Heterologous Overexpression of Sup35 in E. coli Leads to Both Monomer and Complex States

The heterologous overexpression states of prion proteins play a critical role in understanding the mechanisms of prion-related diseases. We report herein the identification of soluble monomer and complex states for a bakers’ yeast prion, Sup35, when expressed in E. coli. Two peaks are apparent with the elution of His-tagged Sup35 by imidazole from a Ni affinity column. Peak I contains Sup35 in both monomer and aggregated states. Sup35 aggregate is abbreviated as C-aggregate and includes a non-fibril complex comprising Sup35 aggregate-HSP90-Dna K, ATP synthase β unit (chain D), 30S ribosome subunit, and Omp F. The purified monomer and C-aggregate can remain stable for an extended period of time. Peak II contains Sup35 also in both monomer and aggregated (abbreviated as S-aggregate) states, but the aggregated states are caused by the formation of inter-Sup35 disulfide bonds. This study demonstrates that further assembly of Sup35 non-fibril C-aggregate can be interrupted by the chaperone repertoire system in E. coli.

Selection and characterisation of single-stranded DNA aptamers for triclosan

<p>Triclosan (TCS) is a chlorinated organic compound which, due to its antibacterial properties in vitro, has found widespread usages in many medical and consumer products such as textiles, plastics and personal care products. Humans are directly and chronically exposed to TCS via dermal and mucosal contact from the use of TCS-formulated products such as soap and toothpaste. TCS is classified as an environmental contaminant by the European Union Water Framework Directive, whose mandatory goal is to develop new and simple-to-use analytical methodologies capable of measuring low concentrations of TCS and that are suitable for high-throughput detection. Synthetically-derived single-stranded oligonucleotides, also known as aptamers, are superior candidates for the development of sensitive and high-throughput biosensing strategies. Biosensors utilising aptamers as molecular recognition elements have showed great promise in a variety of diagnostic and therapeutic applications, especially for the detection of small molecular weight organic compounds such as TCS. The aim of this thesis was to develop aptamers as new capture reagents for TCS, as the first step towards the development of an alternative, user-friendly, diagnostic technique for monitoring TCS in both environmental and biological samples. The objectives of the thesis were to: [i] produce by in vitro selection procedures, TCS binding single-stranded DNA (ssDNA) aptamers; [ii] characterise the selected aptamers and determine their equilibrium dissociation constant (Kd) values and; [iii] evaluate the applicability of the selected aptamers in a biosensing platform. To achieve these objectives, ssDNA aptamers capable of binding TCS were generated in vitro using a sequential approach known as systematic evolution of ligands by exponential enrichment (SELEX). An affinity column-based SELEX strategy together with a variety of SELEX modifications such as negative and counter selections, real-time amplification and fluorescence quantification were explored for finding TCS specific aptamers. A total of 20 TCS aptamers, ten from 8 rounds of a basic-SELEX procedure, and the other ten from 10 rounds of a revised-SELEX procedure were generated. In general, these aptamers showed acceptable levels of sensitivity and specificity to TCS, and the best binding aptamer demonstrated a Kd value of 378 nM. The Kd value is comparable to published Kd values for compounds that share similar chemical structures to TCS. In addition, a novel fluorescent-based imaging method was developed in this dissertation. The method developed provides an alternative approach for monitoring SELEX progression and has the potential to simplify the way to characterise the binding properties of an aptamer to its cognate target. The utility of this method was compared with commonly used methods such as dot blot and fluorescent binding assays. The performance of the new imaging method was superior to the existing methods in terms of accuracy, simplicity and reproducibility. Furthermore, the best binding TCS aptamer was evaluated for its utility in an aptamer-based biosensor. The developed aptasensor, utilising a TCS aptamer as the recognition element and gold nanoparticles (AuNPs) as the signal reporter, was capable of detecting TCS in spiked-water samples at concentrations ranging from 20–750 nM with a visual detection limit of 150 nM. In conclusion, methods were developed to select, refine, and characterise ssDNA aptamers capable of binding to TCS, and these aptamers have the potential to offer a sensitive, simple-to-use, and user-friendly analytical method for TCS detection.</p>

Selection and characterisation of single-stranded DNA aptamers for triclosan

<p>Triclosan (TCS) is a chlorinated organic compound which, due to its antibacterial properties in vitro, has found widespread usages in many medical and consumer products such as textiles, plastics and personal care products. Humans are directly and chronically exposed to TCS via dermal and mucosal contact from the use of TCS-formulated products such as soap and toothpaste. TCS is classified as an environmental contaminant by the European Union Water Framework Directive, whose mandatory goal is to develop new and simple-to-use analytical methodologies capable of measuring low concentrations of TCS and that are suitable for high-throughput detection. Synthetically-derived single-stranded oligonucleotides, also known as aptamers, are superior candidates for the development of sensitive and high-throughput biosensing strategies. Biosensors utilising aptamers as molecular recognition elements have showed great promise in a variety of diagnostic and therapeutic applications, especially for the detection of small molecular weight organic compounds such as TCS. The aim of this thesis was to develop aptamers as new capture reagents for TCS, as the first step towards the development of an alternative, user-friendly, diagnostic technique for monitoring TCS in both environmental and biological samples. The objectives of the thesis were to: [i] produce by in vitro selection procedures, TCS binding single-stranded DNA (ssDNA) aptamers; [ii] characterise the selected aptamers and determine their equilibrium dissociation constant (Kd) values and; [iii] evaluate the applicability of the selected aptamers in a biosensing platform. To achieve these objectives, ssDNA aptamers capable of binding TCS were generated in vitro using a sequential approach known as systematic evolution of ligands by exponential enrichment (SELEX). An affinity column-based SELEX strategy together with a variety of SELEX modifications such as negative and counter selections, real-time amplification and fluorescence quantification were explored for finding TCS specific aptamers. A total of 20 TCS aptamers, ten from 8 rounds of a basic-SELEX procedure, and the other ten from 10 rounds of a revised-SELEX procedure were generated. In general, these aptamers showed acceptable levels of sensitivity and specificity to TCS, and the best binding aptamer demonstrated a Kd value of 378 nM. The Kd value is comparable to published Kd values for compounds that share similar chemical structures to TCS. In addition, a novel fluorescent-based imaging method was developed in this dissertation. The method developed provides an alternative approach for monitoring SELEX progression and has the potential to simplify the way to characterise the binding properties of an aptamer to its cognate target. The utility of this method was compared with commonly used methods such as dot blot and fluorescent binding assays. The performance of the new imaging method was superior to the existing methods in terms of accuracy, simplicity and reproducibility. Furthermore, the best binding TCS aptamer was evaluated for its utility in an aptamer-based biosensor. The developed aptasensor, utilising a TCS aptamer as the recognition element and gold nanoparticles (AuNPs) as the signal reporter, was capable of detecting TCS in spiked-water samples at concentrations ranging from 20–750 nM with a visual detection limit of 150 nM. In conclusion, methods were developed to select, refine, and characterise ssDNA aptamers capable of binding to TCS, and these aptamers have the potential to offer a sensitive, simple-to-use, and user-friendly analytical method for TCS detection.</p>

526 Removal of soluble tumor necrosis factors receptors 1/2 in patients with metastatic solid tumors using immune apheresis

BackgroundTNFα is a cytokine produced by immune cells and by tumor cells. The soluble forms of membrane TNF receptors 1/2 (sTNF-R1/2) act as decoy to neutralize TNFα, and are highly abundant in cancer patients. Elimination of sTNF-R1/2 may therefore unmask endogenous TNFα, to presumably exert anti-neoplastic effects and reverse resistance to immune checkpoint inhibitors. Immune Apheresis (IA) is a procedure designed to specifically capture sTNF-R1/2 from plasma by passing it over an affinity column. Here we employed Immunicom’s LW-02 Immunopheresis® device for removal of sTNF-R1/2 from plasma of cancer patients.MethodsIn cohort A, patients with melanoma, RCC, NSCLC or TNBC refractory to standard therapy were treated with IA only. IA treatment of 2 plasma volumes was done x3/week, for three treatment cycles (4 weeks each) up to a total of 36 treatments. Cohort B patients currently receive concurrent IA and Nivolumab therapy (240mg q2 weeks starting on week 5). sTNF-Rs removal and circulating inflammatory biomarkers were measured by immuno-assays, such as multiplex cytokine detection and mass cytometry. Pre- and post-treatment tumor biopsies were analyzed for tumor markers and TILs by immunohistochemistry.ResultsCohort A included six patients (3 Melanoma and 3 TNBC): three patients completed full study regimen, and three others were withdrawn due to clinical progression. AEs included chills (4/6), fever (2/6), anemia (6/6), central line thrombosis (1/6) and pulmonary embolism (1/6) All were Grade 2 except G3 anemia (1/6). There were no treatment related SAE’s. sTNF-Rs levels were significantly reduced, followed by enhanced detection of TNFα, and IFNγ in some cases. In two patients, CD8 counts and PD-1 and PD-L1 expression were increased. Congruently, blood mass cytometry showed reduction in Treg subsets and differential increase of CD8 subsets following treatment.ConclusionsThe use of Immunicom’s LW-02 Immunopheresis® device in combination with Terumo BCT Spectra Optia Apheresis System is safe and efficient in the removal of sTNF-Rs from blood plasma. Subsequent immuno-assay analyses indicated formation of inflammatory response which may facilitate effects of immunotherapy, yet to be investigated in cohort B.Trial RegistrationNCT04142931Ethics ApprovalSheba Medical Center Ethics Committee, 6136-19ConsentWritten informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal

Analysis of Selected Mycotoxins in Maize from North-West South Africa Using High Performance Liquid Chromatography (HPLC) and Other Analytical Techniques

Contamination of foods by mycotoxins is linked to various health and economic implications. This study evaluated the incidence of mycotoxins in commercial and small-scale maize and evaluated potential health risks for consumers based on South African and international regulations. The sensitivity/specificity of HPLC over other analytical methods used was also ascertained. In total, 100 maize samples were analyzed using immuno-affinity column for extraction and clean-up, thin layer chromatography (TLC), HPLC, and enzyme linked immunosorbent assay (ELISA) for quantification. Results revealed that fumonisinB1 was the most contaminant mycotoxin in both small-scale and commercial samples with incidence rates of 100% and 98.6%, respectively. Aflatoxins contamination occurred at incidences of 26.7% in small-scale and 25.0% in commercial samples. Furthermore, ochratoxin A had high incidence rates of 97.8% and 93.0% and ranged from 3.60–19.44 µg/kg and 1.60–9.89 µg/kg, respectively, in small-scale and commercial samples, while ZEA occurred in 50% and 55% of small-scale and commercial samples, respectively. Results demonstrate that maize, especially from small-scale farmers, may contribute to dietary exposure to mycotoxins. Farmers and consumers should be alerted to the dangers of mycotoxins contamination in maize with resultant health risks. Additionally, HPLC method was also found to be more specific for mycotoxin detection than ELISA.

Cocaine Detection by a Laser-Induced Immunofluorometric Biosensor

The trafficking of illegal drugs by criminal networks at borders, harbors, or airports is an increasing issue for public health as these routes ensure the main supply of illegal drugs. The prevention of drug smuggling, including the installation of scanners and other analytical devices to detect small traces of drugs within a reasonable time frame, remains a challenge. The presented immunosensor is based on a monolithic affinity column with a large excess of immobilized hapten, which traps fluorescently labeled antibodies as long as the analyte cocaine is absent. In the presence of the drug, some binding sites of the antibody will be blocked, which leads to an immediate breakthrough of the labeled protein, detectable by highly sensitive laser-induced fluorescence with the help of a Peltier-cooled complementary metal-oxide-semiconductor (CMOS) camera. Liquid handling is performed with high-precision syringe pumps and microfluidic chip-based mixing devices and flow cells. The biosensor achieved limits of detection of 7 ppt (23 pM) of cocaine with a response time of 90 s and a total assay time below 3 min. With surface wipe sampling, the biosensor was able to detect 300 pg of cocaine. This immunosensor belongs to the most sensitive and fastest detectors for cocaine and offers near-continuous analyte measurement.

An Efficient Heparin Affinity Column Purification Method Coupled with Ultraperformance Liquid Chromatography for the Quantification of Native Lactoferrin in Breast Milk

Lactoferrin (LF) is a bioactive multifunctional protein and found in the highest amounts in human milk. Several methods can be used to quantify LF. However, quantification of native LF has garnered relatively little interest to date. This study aimed to develop a novel efficient two-step method for quantifying native LF in breast milk. During the analysis, LF was first extracted with phosphate buffer (pH 5.0), purified using a heparin affinity column. Subsequently, LF was detected using ultraperformance liquid chromatography (UPLC) at a wavelength of 201 nm. A linear calibration curve was obtained in the range of 5–200 mg/L. The limit of detection and limit of quantitation were 1 mg/L and 5 mg/L, respectively, indicating that the validated method could be employed to quantify LF in breast milk. Compared with previous HPLC methods, this method demonstrated several remarkable advantages, including simple operation, low-cost detection, and high accuracy. Hence, the results demonstrate an efficient method that can be employed commercially to purify and analyze LF in human milk samples.

SARS-CoV-2 Proteins Bind to Hemoglobin and Its Metabolites

(1) Background: coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been linked to hematological dysfunctions, but there are little experimental data that explain this. Spike (S) and Nucleoprotein (N) proteins have been putatively associated with these dysfunctions. In this work, we analyzed the recruitment of hemoglobin (Hb) and other metabolites (hemin and protoporphyrin IX-PpIX) by SARS-Cov2 proteins using different approaches. (2) Methods: shotgun proteomics (LC–MS/MS) after affinity column adsorption identified hemin-binding SARS-CoV-2 proteins. The parallel synthesis of the peptides technique was used to study the interaction of the receptor bind domain (RBD) and N-terminal domain (NTD) of the S protein with Hb and in silico analysis to identify the binding motifs of the N protein. The plaque assay was used to investigate the inhibitory effect of Hb and the metabolites hemin and PpIX on virus adsorption and replication in Vero cells. (3) Results: the proteomic analysis by LC–MS/MS identified the S, N, M, Nsp3, and Nsp7 as putative hemin-binding proteins. Six short sequences in the RBD and 11 in the NTD of the spike were identified by microarray of peptides to interact with Hb and tree motifs in the N protein by in silico analysis to bind with heme. An inhibitory effect in vitro of Hb, hemin, and PpIX at different levels was observed. Strikingly, free Hb at 1mM suppressed viral replication (99%), and its interaction with SARS-CoV-2 was localized into the RBD region of the spike protein. (4) Conclusions: in this study, we identified that (at least) five proteins (S, N, M, Nsp3, and Nsp7) of SARS-CoV-2 recruit Hb/metabolites. The motifs of the RDB of SARS-CoV-2 spike, which binds Hb, and the sites of the heme bind-N protein were disclosed. In addition, these compounds and PpIX block the virus’s adsorption and replication. Furthermore, we also identified heme-binding motifs and interaction with hemin in N protein and other structural (S and M) and non-structural (Nsp3 and Nsp7) proteins.

Recombinant Production and One-Pot Purification for Enhancing Activity of Haloacid Dehalogenase from Bacillus cereus IndB1

In recent years we have witnessed the emergence of organohalogen utilization in various chemical-based industries, particularly polymer-based, agricultural, and pharmaceutical sectors. Despite this, organohalogen compounds are actually very dangerous to the environment, as they are difficult to be naturally degraded and generally toxic to organisms. A green and biocompatible method to overcome this issue is by employing enzymes that could convert organohalogens into non-toxic compounds, such as the class of enzymes known as haloacid dehalogenases. To enhance the activity of haloacid dehalogenase isolated from local strains of Bacillus cereus IndB1, we have developed a recombinant expression system using pET-bcfd1 plasmid in E. coli BL21 (DE3) host cells. Following enzyme production, we also demonstrated a one-pot purification system for the expressed dehalogenase, harnessing the presence of His-tag in the recombinant clones. Purification was carried out using Ni-NTA affinity column chromatography, using imidazole eluent with a concentration gradient of 10 mM to 500 mM. The enzyme activity was tested against the monochloroacetic acid (MCA) substrate according to the Bergmann and Sanik method, and the protein content in the solution was measured using the Bradford method. The purity of the enzyme after one-pot purification was confirmed by SDS-PAGE analyses, showing a single band of 40 kDa in size. Remarkably, the purified haloacid dehalogenase specific activity was increased by 12-fold compared to its crude enzyme extract. Therefore, the expression and purification system developed in this study allow further exploration of dehalogenases from local strains as an efficient catalyst for MCA biodegradation.Keywords: recombinant expression, haloacid dehalogenase, monochloroacetic acid, enzyme purification

Profound structural conservation of chemically cross-linked HIV-1 envelope glycoprotein experimental vaccine antigens

Chemical cross-linking is used to stabilise protein structure with additional benefits of pathogen and toxin inactivation for vaccine use, but its use is restricted by potential induction of local or global structural distortion. This is of particular importance when the protein in question requires a high degree of structural conservation for the purposes of understanding function, or for inducing a biological outcome such as elicitation of antibodies to conformationally-sensitive epitopes. The HIV-1 envelope glycoprotein (Env) trimer is metastable and shifts between different conformational states, complicating its functional analysis and use as a vaccine antigen. Here we have used the hetero-bifunctional zero-length reagent EDC to cross-link two soluble Env trimers, selected well-folded trimers using an antibody affinity column, and transferred this process to good manufacturing practice (GMP) for clinical trial use. Cross-linking enhanced GMP trimer stability to biophysical and enzyme attack, and had broadly beneficial effects on morphology, antigenicity and immunogenicity. Cryo-EM analysis revealed that cross-linking essentially completely retained overall structure with RMSDs between unmodified and cross-linked Env trimers of 0.4 - 0.5 Å. Despite this negligible distortion of global trimer structure we identified individual inter-subunit, intra-subunit and intra-protomer cross-links. Thus, EDC cross-linking maintains protein folding, improves stability, and is readily transferred to GMP, consistent with use of this approach in probing protein structure/function relationships and in the design of vaccines.