Biophysical and functional characterization of N-terminal domain of Human Interferon Regulatory Factor 6.

BACKGROUND: Interferon regulatory factor 6 (IRF6) has a key function in palate fusion during palatogenesis during embryonic development, and mutations in IRF6 cause orofacial clefting disorders. METHODS AND RESULTS: The in silico analysis of IRF6 is done to obtain leads for the domain boundaries and subsequently the sub-cloning of the N-terminal domain of IRF6 into the pGEX-2TK expression vector and successfully optimized the overexpression and purification of recombinant glutathione S-transferase-fused NTD-IRF6 protein under native conditions. After cleavage of the GST tag, NTD-IRF6 was subjected to protein folding studies employing Circular Dichroism and Intrinsic fluorescence spectroscopy at variable pH, temperature, and denaturant. CD studies showed predominantly alpha-helical content and the highest stability of NTD-IRF6 at pH 9.0. A comparison of native and renatured protein depicts loss in the secondary structural content. Intrinsic fluorescence and quenching studies have identified that tryptophan residues are majorly present in the buried areas of the protein and a small fraction was on or near the protein surface. Upon the protein unfolding with a higher concentration of denaturant urea, the peak of fluorescence intensity decreased and red shifted, confirming that tryptophan residues are majorly present in a more polar environment. While regulating IFNß gene expression during viral infection, the N-terminal domain binds to the promoter region of Virus Response Element-Interferon beta (VRE-IFNß). Along with the protein folding analysis, this study also aimed to identify the DNA-binding activity and determine the binding affinities of NTD-IRF6 with the VRE-IFNß promoter region. The protein-DNA interaction is specific as demonstrated by gel retardation assay and the kinetics of molecular interactions as quantified by Biolayer Interferometry showed a strong affinity with an affinity constant (KD) value of 7.96 × 10-10 M. CONCLUSION: NTD-IRF6 consists of a mix of α-helix and ß-sheets that show temperature-dependent cooperative unfolding between 40 °C and 55 °C. Urea-induced unfolding shows moderate tolerance to urea as the mid-transition concentration of urea (Cm) is 3.2 M. The tryptophan residues are majorly buried as depicted by fluorescence quenching studies. NTD-IRF6 has a specific and high affinity toward the promoter region of VRE-IFNß.

Effortless residual adhesive removal: A simple method.

The cheaper surgical dressings often leave residual adhesive, which can be unsightly and uncomfortable. We describe a simple and cost-effective procedure for removing adhesive residue using micropore tape. This technique can be easily performed by anyone, including the patients themselves.

Machine learning assisted hepta band THz metamaterial absorber for biomedical applications.

A hepta-band terahertz metamaterial absorber (MMA) with modified dual T-shaped resonators deposited on polyimide is presented for sensing applications. The proposed polarization sensitive MMA is ultra-thin (0.061 λ) and compact (0.21 λ) at its lowest operational frequency, with multiple absorption peaks at 1.89, 4.15, 5.32, 5.84, 7.04, 8.02, and 8.13 THz. The impedance matching theory and electric field distribution are investigated to understand the physical mechanism of hepta-band absorption. The sensing functionality is evaluated using a surrounding medium with a refractive index between 1 and 1.1, resulting in good Quality factor (Q) value of 117. The proposed sensor has the highest sensitivity of 4.72 THz/RIU for glucose detection. Extreme randomized tree (ERT) model is utilized to predict absorptivities for intermediate frequencies with unit cell dimensions, substrate thickness, angle variation, and refractive index values to reduce simulation time. The effectiveness of the ERT model in predicting absorption values is evaluated using the Adjusted R2 score, which is close to 1.0 for nmin = 2, demonstrating the prediction efficiency in various test cases. The experimental results show that 60% of simulation time and resources can be saved by simulating absorber design using the ERT model. The proposed MMA sensor with an ERT model has potential applications in biomedical fields such as bacterial infections, malaria, and other diseases.

A rapid method for solubilization of chimeric human interferon regulatory factor-1 (IRF-1) protein in Escherichia coli.

Interferon regulatory factor-1 (IRF-1) is a vertebrate transcription factor that plays significant roles in cell cycle regulation, anti-viral response, tumor suppression and immune response. High-level expression of recombinant IRF-1 at 37 °C leads to the formation of insoluble aggregates (insoluble fraction) in Escherichia coli (E. coli), which usually devoid of biological activity. In this study, we use chemical additives such as mannitol, proline, L-arginine and CTAB (cetyl trimethly ammonium bromide) at the recommended concentration during cell lysis to aid in solubility at 37 °C. The use of additives resulted in the increased solubility of the recombinant glutathione S-transferase-linked human IRF-1, with L-arginine being most effective. Here, we developed an efficient process for the manufacturing of soluble IRF-1 with the aid of minimizing the formation of degradation products and optimizing protein purification conditions. This result was further confirmed by western blot with anti-GST and anti-IRF-1 polyclonal antibodies. The functionality of GST-huIRF-1 was attained by elerophoretic mobility shift assay study as a clear band shifting showed with virus response element-Interferon beta (VRE-IFNß) promoter region. Taken together, the biological activity of purified GST-huIRF-1 was also optimized and confirmed by supershift assay concluded that GST-huIRF-1 interacts with the VRE motif of IFNß promoter that reflected to require for IFNß gene regulation. We describe a straightforward approach for the production of absolutely soluble and biologically active IRF-1 in E. coli. This method can be further used for the study of other recombinant proteins and this study will pave way for the analysis of IRF-1 function in vitro.

A highly efficient bilayer graphene/ZnO/silicon nanowire based heterojunction photodetector with broadband spectral response.

This paper presents three self-powered photodetectors namely, p+-bilayer graphene (BLG)/n+-ZnO nanowires (NWs), p+-BLG/n+-Si NWs/p--Si and p+-BLG/n+-ZnO NWs/p--Si. The Silvaco Atlas TCAD software is utilized to characterize the optoelectronic properties of all the devices and is validated by analytical modeling. The proposed dual-junction photodetectors cover broadband spectral response varying from ultraviolet to near-infrared wavelengths. The dual-heterojunction broadband photodetector exhibits photocurrent switching with the rise and fall time of 1.48 and 1.27 ns, respectively. At -0.5 V bias, the highest external quantum efficiency, photocurrent responsivity, specific detectivity, and the lowest noise equivalent power of 71%, 0.28 A W-1, 4.2 × 1012 cmHz1/2 W-1, and 2.59 × 10-17 W, respectively, are found for the dual-heterojunction device with a wavelength of 480 nm at 300 K. The proposed nanowires based photodetectors offer great potential to be utilized as next-generation optoelectronic devices.

Virtual microscopy made economical and effortless using the Foldscope and a smartphone with screen mirroring.

CONTEXT: Virtual Microscopy. AIM: The aim of this study was to demonstrate, as a proof of concept, the integration of a Foldscope, along with a smartphone and screen mirroring devices, into the regular academic teaching program for use with all types of regular slides for economical virtual microscopy. SETTING DESIGN & METHODS: For the purpose of this demonstration, a microscopic slide of a ground section of a tooth, a smartphone (iPhone6), a Foldscope and an Apple TV module were chosen to demonstrate the integration of a low-cost unbreakable microscope along with a smartphone to facilitate immediate viewing, capture, sharing and even projection of the image by screen mirroring, if required, in a classroom setting. CONCLUSION: The Foldscope microscope (Foldscope Instruments, Palo Alto, CA, USA) invented by Manu Prakash is an extremely economical, Origami style, fold to assemble microscope available from popular online retailers at approximately Rupees 500 aimed at simplifying and enabling diagnostics and education worldwide. The Foldscope, integrated with a smartphone, allows for easy screen casting thus proving invaluable as an educational tool by creating an effortless bridge between analogue input and digital output, facilitating instant digitization of slides for viewing, display, communication and storage. This article demonstrates the use of the Foldscope for virtual microscopy in a classroom scenario, by employing the screen mirroring capabilities of a smartphone.

Possible bioremediation of arsenic toxicity by isolating indigenous bacteria from the middle Gangetic plain of Bihar, India.

In middle Gangetic plain, high arsenic concentration is present in water, which causes a significant health risk. Total 48 morphologically distinct arsenite resistant bacteria were isolated from middle Gangetic plain. The minimum inhibitory concentration (MIC) values of arsenite varied widely in the range 1-15 mM of the isolates. On the basis of their MIC, two isolates, AK1 (KY569423) and AK9 (KY569424) were selected. The analysis of the 16S rRNA gene sequence of selected isolates revealed that they are belong to the genus Pseudomonas. The AgNO3 test based microplate method revealed that isolates, AK1 and AK9, have potential in transformation of arsenic species. Further, the presence of aoxR, aoxB and aoxC genes in the both isolated strain AK1 and AK9 was confirmed, which play an important role in arsenic bioremediation by arsenite oxidation. Isolated strains also showed heavy metal resistance against Cr(IV), Ni(II), Co(II), Pb(II), Cu(II), Hg(II), Ag(I) and Cd(II).

Chimeric murine interferon regulatory factor-2 (IRF-2) binds to IRF-E (IRF binding element), VREß (virus response element) but not to VREα1.

Interferon regulatory factor-2 (IRF-2) is a multifunctional transcription factor having gene activation, repression and synergistic effect in conjunction with IRF-1. IRF-2 is also involved in type I IFN signalling by repressing INFß gene. So far, the molecular mechanism of its DNA binding activity remains elusive. We have carried out molecular sub-cloning, expression and electrophoretically mobility shift assay study of chimeric murine IRF-2. Here, we report expression of chimeric murine IRF-2 as GST-IRF-2 fusion protein in Escherichia coli/BL21 cells and demonstrated DNA binding activity by gel retardation technique using radio (32) P-labelled IRF-E motif (GAAAGT)4 , virus response element (VRE) of human INFß and IFNα1 gene. We observed five different masses DNA/GST-IRF-2 complexes (1-5) with IRF-E motif, three different masses DNA/GST-IRF-2 complexes (1-3) with VREß , but we could not observe any complex of DNA/GST-IRF-2 with VREα1 . The specific binding on IRF-E motif was confirmed by carrying out 100-X fold cold competition with (32) P-labelled IRF-E motif. In contrast to specific binding on VREß , we used negative control where we observed no binding complex, but we observed complexes with clones IPTG-induced extract. As far as binding on VREα1 is concerned, we could not observe any complex in negative control as well as in IPTG-inducible clones extract. Chimeric IRF-2 binds with IRF-E motif and VREß but not with VREα1. This study is first of its kind and paves the way to understand the differential DNA binding and molecular mechanism of DNA binding activity of the IRF-2 molecule, which is crucial for its function(s).

Immunogenicity and contraceptive efficacy of Escherichia coli-expressed recombinant porcine zona pellucida proteins.

PROBLEM: To overcome availability of the purified native zona pellucida (ZP) glycoproteins for immunocontraception, porcine ZP3, and ZP4 were expressed in E. coli. METHOD OF STUDY: Purified recombinant proteins were characterized by SDS-PAGE and Western blot, and immunogenicity and contraceptive efficacy determined in FvB/J female mice. RESULTS: Purified ZP3, ZP3 with promiscuous T-cell epitope of tetanus toxoid, ZP4 and ZP4 incorporating promiscuous T-cell epitope of bovine RNase revealed ~44-, ~49-, ~53-, and ~55-kDa bands by SDS-PAGE and Western blot, respectively. Immunization of female mice with recombinant proteins elicited high antibody titers as well as T-cell responses. Immune sera recognized mouse oocyte ZP and also inhibited in vitro fertilization. Immunized mice showed significant decrease in fertility. Recombinant proteins were able to recall memory antibody response in female mice primed with porcine native ZP. CONCLUSION: Availability of recombinant porcine proteins will be useful in the development of contraceptive vaccine.

In vitro plant propagation of Catharanthus roseus and assessment of genetic fidelity of micropropagated plants by RAPD marker assay.

An investigation was carried out to develop an efficient micropropagation protocol for Catharanthus roseus. Experiments were conducted to optimize suitable media for in vitro shoot multiplication and root induction. Out of the different media compared for in vitro shoot multiplication, Murashige and Skoog (MS) medium supplemented with 1 mg/l of 6-benzylaminopurine and 0.2 mg/l α-naphthaleneacetic acid showed better response in terms of the emergence of shoots from axillary buds as well as proliferation and multiplication of shoots. The shoots when placed on half strength of MS medium having 1 mg/l indole 3-butyric acid and 0.25 % charcoal showed cent percent root induction with maximum number of roots per shoot (4.2) as well as maximum root length (1.72 cm). Further, clonal fidelity of the in vitro-raised plants was carried out using randomly amplified polymorphic DNA marker and results indicated that all the tissue culture-derived plants are true-to-type and there were no somaclonal variations among these plants.

Mouse interferon regulatory factor-2: expression, purification and DNA binding activity.

Interferon regulatory factor-2 (IRF-2) is a mammalian transcription factor for Interferon and Interferon inducible genes; biologically, plays an important role in cell growth regulation and has been shown to be a potential oncogene. We have expressed, purified recombinant Murine IRF-2 (349 amino acid) as a GST (Glutathione-S-Transferase)-IRF-2 soluble fusion protein in E. coli XL-1 blue cells. Recombinant GST-IRF-2 was biologically active in terms of its DNA binding activity with IRF-E oligo (GAAAGT)4. GST-alone expressed from the vector did not bind to it. We observed five different molecular mass complexes of GST-IRF-2/DNA (1-5) with IRF-E, which were competed out by 100×-fold molar excess of IRF-E, suggesting that the complexes were specific for IRF-2. Such GAAANN (N=any nucleotide) hexa nucleotides occur in the promoters of many virus and interferon-inducible mammalian genes. Multimeric GAAAGT/C sequences are inducible by virus, IFN, dsRNA and IRF-1/2. Multiple GST-IRF-2/DNA complexes may be helpful to understand the mechanism of DNA binding activity of IRF-2.

A novel DEAD box helicase Has1p from Plasmodium falciparum: N-terminal is essential for activity.

Helicases catalyze the opening of nucleic acid duplexes and are implicated in many nucleic acid metabolic cellular processes that require single stranded DNA or reorganization of RNA structure. Previously we have reported that Plasmodium falciparum genome contains a number of DEAD box helicases. In the present study we report the cloning, expression and characterization of one of the novel members of DEAD box family from P. falciparum. Our results indicate that it is a homologue of Has1p from yeast and it contains DNA and RNA unwinding, nucleic acid-dependent ATPase and RNA binding activities. This enzyme can utilize all the nucleosidetriphosphates (NTPs) and deoxy nucleosidetriphosphates (dNTPs) for its unwinding activity. Using a truncated derivative of this protein we further report that the N-terminal region of the protein is essentially required for its activity. These studies suggest that besides the conserved helicase domain the highly variable N-terminal region also contributes in the activity of the protein.

Replacement of the C-terminal tetrapeptide (314 PAPV 317 to 314 SSSM 317) in interferon regulatory factor-2 alters its N-terminal DNA-binding activity.

Interferon regulatory factor-2 (IRF-2) is an important transcription factor involved in cell growth regulation, immune response and cancer. IRF-2 can function as a transcriptional repressor and activator depending on its DNA-binding activity and protein-protein interactions. We compared the amino acid sequences of IRF-2 and found a C-terminal tetrapeptide (314PAPV317) of mouse IRF-2 to be different (314SSSM317) from human IRF-2. Recombinant GST-IRF-2 with 314PAPV317 (wild type) and 314SSSM317 (mutant) expressed in Escherichia coli were assessed for DNA-binding activity with 32P-(GAAAGT) 4 by electrophoretic mobility shift assay (EMSA). Wild type- and mutant GST-IRF-2 showed similar expression patterns and immunoreactivities but different DNA-binding activities. Mutant (mt) IRF-2 formed higher-molecular-mass, more and stronger DNA-protein complexes in comparison to wild type (wt) IRF-2. Anti-IRF-2 antibody stabilized the DNA-protein complexes formed by both wt IRF-2 and mt IRF-2, resolving the differences. This suggests that PAPV and SSSM sequences at 314-317 in the C-terminal region of mouse and human IRF-2 contribute to conformation of IRF-2 and influence DNA-binding activity of the N-terminal region, indicating intramolecular interactions. Thus, evolution of IRF-2 from murine to human genome has resulted in subtle differences in C-terminal amino acid motifs, which may contribute to qualitative changes in IRF-2-dependent DNA-binding activity and gene expression.