μParaflo™ Biochip for Nucleic Acid and Protein Analysis

Biomolecular interactions between proteins and synthetic surfaces impact diverse biomedical fields. Simple, quantitative, label-free technologies for the analysis of protein adsorption and binding of biomolecules are thus needed. Here, we report the use of a novel type of substrate, poly-p-xylylene coatings prepared by chemical vapor deposition (CVD) polymerization, for surface plasmon resonance enhanced ellipsometry (SPREE) studies and assess the reactive coatings as spatially resolved biomolecular sensing arrays. Prior to use in binding studies, reactive coatings were fully characterized by Fourier transform infrared spectroscopy, electrochemical impedance spectroscopy, and ellipsometry. As a result, the chemical structure, thickness, and homogeneous coverage of the substrate surface were confirmed for a series of CVD-coated samples. Subsequent SPREE imaging and fluorescence microscopy indicated that the synthetic substrates supported detectable binding of a cascade of biomolecules. Moreover, analysis revealed a useful thickness range for CVD films in the assessment of protein and/or antigen-antibody binding via SPREE imaging. With a variety of functionalized end groups available for biomolecule immobilization and ease of patterning, CVD thin films are useful substrates for spatially resolved, quantitative binding arrays.

We have previously identified a 12 nucleotide long sequence element, the TCE, that was demonstrated to be necessary for translational control of expression in the male germ line of Drosophila melanogaster (Schäfer et al., 1990). It is conserved among all seven members of the Mst(3)CGP gene family, that encode structural proteins of the sperm tail. The TCE is invariably located in the 5' untranslated region (UTR) at position +28 relative to the transcription start site. In this paper we analyse the mode of action of this element. We show that protein binding occurs at the TCE after incubation with testis protein extracts from Drosophila melanogaster. While several proteins are associated with the translational control element in the RNA, only one of these proteins directly crosslinks to the sequence element. The binding activity is exclusively observed with testis protein extracts but can be demonstrated with testis extracts from other Drosophila species as well, indicating that regulatory proteins involved in translational regulation in the male germ line are conserved. Although binding to the TCE can occur independent of its position relative to the transcription start site of the in vitro transcripts, its function in vivo is not exerted when shifted further downstream within the 5' UTR of a fusion gene. In addition to being a translational control element the TCE also functions as a transcriptional regulator. Consequently, a DNA-protein complex is also formed at the TCE. In contrast to the RNA-protein complexes we find DNA-protein complexes with protein extracts of several tissues of Drosophila melanogaster.

We have previously shown that in cell extracts from rat striatum, cyclic AMP response element (CRE) binding protein (CREB), rather than AP-1 proteins, preferentially interacts with the CRE-2 element of the proenkephalin second messenger-inducible enhancer, even under conditions in which AP-1 proteins are highly induced. Here we use primary striatal cultures to permit a more detailed analysis of CRE-2 function and protein binding in relevant neural cell types. By transfection we find that in primary striatal cultures, as in transformed cell lines, the CRE-1 and CRE-2 elements are required for significant induction by cyclic AMP. We report that cyclic AMP induction of the proenkephalin gene in striatal cultures is protein synthesis independent, excluding a role for newly synthesized proteins like c-Fos. We also show that cyclic AMP induces CREB phosphorylation and that phosphorylated CREB interacts strongly with CRE-2 and weakly with CRE-1. The predominant protein bound to CRE-1 is not CREB, however, and remains to be identified. Despite some prior predictions, we do not find a role for c-Fos in cyclic AMP regulation of proenkephalin gene expression in neurons.

A guided tour of transcription factor methodologies: Transcription Factors – A Practical Approach edited by D.S. Latchman

To develop a UPLC-MS/MS method for quantifying tigecycline (TIG), meropenem (MER), polymyxin B1 (PB1), and PB2 in human plasma, and analyze their unbound fractions and influencing factors in HSCT patients. Plasma samples from HSCT patients were analyzed using the developed UPLC-MS/MS technique. The method showed high accuracy, precision, and stability. Protein binding rates were 82.4% for TIG, 83.7%-79.3% for PB1, and 86.7%-82.6% for PB2, with lower binding at trough vs. peak concentrations. Preliminary analysis suggests that BMI and serum albumin may influence PB1/PB2 binding in CRE-infected HSCT patients though larger cohorts are needed for confirmation. (clinical trial registration # 2021-0938-01).

Unclassified variants (UV) of BRCA1 can affect normal pre-mRNA splicing. Here, we investigate the UV c.693G>A, a “silent” change in BRCA1 exon 11, which we have found induces aberrant splicing in patient carriers and in vitro. Using a minigene assay, we show that the UV c.693G>A has a strong effect on the splicing isoform ratio of BRCA1. Systematic site-directed mutagenesis of the area surrounding the nucleotide position c.693G>A induced variable changes in the level of exon 11 inclusion/exclusion in the mRNA, pointing to the presence of a complex regulatory element with overlapping enhancer and silencer functions. Accordingly, protein binding analysis in the region detected several splicing regulatory factors involved, including SRSF1, SRSF6 and SRSF9, suggesting that this sequence represents a composite regulatory element of splicing (CERES).

BackgroundHepatitis C Virus (HCV) is an asymptomatic chronic infection with serious clinical consequences. Timely and sensitive detection of HCV RNA is critical for infection control, treatment response follow up. While current technologies, such as PCR or isothermal amplification-based strategies, are specific, they are expensive, labor intensive, time consuming limiting their use in field and small laboratories. ResultsWe introduce a novel technology for detecting nucleic acids, as exemplified by HCV RNA, in clinical specimens. This approach utilizes the crosslinked Enhanced Emission (CEE) phenomena upon mixing fluorescent amino functionalized silica-coated nitrogen-doped carbon dots (N-CDs/SiO2/NH2) with magnetic extracted unamplified HCV RNA showed a significant and immediate fluorescence enhancement. This method was integrated into a semi-automated 3D printed microfluidic chip, wherein the unamplified RNA was mixed with the N-CDs/SiO2/NH2. The assay was also employed on the conventional 96-well plate format. This assay offers high sensitivity with detection limit of 500 IU/ml and 1000 IU/ml for the chip and well plate respectively. The sample-to-result time was <20 min on the chip and is simpler than the amplification-based techniques. Analyzing 141 patient samples yielded sensitivity and specificity of 96.47 % and 98.79 % respectively. SignificanceThis application of N-CDs/SiO2 as nucleic acid fluorescent probes for the first time, offers a versatile, cost-effective, and universal platform for nucleic acids detection. The developed system can be employed in conventional microwell plate-based detection with minimal modifications to current laboratory setups. Additionally, it was integrated into a 3D-printed microfluidic chip enabling enhancement in specificity, sensitivity and accuracy.

Prodigiosin and cycloprodigiosin are tripyrrole red pigmented compounds with medical importance for their anticancer property. In the present investigation, molecular docking studies were performed for both prodigiosin and cycloprodigiosins to evaluate the in- silico anti-inflammatory activity against Cycloxigenase-2 (COX-2) protein as model compound and the data compared with rofecoxib and celcoxid. Cycloprodigiosin showed higher initial potential, initial RMS gradient and potential energy values compared to prodigiosin. Analysis of COX-2 protein and ligand binding revealed that cyclprodigiosin interacted with COX-2 protein amino acid residues of Tyr324, Phe487 and Arg89 while prodigiosin interaction was observed with two amino acids i.e. Leu321 and Tyr324. The computational ligand binding interaction suggested > 45% higher fitness score value for prodigiosin to that of cycloprodigiosin with COX-2 protein while the standard compounds rofecoxib and celecoxid revealed fitness score of 44 and 62, respectively. The prodigiosin ligand revealed the best fitness score compared with the standard drug rofecoxib suggesting the prodigiosin could be effective as the potential inhibitor compound against COX-2 protein and can be evaluated as anti-inflammatory drug molecule using clinical trials.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-2-172) contains supplementary material, which is available to authorized users.

Evaluation of two lanthanide complexes for qualitative and quantitative analysis of target proteins via partial least squares analysis

The development of effective malaria vaccines remains a global health priority. In addition to an effective vaccine, there is urgent demand for effective delivery technologies that can be easily deployed. The need for effective vaccine delivery tools is particularly pertinent in resource-poor settings where access to healthcare is limited. Microneedles are micron-scale structures that offer distinct advantages for vaccine delivery by efficiently targeting skin-resident immune cells, eliminating injection-associated pain, and improving patient compliance. Here, we developed and characterized a candidate malaria vaccine loaded and deployed using dissolvable microneedle arrays. Of note, a newly indicated human-relevant antigen was employed, Plasmodium falciparum surface protein P47. P47 and a potent toll-like receptor (TLR9) agonist vaccine adjuvant, CpG, were fabricated into microneedles using a gelatin polymer. Protein binding, ELISA, and fluorescence analysis confirmed the molecular structure, and the function of the P47 antigen and CpG was maintained after fabrication, storage, and release from microneedles. In cell culture, the cargo released from the microneedle arrays triggered TLR9 signaling and activated primary dendritic cells at levels similar to native, unincorporated vaccine components. Together, these studies demonstrate the potential of microneedles as an easily deployable strategy for a P47-based malaria vaccine.

The two SH2 (Src homology domain 2) domains present in phospholipase C-gamma1 (PLC-gamma1) were assayed for their capacities to recognize the five autophosphorylation sites in the epidermal growth factor receptor. Plasmon resonance and immunological techniques were employed to measure interactions between SH2 fusion proteins and phosphotyrosine-containing peptides. The N-SH2 domain recognized peptides in the order of pY1173 > pY992 > pY1068 > pY1148 >> pY1086, while the C-SH2 domain recognized peptides in the order of pY992 > pY1068 > pY1148 >> pY1086 and pY1173. The major autophosphorylation site, pY1173, was recognized only by the N-SH2 domain. Contributions of the N-SH2 and C-SH2 domains to the association of the intact PLC-gamma1 molecule with the activated epidermal growth factor (EGF) receptor were assessed in vivo. Loss of function mutants of each SH2 domain were produced in a full-length epitope-tagged PLC-gamma1. After expression of the mutants, cells were treated with EGF and association of exogenous PLC-gamma1 with EGF receptors was measured. In this context the N-SH2 is the primary contributor to PLC-gamma1 association with the EGF receptor. The combined results suggest an association mechanism involving the N-SH2 domain and the pY1173 autophosphorylation site as a primary event and the C-SH2 domain and the pY992 autophosphorylation site as a secondary event.

A microfluidic system for the separation and detection of E. coli O157:H7 in soil sample using ternary interactions between humic acid, bacteria, and a hydrophilic surface

We present a new technique for determining RNA:DNA ratios in larval and juvenile fish that relies on the rapid homogenization of samples with salts, detergents, and proteinase K and the detection of DNA and RNA with specific fluorescent dyes. Samples are not purified before assay of nucleic acids. The technique was designed for use in 96 or 384 well plates and required only 50 μL of sample per well. The detection of RNA and DNA is linear over 500 ng and is sensitive to a nucleic acid content as little as that within 50 μg of tissue. The new technique was used to characterize the RNA:DNA ratio in the white muscle of juvenile mummichogs Fundulus heteroclitus and larval winter flounder Pleuronectes americanus. This technique was compared with other techniques that use fluorescent dyes to determine the RNA:DNA ratio in fish samples.

Janus kinase 2 (JAK2) initiates signaling from several cytokine receptors and is required for biological responses such as erythropoiesis. JAK2 activity is controlled by regulatory proteins such as Suppressor of Cytokine Signaling (SOCS) proteins and protein tyrosine phosphatases. JAK2 activity is also intrinsically controlled by regulatory domains, where the pseudokinase (JAK homology 2, JH2) domain has been shown to play an essential role. The physiological role of the JH2 domain in the regulation of JAK2 activity was highlighted by the discovery of the acquired missense point mutation V617F in myeloproliferative neoplasms (MPN). Hence, determining the precise role of this domain is critical for understanding disease pathogenesis and design of new treatment modalities. Here, we have evaluated the effect of inter-domain interactions in kinase activity and substrate specificity. By using for the first time purified recombinant JAK2 proteins and a novel peptide micro-array platform, we have determined initial phosphorylation rates and peptide substrate preference for the recombinant kinase domain (JH1) of JAK2, and two constructs comprising both the kinase and pseudokinase domains (JH1-JH2) of JAK2. The data demonstrate that (i) JH2 drastically decreases the activity of the JAK2 JH1 domain, (ii) JH2 increased the Km for ATP (iii) JH2 modulates the peptide preference of JAK2 (iv) the V617F mutation partially releases this inhibitory mechanism but does not significantly affect substrate preference or Km for ATP. These results provide the biochemical basis for understanding the interaction between the kinase and the pseudokinase domain of JAK2 and identify a novel regulatory role for the JAK2 pseudokinase domain. Additionally, this method can be used to identify new regulatory mechanisms for protein kinases that provide a better platform for designing specific strategies for therapeutic approaches.

The interactions of N4-octadecyl-1-beta-D-arabinofuranosylcytosine (NOAC), a lipophilic derivative of 1-beta-D-arabinofuranosylcytosine (ara-C), were studied in vitro with human blood components. Binding of NOAC incorporated into liposomes to erythrocytes (Ec) was saturated at 63 nmol/10(9) Ec and binding analysis resulted in a weak affinity of 3 x 10(3) liters/mol and 4 x 10(7) binding sites per Ec. The Ec partition coefficient D(Ec) was approximately 4, which demonstrates the high accumulation of NOAC in Ec membranes. The calculated fraction f(b) of drug bound to plasma proteins was 30%. Analysis of serum protein binding of NOAC was done by density gradient ultracentrifugation and agarose gel electrophoresis. Liposomal NOAC was distributed to low-density lipoproteins (LDL) at 36%, to high-density lipoproteins at 21%, to albumin and other proteins at 12% and to very-low-density lipoproteins at 5%. Comparable results were obtained for the analog N4-hexadecyl-1-beta-D-arabinofuranosylcytosine and when the drugs were dissolved in dimethyl sulfoxide. The biodistribution of liposomal NOAC in ICR mice after intravenous application revealed a biphasic blood concentration versus time curve with a distribution half-life t1/2alpha of 23 min and an elimination half-life t1/2beta of 7 h. The drug was distributed mainly into the liver with an organ load of 69% and with an elimination half-life of 8 h. The strong affinity of NOAC to LDL might be exploited for the enhanced uptake of the drug in tumor cells expressing high numbers of LDL receptor molecules.