Sensitive protein detection using site-specifically oligonucleotide-conjugated nanobodies

High-quality affinity probes are critical for sensitive and specific protein detection, in particular to detect protein biomarkers at early phases of disease development. Clonal affinity reagents can offer advantages over the commonly used polyclonal antibodies (pAbs) in terms of reproducibility and standardization of such assays. In particular, clonal reagents offer opportunities for site-directed attachment of exactly one modification per affinity reagent at a site designed not to interfere with target binding to help standardize assays. The proximity extension assays (PEA) is a widely used protein assay where pairs of protein-binding reagents are modified with oligonucleotides (oligos), so that their proximal binding to a target protein generates a reporter DNA strand for DNA-assisted readout. The assays have been used for high-throughput multiplexed protein detection of up to a few thousand different proteins in one or a few microliters of plasma. Here we explore nanobodies (Nb) as an alternative to polyclonal antibodies pAbs as affinity reagents for PEA. We describe an efficient site-specific approach for preparing high-quality oligo-conjugated Nb probes via Sortase A (SrtA) enzyme coupling. The procedure allows convenient removal of unconjugated affinity reagents after conjugation. The purified high-grade Nb probes were used in PEA and the reactions provided an efficient means to select optimal pairs of binding reagents from a group of affinity reagents. We demonstrate that Nb-based PEA for interleukin-6 (IL6) detection can augment assay performance, compared to the use of pAb probes. We identify and validate Nb combinations capable of binding in pairs without competition for IL6 antigen detection by PEA.

Purification of synchronized E. coli transcription elongation complexes by reversible immobilization on magnetic beads

Synchronized transcription elongation complexes (TECs) are a fundamental tool for in vitro studies of transcription and RNA folding. Transcription elongation can be synchronized by omitting one or more NTPs from an in vitro transcription reaction so that RNA polymerase can only transcribe to the first occurrence of the omitted nucleotide(s) in the coding DNA strand. This approach was developed over four decades ago and has been applied extensively in biochemical investigations of RNA polymerase enzymes, but has not been optimized for RNA-centric assays. In this work, we describe the development of a system for isolating synchronized TECs from an in vitro transcription reaction. Our approach uses a custom 5′ leader sequence, called C3-SC1, to reversibly capture synchronized TECs on magnetic beads. We first show that complexes isolated by this procedure, called C3-SC1TECs, are >95% pure, >98% active, highly synchronous (94% of complexes chase in <15s upon addition of saturating NTPs), and compatible with solid-phase transcription; the yield of this purification is ~8%. We then show that C3-SC1TECs perturb, but do not interfere with, the function of ZTP-sensing and ppGpp-sensing transcriptional riboswitches. For both riboswitches, transcription using C3-SC1TECs improved the efficiency of transcription termination in the absence of ligand but did not inhibit ligand-induced transcription antitermination. Given these properties, C3-SC1TECs will likely be useful for developing biochemical and biophysical RNA assays that require high-performance, quantitative bacterial in vitro transcription.

Auger Emitter Conjugated PARP Inhibitor for Therapy in Triple Negative Breast Cancers: A Comparative In-Vitro Study

PARP1 inhibitors (PARPi) are currently approved for BRCAmut metastatic breast cancer, but they have shown limited response in triple negative breast cancer (TNBC) patients. Combination of an Auger emitter with PARPis enables PARP inhibition and DNA strand break induction simultaneously. This will enhance cytotoxicity and additionally allow a theranostic approach. This study presents the radiosynthesis of the Auger emitter [125I] coupled olaparib derivative: [125I]-PARPi-01, and its therapeutic evaluation in a panel of TNBC cell lines. Specificity was tested by a blocking assay. DNA strand break induction was analysed by γH2AX immunofluorescence staining. Cell cycle analysis and apoptosis assays were studied using flow cytometry in TNBC cell lines (BRCAwt/mut). Anchorage independent growth potential was evaluated using soft agar assay. [125I]-PARPi-01 showed PARP1-specificity and higher cytotoxicity than olaparib in TNBC cell lines irrespective of BRCA their status. Cell lines harbouring DNA repair deficiency showed response to [125I]-PARPi-01 monotherapy. Combined treatment with Dox-NP further enhanced therapeutic efficiency in metastatic resistant BRCAwt cell lines. The clonogenic survival was significantly reduced after treatment with [125I]-PARPi-01 in all TNBC lines investigated. Therapeutic efficacy was further enhanced after combined treatment with chemotherapeutics. [125I]-PARPi-01 is a promising radiotherapeutic agent for low radiation dosages, and mono/combined therapies of TNBC.

Phytochemistry and Antigenotoxic Properties of Six Ethnobotanically Important Members From the Family Zingiberaceae

Genotoxicity is considered as a potential cause of various diseases including cancer. During the last decade, herbal extracts attained a great deal of attention due to its safe and effective applications against various DNA damaging agents. However, the mechanism of DNA strand breaks by various mutagens and genotoxins is often correlated with the generation of Reactive Oxygen Species (ROS). Herbal extracts constitute a number of phytochemicals and those are reported to have considerable antioxidant properties, which are in turn capable of neutralizing ROS mediated DNA damage. The botanical family Zingiberaceae is reported to have significant antioxidant and antigenotoxic potential by various researchers. Among a number of species belonging to this family, six species, namely Alpinia galanga, A. zerumbet, Curcuma amada, C. caesia, Zingiber officinale, and Z. zerumbet, attract notable attention due to their remarkable ethnobotanical and medicinal importance. This chapter deals with phytochemical composition, antioxidant, and antigenotoxic properties of these six Zingiberaceous plant extracts.

Characterization of Butachlor Degradation by A Molybdenum-Reducing and Aniline-degrading Pseudomonas sp.

Butachlor is a chloroacetanilide herbicide that is selective in action and commonly used for pre-emergence control of weeds. It is believed to have range of toxicity from acute to chronic and also can cause DNA strand breaks and chromosomal aberrations in humans. This study was aimed at characterizing the potential of previously isolated bacteria for butachlor degradation. The isolates from culture collection, labelled A-F were screened for butachlor degradation on Bushnell Hass agar media with butachlor as a sole carbon source. Isolate A (molybdenum-reducing and aniline-degrading Pseudomonas sp.) was observed to grow best and tolerated the highest concentration of butachlor supplemented in the media after 72 h of incubation at 37 ℃. Characterization study revealed that the Pseudomonas sp. can utilize and grow with butachlor at optimum pH between 6.0 - 6.5, temperature between 30 – 37 ℃ and can tolerate up to 600 mg/L butachlor concentration with increase in growth as inoculum size increases. Additionally, this bacterial strain shows no lag phase regardless of the concentration of the herbicide used and reach its maximum growth after 24 h of incubation. The ability of this isolate to tolerate and utilize butachlor as sole carbon source makes it suitable for future bioremediation of this toxicant.

Principles for assessing the genotoxicity of carbon nanomaterials in vitro (on the example of carbon nanotubes) (literature review)

Introduction. Genotoxicity of nanomaterials (NM) is becoming a major concern when investigating new NM for their safety. Each mutagen is considered to be potentially carcinogenic, therefore a genotoxicity assessment is necessary. However, a clear strategy for assessing the genotoxic effect of NM has not yet been developed. Material and methods. The material for the analysis have included literature sources from the bibliographic databases PubMed, Scopus, RSCI. Results. Physicochemical characterization of NM is carried out using high-resolution microscopic and light scattering methods. Before testing for genotoxicity, it is necessary to know the cytotoxicity of the tested NM in order to select the appropriate concentration range. The most important and significant tests are based on the cell viability. MTT assay is a colorimetric test that evaluates the metabolic activity of cells. In addition, viability can be determined using microscopy, flow cytometry, determination of lactate dehydrogenase. Genotoxicity evaluation can be carried out only after the preliminary steps. The strategy should include genotoxicity endpoints: DNA damage, gene mutations, chromosomal damage. The in vitro mammalian gene mutation test, usually performed using mouse lymphoma cells, detects a wide range of genetic damage, including gene deletions. The most common test for detecting chromosomal damage is an in vitro micronucleus assay. DNA strand breaks are most often assessed using the comet DNA assay. Conclusion. Compulsory stages in the study of the genotoxicity of nanomaterials should be preliminary studies, including physicochemical characterization and assessment of cytotoxicity, as well as the study of the endpoints of genotoxicity and potential mechanisms.

A nanopore interface for high bandwidth DNA computing

DNA has emerged as a powerful substrate for programming information processing machines at the nanoscale. Among the DNA computing primitives used today, DNA strand displacement (DSD) is arguably the most popular, with DSD-based circuit applications ranging from disease diagnostics to molecular artificial neural networks. The outputs of DSD circuits are generally read using fluorescence spectroscopy. However, due to the spectral overlap of typical small-molecule fluorescent reporters, the number of unique outputs that can be detected in parallel is limited, requiring complex optical setups or spatial isolation of reactions to make output bandwidths scalable. Here, we present a multiplexable sequencing-free readout method that enables real-time, kinetic measurement of DSD circuit activity through highly parallel, direct detection of barcoded output strands using nanopore sensor array technology (Oxford Nanopore Technologies' MinION device). We show that engineered reporter probes can be detected and classified with high accuracy at the single-molecule level directly from raw nanopore signals using deep learning. We then demonstrate this method's utility in multiplexed detection of clinically relevant microRNA sequences. These results increase DSD output bandwidth by an order of magnitude over what is possible with fluorescence spectroscopy, laying the foundations for a new paradigm in DNA circuit readout and programmable multiplexed molecular diagnostics using portable nanopore devices.

Genotoxic and histopathological alterations in rats exposed to herbal liquors

The increase in consumption of herbal liquors in Nigeria is a cause for alarm. These are consumed with the mis-conception that they are without toxic effects. The aim of this study is to investigate the genotoxic and histopathological alterations in rats exposed to herbal liquors. Female rats were exposed to herbal liquors for 6 weeks. The histopathological and genotoxic evaluation were done to assess extent of damage. Pathological examination revealed incidences of aggregates chronic inflammatory cell infiltrates in the heart of Jedi treated group while the heart of the other groups had no abnormalities. The histologic sections of the kidney tissue revealed congested vessels while the lung showed reduction in air filled alveolar spaces with infiltration of alveoli and interstitium by aggregates of inflammatory cells indnadicating moderate to severe pulmonary inflammation. Histologic sections of lung tissue in rats treated with herbal liquors reveals congestion of pulmonary vessels and interstitial hemorrhages. Genotoxic evaluation of rat lymphocytes exposed to herbal liquors via comet assay shows that rats administered with the different herbal liquors developed significant (p < 0.05) as revealed in the % DNA in tail, % DNA in head, olive moment, tail length and tail moment which indicates the presence of DNA strand breaks and a marker for oxidative DNA damage. This result reveals that herbal liquors contain substances that produce reactive oxygen species that have pathological effect on certain organs as well as inducing DNA strand breaks that could compromise the integrity of the DNA which can lead to mutation.

ZINC CONTAMINATION IS AN UNDERESTIMATED RISK TO AMPHIBIANS: TOXICITY EVALUATION IN TADPOLES OF FEJERVARYA LIMNOCHARIS

Aquatic environments are often contaminated with zinc. Amphibian tadpoles are likely to be exposed to high concentrations of zinc present in these environments. We determined the acute and sub-chronic toxicity of ZnCl2 on Fejervarya limnocharis tadpoles under laboratory conditions. The LC50 values of ZnCl2 were found to be 5.81, 4.32, 3.79 and 3.61 mg/L at 24, 48, 72 and 96 h of exposure respectively. Long-term exposure to sub-lethal concentrations of ZnCl2 induced significant mortality in concentration and time dependent manner. Sub-lethal ZnCl2 exposure significantly altered survival, body length and body weight at metamorphosis. Micronucleus test and comet assay indicated the genotoxic potential of ZnCl2. Significant increase in DNA strand break was observed following ZnCl2 exposure equivalent to 1% of the of 24 h LC50 value. The findings indicate possible adverse to tadpoles inhabiting aquatic environments contaminated with zinc. In addition, the findings may be extrapolated to aquatic organisms of similar torphic status.

HELQ is a dual-function DSB repair enzyme modulated by RPA and RAD51

DNA double-stranded breaks (DSBs) are deleterious lesions, and their incorrect repair can drive cancer development1. HELQ is a superfamily 2 helicase with 3′ to 5′ polarity, and its disruption in mice confers germ cells loss, infertility and increased predisposition to ovarian and pituitary tumours2–4. At the cellular level, defects in HELQ result in hypersensitivity to cisplatin and mitomycin C, and persistence of RAD51 foci after DNA damage3,5. Notably, HELQ binds to RPA and the RAD51-paralogue BCDX2 complex, but the relevance of these interactions and how HELQ functions in DSB repair remains unclear3,5,6. Here we show that HELQ helicase activity and a previously unappreciated DNA strand annealing function are differentially regulated by RPA and RAD51. Using biochemistry analyses and single-molecule imaging, we establish that RAD51 forms a complex with and strongly stimulates HELQ as it translocates during DNA unwinding. By contrast, RPA inhibits DNA unwinding by HELQ but strongly stimulates DNA strand annealing. Mechanistically, we show that HELQ possesses an intrinsic ability to capture RPA-bound DNA strands and then displace RPA to facilitate annealing of complementary sequences. Finally, we show that HELQ deficiency in cells compromises single-strand annealing and microhomology-mediated end-joining pathways and leads to bias towards long-tract gene conversion tracts during homologous recombination. Thus, our results implicate HELQ in multiple arms of DSB repair through co-factor-dependent modulation of intrinsic translocase and DNA strand annealing activities.