monooxygenases were measured in the An. epiroticus and An. subpictus populations. In fully suscepti- ble strains of An. gambiae s.s. and An. albimanus, the monooxygenase levels were also lower than the levels measured in the VHBA An. minimus s.s. population [19,36]. Additionally, WHO bioassays carried out in 2004 on 1–2 days old mosquitoes of the VHBA An. minimus s.s. population showed reduced mortality against the type II pyrethroids (alpha-cypermethrin 96% and lambda-cyha- lothrin 94% mortality) and the non-ester pyrethroid etofenprox (95% mortality). Taking this into account, it is likely that the pyrethroid resistance in An. minimus s.l. could be conferred to an increased detoxification by both P450 monooxygenases and esterases, whereas in An. epiroticus and An. subpictus the pyrethroid resistance could be conferred to an esterase mediated detoxification. How- ever, additional WHO bioassays performed on 1–2 days old An. epiroticus mosquitoes of VBLA and VBLB (Mekong Delta) revealed a low mortality against the non-ester pyre- throid etofenprox . This means that it is likely that beside an esterase mediated detoxification also other pyrethroid resistance mechanisms are involved in An. epiroticus of the Mekong Delta.
14 Read more
The chemo-protective effects of chitosan were evaluated on male and female mice treated with Cyclophosphamide as strong cytotoxic agent by measuring standard biochemical markers. Table 9, 10 illustrated the levels of SOD, GOT, GPT, Glucose, Cholesterol and Triglycerides of experimental male and female subgroups. The results showed that the estimated biomarkers activities in male and female different subgroups had almost the similar significant different between the treated and control subgroups.
18 Read more
The melon aphid, Aphis gossypii Glover (Hemiptera: Aphididae), is the most important cosmopolitan, highly polyphagous aphid species that infests 200 economically important crops worldwide . Damage occurs as a result of direct feeding on the plant phloem sap, honeydew deposition and physiological disorders. It is the most versatile insect vector capable of transmitting >75 plant viruses . It can easily reach outbreak population le- vels when environmental conditions are suitable and is regulated by their natural enemies in nature. However, chemical control using insecticides remains the main method of melon aphid control  and means of virus management in the absence of resistant varieties. Due to widespread intensive application of organophosphates (OPs), melon aphids have developed high levels of resistance ensuing in eventual control failures and reduced crop yield  . It is considered to develop elevated levels of resistance to OPs in a relatively short period of time due to its high reproductive potential by means of parthenogenesis and likelihood to develop resistance   . Due to which it is one among top 12 resistant arthropods with reported resistance to 49 unique com- pounds . In addition, the ability of this aphid species to travel over long distances could widely distribute re- sistant populations dispersing pathogenic viruses across host plants ensuing in huge crop loss . Thus, resis- tance problem in A. gossypii has become a greater concern around world year by year and it is the most difficult case to manage. In the last three decades, remarkable strides have been made in understanding pesticide resis- tance in arthropods especially aphids. However, there are no reports available on the underlying mechanisms conferring OP resistance in Indian populations of A. gossypii. Among the OPs, dimethoate remains the man- agement choice in aphid and aphid transmitted virus control for farmers and seed industries for the past 40 years . Although effective for several years, a decrease in OP efficacy has been reported in populations collected from South India especially Guntur where this insecticide was applied intensively for many years . It is an- ticipated that melon aphids have developed resistance to dimethoate that has been applied intensively for four decades and if so, biochemical basis behind such resistance needs to be determined and documented.
16 Read more
All of the isolates of C. jejuni, C. coli, C. lari, or C. upsaliensis tested could be identified by the LiPA. For only 9 (2.8%) of 320 isolates tested did the LiPA yield results discordant with those of conventional methods. Three of these samples con- tained C. jejuni as well as C. coli. In contrast to conventional identification methods, the LiPA allows sensitive detection of cultures, and thus infections, with multiple species. Purification of single colonies from several primary cultures resulted in unequivocal detection of only a single Campylobacter species, indicating that initial detection of two species was not due to cross-reactivity of the probes on the strip. In five of the re- maining six discrepant cases, the PCR-LiPA results were con- firmed by an alternative molecular method, and in one case, the isolate probably contained a mixture of strains. Our find- ings strongly confirm previous observations (3, 9) that conven- tional biochemical identification methods, such as hippurate hydrolysis and resistance to cephalothin and nalidixic acid, are not always accurate for identification of Campylobacter species. Taken together, this study indicates that the specificity of the PCR-LiPA method is virtually 100%.
Table 3 explains primers sequences, target genes, amplicon sizes and cycling conditions which used in preparation of DNA. Our results in Tables 1 and 2 showed the characterization of E. coli isolated from mastitic milk by chemical tests which differantiate it from other cause of mastitis and other enterobactereacae. Table 1 determines the strain of E. coli by serotypes of E. coli isolated from clinical mastitis cow. Table 4 showed that virulant genes present in strains O44eae, O44eae and Stx1, O55, O26eae, O114, O146 eae and Stx2, O158, O125. Biochemical changes associated to infection appear in cows that infected with mastitis Table 5 showed biochemical changes associated to E. coli infection in serum of cows while Tabie 6 showed inflamatory response associated to E. coli infection and immunity response. Figure 1 showed abnormal changes in teat infected with E. coli showed inflamed and redness teat when compare with normal teat in other figure while Figure 2 agrose gel electrophoresis showed Intamin (eaeA, Stx1 and Stx2) genes from extracted DNA of E.coli serogroup (O55, O26, O114, O146 O158, and O125).
mediated Cp promoter activation and LCL cell growth by recruiting a repressor, inhibiting an EBNA2-associated activa- tor, or by altering the interaction of RBP-J with DNA. Al- though EBNA3A aa 240 to 300 and 386 to 410 do not affect EBNA3A association with RBP-J or repression of EBNA2 through RBP-J in transient assays, these residues may alter interactions through RBP-J at specific promoters or be a scaffold for a new regulatory factor. Transcriptional regulation through RBP-J can be determined by other transcription fac- tors that regulate individual promoters. In contrast to the Cp promoter, EBNA3A coactivates with EBNA2 at the EBV LMP1 promoter (27). EBNA3A sequences responsible for this effect have not been identified. Further, neither EBNA nor LMP1 expression was altered following EBNA3A inactivation in the EBNA3AHT LCLs. Thus, the biochemical roles of aa 240 to 300, 300 to 386, and 386 to 410 are uncertain.
The trend towards assay miniaturization arose simultaneously with move towards automation as a direct need to reduce development cost. Although at present most HTS is still carried out in 96-well plate format, the move towards 384-well and higher density plate formats is well under construction. Instrumentation for accurate, low-volume dispensing into 384-well plates is commercially available, so are sensitive plate-readers that accommodate this format. Many of the HTS studies are carried out in 384-well plates; yet, reformatting of 96-well compound plates into the higher density format can become a significantly difficult to implementing screens in this mode. Researchers have implemented their recombinase/luciferase reporter system for use in 864-well plates. As few as 560 cells per assay well were sufficient to measure dose response curves for ligand binding to the Glucocorticoid receptor. Some scientists have carried out luciferase reporter gene assays in human T-cells using a 1,536-well (3 microliter) plate format. The evolution and implementation of microplate-based screening in smaller volume, higher density formats (1,536-well plates and beyond) are challenged by numerous technical obstacles. Some assays may be difficult to implement in these formats due to sensitivity to final dimethylsulfoxide (DMSO) concentration as this a versatile and frequently used reagent which may build up additional cost.
To test further the hypothesis that srPSTVds are derived from structured PSTVd RNAs, we performed biochemical ex- periments to determine whether structured PSTVd RNAs can indeed be cleaved by DCLs. Partial purification of Arabidopsis DCLs has been reported (64). We observed that PSTVd could replicate in leaf protoplasts of Arabidopsis and that PSTVd- specific small RNAs accumulated in the infected protoplasts (Fig. 3A). Therefore, Arabidopsis possesses enzymatic activi- ties for the biogenesis of srPSTVds. We used chromatography to identify Arabidopsis cell extract fractions that contained DCL activities, as revealed by the processing of mir319 pre- cursor RNA into small RNAs of ⬃ 21 nt (Fig. 3B). The linear, plus-PSTVd RNA, which folds into a rod-like secondary struc- ture similar to that of the circular genomic RNAs (8, 103), was also processed into small RNAs of ⬃ 21 nt in such fractions (Fig. 3B). In contrast, incubation of ssGFP RNAs did not yield any small RNAs or any other types of cleavage products (Fig. 3B). Therefore, production of small RNAs from the substrate RNAs in this system was not attributed to random nuclease activities. These results provide biochemical evidence that the structured PSTVd RNAs can indeed serve as substrates of DCL activities.
15 Read more
The effect of M50I with R263K on susceptibility to INSTIs The integrase coding region of HIV-1 contains similar levels of natural variation as that seen in protease . Fur- thermore, the pre-existing polymorphism L63P which emerges during treatment with PIs has been shown to be compensatory when combined with a primary resistance mutation . We therefore wanted to determine if the addition of M50I, a natural polymorphism in integrase, to R263K affected susceptibility to DTG, RAL, and EVG in strand-transfer assays. Analysis with the competitive inhib- ition model was used to generate values of relative V max