lncRNA genes can be genic or "intergenic". "Genic" RNAs can be further divided into six biotypes. Through genome-wide analysis of a publicly available data set on corticogenesis, we found that the ...divergent lncRNA (XH) biotype, comprising the lncRNA and the coding gene being in opposite directions in a head-to-head manner, was most prominent during neural commitment. Within this biotype, a coding gene/divergent RNA pair of the BASP1 gene and the uncharacterized RNA loc285696 (hitherto referred as BASP1-AS1) formed a major HUB gene during neuronal differentiation. Experimental validation during the in vitro differentiation of human neural progenitor cells (hNPCs) showed that BASP1-AS1 regulates the expression of its adjacent coding gene, BASP1. Both transcripts increased sharply on the first day of neuronal differentiation of hNPCs, to fall steadily thereafter, reaching very low levels in differentiated neurons. BASP1-AS1 RNA and the BASP1 gene formed a molecular complex that also included the transcription factor TCF12. TCF12 is coded by the DYX1 locus, associated with inherited dyslexia and neurodevelopmental defects. Knockdown of BASP1-AS1, BASP1, or TCF12 impaired the neuronal differentiation of hNPCs, as seen by reduction in DCX and TUJ1-positive cells and by reduced neurite length. There was also increased cell proliferation. A common set of critical genes was affected by the three molecules in the complex. Our study thus identified the role of the XH biotype and a novel mediator of neuronal differentiation-the complex of BASP1-AS1, BASP1, and TCF12. It also linked a neuronal differentiation pathway to inherited dyslexia.
Neutralizing monoclonal antibodies are being found to be increasingly useful in viral infections. In hepatitis B infection, antibodies are proven to be useful for passive prophylaxis. The preS1 ...region (21-47a.a.) of HBV contains the viral hepatocyte-binding domain crucial for its attachment and infection of hepatocytes. Antibodies against this region are neutralizing and are best suited for immune-based neutralization of HBV, especially in view of their not recognizing decoy particles. Anti-preS1 (21-47a.a.) antibodies are present in serum of spontaneously recovered individuals. We generated a phage-displayed scFv library using circulating lymphocytes from these individuals and selected four preS1-peptide specific scFvs with markedly distinct sequences from this library. All the antibodies recognized the blood-derived and recombinant preS1 containing antigens. Each scFv showed a discrete binding signature, interacting with different amino acids within the preS1-peptide region. Ability to prevent binding of the preS1 protein (N-terminus 60a.a.) to HepG2 cells stably expressing hNTCP (HepG2-hNTCP-C4 cells), the HBV receptor on human hepatocytes was taken as a surrogate marker for neutralizing capacity. These antibodies inhibited preS1-hepatocyte interaction individually and even better in combination. Such a combination of potentially neutralizing recombinant antibodies with defined specificities could be used for preventing/managing HBV infections, including those by possible escape mutants.
Hypoxic ischemic injury to the fetal and neonatal brain is a leading cause of death and disability worldwide. Although animal and culture studies suggest that glutamate excitotoxicity is a primary ...contributor to neuronal death following hypoxia, the molecular mechanisms, and roles of various neural cells in the development of glutamate excitotoxicity in humans, is not fully understood. In this study, we developed a culture model of human fetal neural stem cell (FNSC)-derived astrocytes and examined their glutamate uptake in response to hypoxia. We isolated, established, and characterized cultures of FNSCs from aborted fetal brains and differentiated them into astrocytes, characterized by increased expression of the astrocyte markers glial fibrillary acidic protein (GFAP), excitatory amino acid transporter 1 (EAAT1) and EAAT2, and decreased expression of neural stem cell marker Nestin. Differentiated astrocytes were exposed to various oxygen concentrations mimicking normoxia (20% and 6%), moderate and severe hypoxia (2% and 0.2%, respectively). Interestingly, no change was observed in the expression of the glutamate transporter EAAT2 or glutamate uptake by astrocytes, even after exposure to severe hypoxia for 48 h. These results together suggest that human FNSC-derived astrocytes can maintain glutamate uptake after hypoxic injury and thus provide evidence for the possible neuroprotective role of astrocytes in hypoxic conditions.
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•Zinc tetraphenylporphyrin-2-nitroflurne system is studied for electron transfer.•Observations indicate significant intermolecular photoinduced electron transfer.•Observed ...fluorescence quenching rates are in good agreement with Marcus theory.•Femtosecond transient absorption data show formation of charge separated state.
The possibility of intermolecular photoinduced ET (PET) is investigated in a novel donor–acceptor (D–A) system consisting of zinc tetraphenylporphyrin (ZnTPP) as donor and 2-nitrofluorene (2NF) as acceptor upon photoexcitation of the former moieties in polar solvent acetonitrile (ACN). The observed values (7.23×109L/(mols) and 7.04×109L/(mols)) of the fluorescence quenching rate constant from steady state and time-resolved data, respectively, are in good agreement with that (5.65×109L/(mols)) obtained from Marcus theory. Excited state absorption (ESA) from the S2 state of Soret excited ZnTPP to higher electronic states gives rise to broad transient absorption spectra in 560–700nm region. Presence of 2NF leads to a faster decay (up to 100ps time delay) of the ESA signal of ZnTPP at a probe wavelength of 620nm. This indicates ultrafast ET from the S2 state (zero vibrational level) as well as the higher vibrational levels of the S1 state of ZnTPP to 2NF. Moreover, transient absorption kinetics at longer time delays (up to 7ns) at a probe wavelength of 620nm show the formation of a charge separated state due to ET from the S1 state (zero vibrational level) of the Soret excited ZnTPP to 2NF.
The Sonic hedgehog (Shh) signaling pathway is critical for cell growth and differentiation. Impairment of this pathway can result in both birth defects and cancer. Despite its importance in cancer ...development, the Shh pathway has not been thoroughly investigated in tumorigenesis of brain tumors. In this study, we sought to understand the regulatory roles of GLI1, the immediate downstream activator of the Shh signaling pathway on its downstream target genes PTCH1, Cyclin D2, Plakoglobin, NKX2.2 and PAX6 in medulloblastoma and astrocytic tumors.
We silenced GLI1 expression in medulloblastoma and astrocytic cell lines by transfection of siRNA against GLI1. Subsequently, we performed RT-PCR and quantitative real time RT-PCR (qRT-PCR) to assay the expression of downstream target genes PTCH1, Cyclin D2, Plakoglobin, NKX2.2 and PAX6. We also attempted to correlate the pattern of expression of GLI1 and its regulated genes in 14 cell lines and 41 primary medulloblastoma and astrocytoma tumor samples. We also assessed the methylation status of the Cyclin D2 and PTCH1 promoters in these 14 cell lines and 58 primary tumor samples.
Silencing expression of GLI1 resulted up-regulation of all target genes in the medulloblastoma cell line, while only PTCH1 was up-regulated in astrocytoma. We also observed methylation of the cyclin D2 promoter in a significant number of astrocytoma cell lines (63%) and primary astrocytoma tumor samples (32%), but not at all in any medulloblastoma samples. PTCH1 promoter methylation was less frequently observed than Cyclin D2 promoter methylation in astrocytomas, and not at all in medulloblastomas.
Our results demonstrate different regulatory mechanisms of Shh-GLI1 signaling. These differences vary according to the downstream target gene affected, the origin of the tissue, as well as epigenetic regulation of some of these genes.
Bismuth sulfide (Bi
2
S
3
) thin films were electrodeposited from non-aqueous dimethyl sulfoxide medium containing Bi(NO
3
)
3
and thiourea as the precursor salts, triethanol amine as the complexing ...agent, and TritonX-100 as the surface active agent. The prepared films were subjected to rigorous experimentation in order to validate their potential candidature for solar cells. The films exhibited band gap energy of ∼1.3 eV and resistivity of the order of 2 × 10
6
Ω cm at room temperature as was obtained from UV–Vis spectroscopy and four-probe measurements, respectively. Scanning electron microscopy, transmission electron microscopy, X-ray diffraction, and energy dispersive analysis of X-ray were employed to reveal the morphology, structure, and chemical composition of the film matrix. The Bi
2
S
3
films were found to be non-decomposable up to the temperature of 1,000 °C with the help of thermogravimetry–differential thermal analysis. The Nyquist and Mott–Schottky plots derived from electrochemical impedance spectroscopy measurements provided important information regarding electrical and semiconducting properties of the films. The
n
-type film with a donor density of the order of ∼10
23
m
−3
displayed reasonable photoactivity under illumination and is recommended as a promising candidate for potential photoelectrochemical applications.
Progression of human immunodeficiency virus type-1 (HIV-1) infection in children is faster than adults. HIV-1 subtype C is responsible for more than 50% of the infections globally and more than 90% ...infections in India. To date, there is no effective vaccine against HIV-1. Recent animal studies and human Phase I trials showed promising results of the protective effect of anti-HIV-1 broadly neutralizing antibodies (bnAbs). Interaction between CD4 binding site (CD4bs) on the HIV-1 envelope glycoprotein and CD4 receptor on the host immune cells is the primary event leading to HIV-1 infection. The CD4bs is a highly conserved region, comprised of a conformational epitope, and is a potential target of bnAbs such as VRC01 that is presently under human clinical trials. Recombinant scFvs can access masked epitopes due to their small size and have shown the potential to inhibit viral replication and neutralize a broad range of viruses. Pediatric viruses are resistant to many of the existing bnAbs isolated from adults. Therefore, in this study, pooled peripheral blood mononuclear cells from 9 chronically HIV-1 subtype C infected pediatric cross-neutralizers whose plasma antibodies exhibited potent and cross-neutralizing activity were used to construct a human anti-HIV-1 scFv phage library of 9 × 10
individual clones. Plasma mapping using CD4bs-specific probes identified the presence of CD4bs directed antibodies in 4 of these children. By extensive biopanning of the library with CD4bs-specific antigen RSC3 core protein, we identified two cross-neutralizing scFv monoclonals 2B10 and 2E4 demonstrating a neutralizing breadth and GMT of 77%, 17.9 µg/ml and 32%, 51.2 µg/ml, respectively, against a panel of 49 tier 1, 2 and 3 viruses. Both scFvs competed with anti-CD4bs bnAb VRC01 confirming their CD4bs epitope specificity. The 2B10 scFv was effective in neutralizing the 7 subtype C and subtype A pediatric viruses tested. Somatic hypermutations in the VH gene of scFvs (10.1-11.1%) is comparable with that of the adult antibodies. These cross-neutralizing CD4bs-directed scFvs can serve as potential reagents for passive immunotherapy. A combination of cross-neutralizing scFvs of diverse specificities with antiretroviral drugs may be effective in suppressing viremia at an early stage of HIV-1 infection and prevent disease progression.
Production of properly folded, functional recombinant antibodies in a prokaryotic system is governed by multiple factors like codon usage, plasmid copy number, upstream elements such as leader ...sequence, mRNA stability and presence of tightly controlled promoters. Here we present a strategy for enhanced production of the functional scFv in
Escherichia coli by codon optimization. We have previously reported the generation of humanized scFv form of a potentially neutralizing mouse monoclonal antibody (5S) to the Hepatitis B surface antigen. However, the expression level of 5S-scFv in
E. coli was fairly low which was possibly due to the presence of rare codons. In the native 5S-scFv gene, almost 58% of codons showed poor codon bias with varying degrees of rare occurrence in the
E. coli genes. We therefore designed a synthetic gene encoding the 5S-scFv protein by using
E. coli preferred codon usage. The codon-optimized scFv gene was further cloned into a T7 expression system with a C-terminus His-tag and expressed as a soluble protein mainly in the periplasm. The scFv was both purified by IMAC and detected on Western blot with this His-tag. Using the codon optimization strategy, we were able to achieve a more than 100-fold increased periplasmic expression of soluble scFv. Further, the purified scFv was stable and retained its antigen-binding affinity and epitope specificity. Interestingly, based on secondary structure prediction, we observed that the mRNA secondary structure, including that of the 5′-end, may not have a significant role in the increased expression of this optimized gene.
Production of human monoclonal antibodies that exhibit broadly neutralizing activity is needed for preventing HIV-1 infection, however only a few such antibodies have been generated till date. ...Isolation of antibodies by the hybridoma technology is a cumbersome process with fewer yields. Further, the loss of unstable or slowly growing clones which may have unique binding specificities often occurs during cloning and propagation and the strongly positive clones are often lost. This has been avoided by the process described in this paper, wherein, by combining the strategy of EBV transformation and recombinant DNA technology, we constructed human single chain variable fragments (scFvs) against the third variable region (V3) of the clade C HIV-1 envelope.
An antigen specific phage library of 7000 clones was constructed from the enriched V3- positive antibody secreting EBV transformed cells. By ligation of the digested scFv DNA into phagemid vector and bio panning against the HIV-1 consensus C and B V3 peptides followed by random selection of 40 clones, we identified 15 clones that showed V3 reactivity in phage ELISA. DNA fingerprinting analysis and sequencing showed that 13 out of the 15 clones were distinct. Expression of the positive clones was tested by SDS-PAGE and Western blot. All the 13 anti-V3 scFvs showed cross-reactivity against both the clade C and B V3 peptides and did not show any reactivity against other unrelated peptides in ELISA. Preliminary neutralization assays indicated varying degrees of neutralization of clade C and B viruses. EBV transformation, followed by antigen selection of lines to identify specific binders, enabled the selection of phage from un-cloned lines for scFv generation, thus avoiding the problems of hybridoma technology. Moreover, as the clones were pretested for antigen binding, a comparatively small library sufficed for the selection of a considerable number of unique antigen binding phage. After selection, the phage clones were propagated in a clonal manner.
This strategy can be efficiently used and is cost effective for the generation of diverse recombinant antibodies. This is the first study to generate anti-V3 scFvs against HIV-1 Clade C.
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