Plasmid Deoxyribonucleic Acid Research Articles

Arginine and Histidine-modified Layered Double Hydroxides Facilitate Transgene Expression in Cancer Cells in Vitro

Layered double hydroxides (LDHs) have interesting properties and structures that enable them to carry nucleic acids, such as deoxyribonucleic acid (DNA). This study synthesized LDHs using the co-precipitation method and functionalized with the amino acids arginine (Arg) and histidine (His) to promote proton-sponge activity for enhanced transgene expression. The LDHs were characterized using X-ray diffraction (XRD), transmission electron microscopy (TEM), and nanoparticle tracking analysis (NTA). The interaction of the LDHs with the reporter gene plasmid DNA (<em>pCMV-Luc DNA</em>) was determined using agarose gel electrophoresis. Cytotoxicity and transgene expression was assessed using the 3-(4, 5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide (MTT) and luciferase reporter gene assay in the human embryonic kidney (HEK293), colorectal carcinoma (Caco-2) and hepatocellular carcinoma (HepG2) cells. The DNA: LDH complexes were relatively non-cytotoxic to all cells, and the highest transgene expression was achieved in the HEK293 cells exhibiting the most significant degree of transfection, followed by the Caco-2 cells. The His-LDH complexes displayed more than a two-fold increase in transfection than the Arg-LDHs, especially in the HEK293 cells at the optimal binding ratio. The non-functionalized LDHs demonstrated high transfection, which exceeded that of the His-LDH and Arg-LDH by 20% and 30%, respectively, in the Caco-2 cells. Little difference was noted in the HepG2 cells, which presented with the lowest transfection. These LDHs have demonstrated the potential to bind, protect, and efficiently deliver pDNA <em>in vitro</em>.

Conformation-dependent UV inactivation efficiency of a conjugative, multi-drug resistant plasmid

A conjugative, multi-drug-resistant plasmid was irradiated in-vivo and in-vitro with a 265-nm UV-light emitting diode (UV-LED) to investigate the gene inactivation efficiency of a plasmid deoxyribonucleic acid (pDNA) carrying DNA transfer and replication genes. The clinical-isolate 60 kb RP4 plasmid of the IncPα group containing the traG gene, was irradiated intracellularly in E. coli DH5α and extracellularly in a water medium at pH 8.5. Real-time quantitative polymerase chain reaction (RT-qPCR) measurements of the UV-fluence response gene inactivation rate constants revealed a decreasing pattern via a pseudo-1st-order inactivation kinetics in all forms examined. Our findings showed that the intracellular-supercoiled conformation, with k = 6.1 × 10-3 cm2/mJ, has the lowest UV susceptibility (lowest inactivation rate). UV absorbance measurements and a computational approach showed that the host's RNA provides the photo-shielding, demonstrating this high UV resistance. When UV exposure was measured in-vitro, the condensed DNA exhibited a self-shielding effect over supercoiled and denatured DNA due to the hypochromic-hyperchromic effects. This study has shown that large-sized conjugative plasmids with conformation-dependent UV/UV-LED-based gene inactivation play a significant role in preventing the spread of antibiotic resistance.

Development of Specialized Microelectrode Arrays with Local Electroporation Functionality

When a cell or tissue is exposed to a pulsed electric field (100–1000 V/cm), the cellular membrane permeabilizes for biomolecules that cannot pass an intact cellular membrane. During this electropermeabilization (EP), plasmid deoxyribonucleic acid sequences encoding therapeutic or regulatory genes can enter the cell, which is called gene electrotransfer (GET). GET using micro-/nano technology provides higher spatial resolution and operates with lower voltage amplitudes compared to conventional bulk EP. Microelectrode arrays (MEAs), which are usually used for the recording and stimulation of neuronal signals, can be utilized for GET as well. In this study, we developed a specialized MEA for local EP of adherent cells. Our manufacturing process provides a most flexible electrode and substrate material selection. We used electrochemical impedance spectroscopy to characterize the impedance of the MEAs and the impact of an adherent cellular layer. We verified the local EP functionality of the MEAs by loading a fluorophore dye into human embryonic kidney 293T cells. Finally, we demonstrated a GET with a subsequent green fluorescent protein expression by the cells. Our experiments prove that a high spatial resolution of GET can be obtained using MEAs.

High‐Throughput CRISPR/Cas9 Mediated Gene Editing of Primary Human T Cells in a Microfluidic Device for Cellular Therapy Manufacturing

AbstractAutologous cellular therapies have been highly successful in treating hematological cancers and have the potential to be used for a variety of indications. Manufacturing these therapies rapidly and at low cost remains a major challenge. A key bottleneck in cellular therapy manufacturing is genetic modification of target cells, which is often done using viral vectors. Because vectors are expensive to develop and produce, non‐viral gene transfer using electroporation is emerging as a preferred transfection method for next‐generation therapies. However, most commercial electroporation systems are built for research use rather than large‐scale clinical manufacturing. The microfluidic, continuous‐flow electroporation device presented here offers several advantages including large‐scale and high throughput processing, high performance, and the potential for automation. It transfects primary human T cells with Cas9‐guide ribonucleic acid (RNA) ribonucleoprotein complexes (RNP) and messenger RNA (mRNA) with up to 99–100% efficiency and minimal impact on viability. In addition, this device transfects 3.5 kbp plasmid deoxyribonucleic acid with up to 86% efficiency after preliminary optimization studies. A single microchannel can deliver a total cellular processing throughput of up to 9.6 billion per hour. The combination of high throughput and high performance enables the scale of processing required for future “off‐the‐shelf” allogeneic cellular therapies.

An injectable hydrogel to disrupt neutrophil extracellular traps for treating rheumatoid arthritis

Rheumatoid arthritis (RA), an autoimmune disease, is characterized by inflammatory cell infiltration that damages cartilage, disrupts bone, and impairs joint function. The therapeutic efficacy of RA treatments with the severely affected side remains unsatisfactory despite current treatment methods that primarily focus on anti-inflammatory activity, largely because of the complicatedly pathological mechanisms. A recently identified mechanism for RA development involves the interaction of RA autoantibodies with various proinflammatory cytokines to facilitate the formation of neutrophil extracellular traps (NETs), which increased inflammatory responses to express inflammatory cytokines and chemokines. Therefore, NETs architecture digestion may inhibit the positive-feedback inflammatory signal pathway and lessen joint damage in RA. In this work, deoxyribonuclease I (DNase) is connected to oxidized hyaluronic acid (OHA) via Schiff base reaction to extend the half-life of DNase. The modification does not influence the DNase activity for plasmid deoxyribonucleic acid hydrolysis and NETs’ architecture disruption. Carboxymethyl chitosan is crosslinked with DNase-functionalised OHA (DHA) to form an injectable, degradable, and biocompatible hydrogel (DHY) to further strengthen the adhesive capability of DHA. Importantly, the collagen-induced arthritis model demonstrates that intra-articular injection of DHY can significantly reduce inflammatory cytokine expression and alleviate RA symptoms, which can be significantly improved by combining methotrexate. Here, a DNase-functionalised hydrogel has been developed for RA treatment by constantly degrading the novel drug target of NETs to decrease inflammatory response in RA.

Biological active compounds and biological activities of the foam used in the traditional kerebiç dessert

In the present work, foam and water extracts were obtained from Gypsophila root, which forms the foam part of the dessert known as kerebiç. The bioactive compounds of samples were analysed with Fourier transform infrared (FT-IR) and high-performance liquid chromatography (HPLC). To determine their antioxidant properties, ferric-reducing antioxidant power (FRAP) method, cupric-reducing antioxidant capacity (CUPRAC) method, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate (ABTS) method, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging method, and total antioxidant activity method were performed. The pUC18 plasmid DNA was used to investigate the effect of the samples on the deoxyribonucleic acid (DNA). In addition, the inhibitory properties of the samples against key enzymes (acetyl and butyryl-cholinesterases) involved in the pathogenesis of diseases were investigated. Based on the results of FT-IR analysis, terpenes and sugars were detected in the extracts. Based on the HPLC results, glucose, fructose, maleic acid, acetic acid, acetoin, and 2,3 butanediol were detected in the extracts. Antioxidant results showed that extracts had similar and moderate activities as compared to the standard antioxidants. It was observed that the extracts preserved the stable structure of pUC18 plasmid DNA, but could not remove the scavenging effect of peroxide. In addition, it was determined that the extracts showed a stronger inhibitory effect on acetylcholinesterase (AChE) and butyryl-cholinesterases (BChE) than galantamine, which was used as the control.

Design of Smart Nanodiamonds: Introducing pH Sensitivity to Improve Nucleic Acid Carrier Efficiency of Diamoplexes.

The mechanism of cellular uptake and intracellular fate of nanodiamond/nucleic acid complexes (diamoplexes) are major determinants of its performance as a gene carrier. Our group designed lysine-nanodiamonds (K-NDs) as vectors for nucleic acid delivery. In this work, we modified the surface of K-NDs with histidine to overcome endo-lysosomal entrapment diamoplexes, the major rate limiting step in gene transfer. Histidine is conjugated onto the NDs in two configurations: lysyl-histidine-NDs (HK-NDs) where histidine is loaded on 100% of the lysine moieties and lysine/lysyl-histidine-NDs (H50K50-NDs) where histidine is loaded on 50% of the lysine moieties. Both HK-NDs and H50K50-NDs maintained the optimum size distribution (i.e., <200 nm) and a cationic surface (zeta potential > 20 mV), similar to K-NDs. HK-NDs binds plasmid deoxyribonucleic acid (pDNA) and small interfering ribonucleic acid (siRNA) forming diamoplexes at mass ratios of 10:1 and 60:1, respectively. H50K50-NDs significantly improved nucleic acid binding, forming diamoplexes at a 2:1 mass ratio with pDNA and a 30:1 mass ratio with siRNA, which are at values similar to the K-NDs. The amount of histidine on the surface also impacted the interactions with mammalian cells. The HK-NDs reduced the cell viability by 30% at therapeutic concentrations, while H50K50-NDs maintained more than 90% cell viability, even at the highest concentrations. H50K50-NDs also showed highest cellular uptake within 24 h, followed by K-NDs and HK-NDs. Most functionalized NDs show cellular exit after 5 days, leaving less than 10% of cells with internalized diamonds. The addition of histidine to the ND resulted in higher transfection of anti-green fluorescent protein siRNA (anti-GFP siRNA) with the fraction of GFP knockdown being 0.8 vs. 0.6 for K-NDs at a mass ratio of 50:1. H50K50-NDs further improved transfection by achieving a similar fraction of GFP knockdown (0.8) at a lower mass ratio of 30:1. Overall, this study provides evidence that the addition of histidine, a pH-modulating entity in the functionalization design at an optimized ratio, renders high efficiency to the diamoplexes. Further studies will elucidate the uptake mechanism and intracellular fate to build the relationship between physicochemical characteristics and biological efficacy and create a platform for solid-core nanoparticle-based gene delivery.

Inactivation behavior and intracellular changes in Escherichia coli during electro-oxidation process using Ti/Sb–SnO2/PbO2 anode: Elucidation of the disinfection mechanism

This study investigates the behavior and intracellular changes in Escherichia coli (model organism) during electro-oxidation with Ti/Sb–SnO2/PbO2 anode in a chlorine free electrochemical system. Preliminary studies were conducted to understand the effect of initial E. coli concentration and applied current density on disinfection. At an applied current density 30 mA cm−2, 7 log reduction of E. coli was achieved in 75 min. The role of reactive oxygen species’ (ROS) in E.coli disinfection was evaluated, which confirmed hydroxyl (•OH) radical as the predominant ROS in electro-oxidation. Observations were carried out at cell and molecular level to understand E.coli inactivation mechanism. Scanning electron microscopy images confirmed oxidative damage of the cell wall and irreversible cell death. Intracellular and extracellular protein quantification and genetic material release further confirmed cell component leakage due to cell wall rupture and degradation due to •OH radical interaction. Change in cell membrane potential suggests the colloidal nature of E. coli cells under applied current density. Plasmid deoxyribonucleic acid degradation study confirmed fragmentation and degradation of released genetic material. Overall, effective disinfection could be achieved by electro-oxidation, which ensures effective inactivation and prevents regrowth of E. coli. Disinfection of real wastewater was achieved in 12 min at an applied current density 30 mA cm−2. Real wastewater study further confirmed that effective disinfection is possible with a low cost electrode material such as Ti/Sb–SnO2/PbO2. Energy consumed during disinfection was determined to be 4.978 kWh m−3 for real wastewater disinfection at applied current density 30 mA cm−2. Cost of operation was estimated and stability of the electrode was studied to evaluate the feasibility of large scale operation. Relatively low energy and less disinfection time makes this technology suitable for field scale applications.

Membrane-wrapped nanoparticles for nucleic acid delivery.

There is an unmet need for carriers that can deliver nucleic acids (NAs) to cancer cells and tumors to perpetuate gene regulation and manage disease progression. Membrane-wrapped nanoparticles (NPs) can be loaded with exogenously designed nucleic acid cargoes, such as plasmid deoxyribonucleic acid (pDNA), messenger ribonucleic acid (mRNA), small interfering RNA (siRNA), microRNA (miRNA), and immunostimulatory CpG oligodeoxynucleotides (CpG ODNs), to mitigate challenges presented by NAs' undesirable negative charge, hydrophilicity, and relatively large size. By conjugating or encapsulating NAs within membrane-wrapped NPs, various physiological barriers can be overcome so that NAs experience increased blood circulation half-lives and enhanced accumulation in intended sites. This review discusses the status of membrane-wrapped NPs as NA delivery vehicles and their advancement in gene regulation for cancer management in vitro and in vivo. With continued development, membrane-wrapped NPs have great potential as future clinical tools to treat cancer and other diseases with a known genetic basis.

Molecular Level Study and Characterization of Plasmid Deoxyribonucleic Acid Profile of Acinetobacter baumannii Isolated from Diabetic Foot Ulcers

The main objective is to study the genotypic and characterization of plasmid deoxyribonucleic acid content of resistant isolates in Erbil city. A total of 39 Acinetobacter baumannii were isolated from 102 diabetic foot ulcer patients, whose ages ranged from 37 to 88 y, attending various medical wards of hospitals in Erbil city, Iraq, between May 2020 and March 2021. Isolates were tested for the presence of extended-spectrum beta-lactamases and metallo beta-lactamases. The presence and prevalence of multidrug-resistant isolates namely blaOXA-51- like, blaOXA-23-like, ISAba1 and outer membrane protein A expression of oxacillinase carbapenemases gene and biofilm formation respectively were screened on the molecular level. Finally, plasmid deoxyribonucleic acid profile was determined and the biofilm-forming capacity was measured. Most isolates showed high resistance to different antimicrobial agents tested, the most effective agents were colistin and polymyxin B. 12 (30.76 %) isolates were tested positive for extended-spectrum beta-lactamases, while all 39 (100 %) were metallo beta-lactamase producers. 27 (69.23 %) of isolates were biofilm producers, while 12 (30.76 %) were nonbiofilm producers. The blaOXA-51-like, blaOXA-23-like, ISAba1 and outer membrane protein A genes were detected in all 39 (100 %) isolates. The genes encoding resistance to certain antibiotics, in addition to genes that coding for extended-spectrum beta-lactamase and biofilm formation were located on plasmids. Analysis of plasmid deoxyribonucleic acid content revealed that 25 (64.10 %) isolates harbored from one to several plasmid bands ranging from 1 kilo base pairs to more than 10 kilo base pairs. No thermo-sensitive plasmids were detected. Due to the emergence of multidrug-resistance, Acinetobacter baumannii is evolving into a global health threat. Carbapenems are effective therapeutic agents for Acinetobacter baumannii nosocomial infections. Before deciding the course of treatment, it is important to consider the aforementioned interaction since the presence of multiple species may worsen the infection.

Moscow State University of Medicine and Dentistry named after A.I. Evdokimov

Relevance. The effect of a tissue engineering construct based on synthetic octacalcium phosphate activated with plasmid DNA with vascular endothelial growth factor gene on bone morphogenesis at the jaw defect sites of patients was studied. It is shown that the studied osteoplastic material stimulates osteosynthesis pathways already at early stages, and xenogenic hydroxyapatite, triggers osteogenesis processes with considerable delay and does not have time to form a full-fledged bone structure by 6 months.Aim. Evaluate the dynamics of reparative osteogenesis based on the results of histomorphometric diagnostics in patients with defects in the jaw bones of various configurations and lengths with an implanted bone matrix based on synthetic octacalcium phosphate activated with plasmid deoxyribonucleic acid with genes of vascular endothelial growth factor.Materials and methods. Histomorphological examination of bone tissue biopsy of jaws was carried out in 50 patients of both genders, who needed additional volume of bone tissue to install dental implantation. Patients were divided into 2 groups by type of grafted material. 6 months after the surgery, bone biopsies were taken from the bone sites at the stage of implant placement. Histomorphological patterns and histomycrophotograms were studied in bone biopcies.Results. In bone tissue biopsies of patients implanted with a tissue engineering construct based on synthetic octacalcium phosphate activated with plasmid DNA with vascular endothelial growth factor gene, it was revealed that after 6 months there was prevaluation of mature bone tissue (42,71%), with the proportion of differentiated plate bone tissue being &lt; 90%.Conclusions. Histomorphometric analysis showed that in patients implanted with a tissue engineering construct based on synthetic octacalcium phosphate activated with plasmid DNA with vascular endothelial growth factor gene, after 6 months, early rearrangement of bone tissue into a mechanically dense and highly mineralized structure was detected.

Facile Synthesis of N-Doped Graphene Quantum Dots as Novel Transfection Agents for mRNA and pDNA.

In the wake of the coronavirus disease 2019 (COVID-19) pandemic, global pharmaceutical companies have developed vaccines for the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Some have adopted lipid nanoparticles (LNPs) or viral vectors to deliver the genes associated with the spike protein of SARS-CoV-2 for vaccination. This strategy of vaccination by delivering genes to express viral proteins has been successfully applied to the mRNA vaccines for COVID-19, and is also applicable to gene therapy. However, conventional transfection agents such as LNPs and viral vectors are not yet sufficient to satisfy the levels of safety, stability, and efficiency required for the clinical applications of gene therapy. In this study, we synthesized N-doped graphene quantum dots (NGQDs) for the transfection of various genes, including messenger ribonucleic acids (mRNAs) and plasmid deoxyribonucleic acids (pDNAs). The positively charged NGQDs successfully formed electrostatic complexes with negatively charged mRNAs and pDNAs, and resulted in the efficient delivery and transfection of the genes into target cells. The transfection efficiency of NGQDs is found to be comparable to that of commercially available LNPs. Considering their outstanding stability even at room temperature as well as their low toxicity, NGQDs are expected to be novel universal gene delivery platforms that can outperform LNPs and viral vectors.

Fast and Flexible mRNA Vaccine Manufacturing as a Solution to Pandemic Situations by Adopting Chemical Engineering Good Practice—Continuous Autonomous Operation in Stainless Steel Equipment Concepts

SARS-COVID-19 vaccine supply for the total worldwide population has a bottleneck in manufacturing capacity. Assessment of existing messenger ribonucleic acid (mRNA) vaccine processing shows a need for digital twins enabled by process analytical technology approaches in order to improve process transfer for manufacturing capacity multiplication, a reduction in out-of-specification batch failures, qualified personal training for faster validation and efficient operation, optimal utilization of scarce buffers and chemicals and speed-up of product release by continuous manufacturing. In this work, three manufacturing concepts for mRNA-based vaccines are evaluated: Batch, full-continuous and semi-continuous. Technical transfer from batch single-use to semi-continuous stainless-steel, i.e., plasmid deoxyribonucleic acid (pDNA) in batch and mRNA in continuous operation mode, is recommended, in order to gain: faster plant commissioning and start-up times of about 8–12 months and a rise in dose number by a factor of about 30 per year, with almost identical efforts in capital expenditures (CAPEX) and personnel resources, which are the dominant bottlenecks at the moment, at about 25% lower operating expenses (OPEX). Consumables are also reduceable by a factor of 6 as outcome of this study. Further optimization potential is seen at consequent digital twin and PAT (Process Analytical Technology) concept integration as key-enabling technologies towards autonomous operation including real-time release-testing.

Nucleic acid vaccines and CpG oligodeoxynucleotides for allergen immunotherapy.

Molecular forms of allergen-specific immunotherapy (AIT) are continuously emerging to improve the efficacy of the treatment, to shorten the duration of protocols and to prevent any side effects. The present review covers the recent progress in the development of AIT based on nucleic acid encoding allergens or CpG oligodeoxynucleotides (CpG-ODN). Therapeutic vaccinations with plasmid deoxyribonucleic acid (DNA) encoding major shrimp Met e 1 or insect For t 2 allergen were effective for the treatment of food or insect bite allergy in respective animal models. DNA expressing hypoallergenic shrimp tropomyosin activated Foxp3+ T regulatory (Treg) cells whereas DNA encoding For t 2 down-regulated the expression of pruritus-inducing IL-31. Co-administrations of major cat allergen Fel d 1 with high doses of CpG-ODN reduced Th2 airway inflammation through tolerance induction mediated by GATA3+ Foxp3hi Treg cells as well as early anti-inflammatory TNF/TNFR2 signaling cascade. Non-canonical CpG-ODN derived from Cryptococcus neoformans as well as methylated CpG sites present in the genomic DNA from Bifidobacterium infantis mediated Th1 or Treg cell differentiation respectively. Recent studies on plasmid DNA encoding allergens evidenced their therapeutic potential for the treatment of food allergy and atopic dermatitis. Unmethylated or methylated CpG-ODNs were shown to activate dose-dependent Treg/Th1 responses. Large clinical trials need to be conducted to confirm these promising preclinical data. Moreover, tremendous success of messenger ribonucleic acid (mRNA) vaccines against severe acute respiratory syndrome coronavirus 2 must encourage as well the re-exploration of mRNA vaccine platform for innovative AIT.

Ultrasound-Targeted Microbubble Destruction Mediates Gene Transfection for Beta-Cell Regeneration and Glucose Regulation.

Ultrasound-targeted microbubble destruction (UTMD) mediates gene transfection with high biosafety and thus has been promising toward treatment of type 1 diabetes. However, the potential application of UTMD in type 2 diabetes (T2D) is still limited, due to the lack of systematic design and dynamic monitoring. Herein, an efficient gene delivery system is constructed by plasmid deoxyribonucleic acid (DNA) encoding glucagon-like peptide 1 (GLP-1) in ultrasound-induced microbubbles, toward treatment of T2D in macaque. The as designed UTMD afforded enhancement of cell membrane penetration and GLP-1 expression in macaque, which is characterized by ultrasound-guided biopsy to monitor the dynamic process of islet cells for 6months. Also, improvement of pancreatic beta cell regeneration, and regulation of plasma glucose in macaque with T2D is achieved. The approach would serve as promising alternatives for the treatment of T2D.

Digital Twin of mRNA-Based SARS-COVID-19 Vaccine Manufacturing towards Autonomous Operation for Improvements in Speed, Scale, Robustness, Flexibility and Real-Time Release Testing

Supplying SARS-COVID-19 vaccines in quantities to meet global demand has a bottleneck in manufacturing capacity. Assessment of existing mRNA (messenger ribonucleic acid) vaccine processing shows the need for digital twins enabled by process analytical technology approaches to improve process transfers for manufacturing capacity multiplication, reduction of out-of-specification batch failures, qualified personnel training for faster validation and efficient operation, optimal utilization of scarce buffers and chemicals, and faster product release. A digital twin of the total pDNA (plasmid deoxyribonucleic acid) to mRNA process is proposed. In addition, a first feasibility of multisensory process analytical technology (PAT) is shown. Process performance characteristics are derived as results and evaluated regarding manufacturing technology bottlenecks. Potential improvements could be pointed out such as dilution reduction in lysis, and potential reduction of necessary chromatography steps. 1 g pDNA may lead to about 30 g mRNA. This shifts the bottleneck towards the mRNA processing step, which points out co-transcriptional capping as a preferred option to reduce the number of purification steps. Purity demands are fulfilled by a combination of mixed-mode and reversed-phase chromatography as established unit operations on a higher industrial readiness level than e.g., precipitation and ethanol-chloroform extraction. As a final step, lyophilization was chosen for stability, storage and transportation logistics. Alternative process units like UF/DF (ultra-/diafiltration) integration would allow the adjustment of final concentration and buffer composition before lipid-nano particle (LNP) formulation. The complete digital twin is proposed for further validation in manufacturing scale and utilization in process optimization and manufacturing operations. The first PAT results should be followed by detailed investigation of different batches and processing steps in order to implement this strategy for process control and reliable, efficient operation.

Characterisation of Plasmids in Multidrug Resistant Uropathogenic Gram-negative Bacterial Isolates

Introduction: Urinary Tract Infections (UTI) are one of the commonest conditions for which people seek medical care with an estimated 150 million episodes per annum worldwide. An unprecedented upsurge in the rate of development of antimicrobial resistance has reduced the therapeutic options leading to increased morbidity, prolonged hospital stays, development of complications. Majority of these infections are attributable to Gram negative bacteria which have now acquired resistance to almost all classes of antibiotics. Aim: To analyse the plasmid-mediated drug resistance and characterise the major plasmid families that are in circulation. Materials and Methods: A cross-sectional study comprising of a total of 95 non consecutive multidrug-resistant gram-negative bacterial isolates were subjected to Plasmid based replicon typing from January 2017 to June 2018. The 18 major replicons were divided in five multiplex and three uniplex Polymerase Chain Reaction (PCR) formats and the samples were subjected for plasmid characterisation and further sequencing of the plasmid Deoxyribonucleic Acid (DNA). The data obtained was analysed by Microsoft Excel software. Results: Escherichia coli, accounted for maximum n=51 (53.7%), Klebsiella pneumoniae n=19 (20%), Citrobacter sp n=11 (11.6%), miscellaneous gram negative n=14 (14.7%) The isolates exhibited a high degree of resistance to almost all tested antibiotics, sparing a few like Fosfomycin, Chloramphenicol, Imipenem, Amikacin. A total of 154 different plasmid families were detected from the 95 isolates. FIB replicon (24%), FIA (21%), F, W (20%), FIC, B/O (14%), Y (12%), I1 replicon (10.5%) were the major plasmid families detected in the present study. Conclusion: Many isolates exhibited the presence of more than one Incompatibility (Inc.) group plasmids, conferring multidrug resistance to the isolates. The study highlights the need for further research to study the association between plasmid families and their respective antibiotic resistance profiles for a given geographical niche and the need to devise further methods to target these epidemic plasmids.

Ultraviolet-induced in situ gold nanoparticles for point-of-care testing of infectious diseases in loop-mediated isothermal amplification.

The present study investigated ultraviolet-induced in situ gold nanoparticles (AuNPs) coupled with loop-mediated isothermal amplification (LAMP) for the point-of-care testing (POCT) of two major infectious pathogens, namely, Coronavirus (COVID-19) and Enterococcus faecium (E. faecium spp.). In the process, gold ions in a gold chloride (HAuCl4) solution were reduced using trisodium citrate (Na3Ct), a reducing agent, and upon UV illumination, red-colored AuNPs were produced in the presence of LAMP amplicons. The nitrogenous bases of the target deoxyribonucleic acid (DNA) acted as a physical support for capturing gold ions dissolved in the sample. The high affinity of gold with the nitrogenous bases enabled facile detection within 10 min, and the detection limit of COVID-19 plasmid DNA was as low as 42 fg μL-1. To ensure POCT, we designed a portable device that contained arrays of reagent chambers and detection chambers. In the portable device, colorimetric reagents such as HAuCl4 and Na3Ct were contained in the reagent chambers; these reagents were subsequently transferred to the detection chambers where LAMP amplicons were present and thus allowed convenient sample delivery and multiplex detection. Owing to the high sensitivity of the in situ AuNPs, simplicity of portable device fabrication, and rapid colorimetric detection, we strongly believe that the fabricated portable device could serve as a kit for rapid POCT for instantaneous detection of infectious diseases, and could be readily usable at the bedside.

Modified chitosan microparticles to molecular diagnostic of Chagas disease

Background: Chagas disease is caused by the protozoan Trypanosoma cruzi (T.cruzi), is a major public health problem in Latin America and an emerging threat in worldwide. Direct diagnosis is mainly based on the microscopic observation of the parasite but has low sensitivity mainly during the chronic phase; molecular techniques increase the sensitivity, but still limited. Nanobiotechnology is latest advance in biomedicine, recently the innovation of natural polymers is has been explored. The novel use of micro and/or nanoplatforms can be used to increase the sensitivity. The objective of the study was to develop different modified chitosan microparticles with capacity of adsorption of deoxyribonucleic acid (DNA) for the molecular diagnosis of Chagas disease. Methods and materials: Different microparticles of chitosan were synthesized and characterized, the DNA adsorption procedure was optimized evaluating parameters (type, pH of the medium, concentration, time and the desorption method) The limit of detection of plasmid DNA and T. cruzi DNA was determined. The sensitivity and specificity in the detection of kinetoplast DNA (kDNA) and satellite DNA (satDNA) was determined in urine samples of experimentally infected animals obtained at 14 and 45 d post-infection (dpi). Results: The results showed the glutaraldehyde crosslinked chitosan microparticles has the highest DNA adsorption. Using these microparticles the best conditions were obtained with: 1% glutaraldehyde, pH at 6.9, contact time of 60 m and EDTA-SDS as a desorption buffer. The detection limit of plasmid DNA was 101 copies/mL and T.cruzi DNA was 10-1 parasites/mL. Higher sensitivity was obtained with kDNA (71%) compared to satDNA (25%). The sensitivity in urine samples of infected animals at 14 dpi was 42% using ADNsat target and 78% with kDNA target; at 45 dpi with ADNsat target was 7% and with the ADNk target was 64%. High specificity (100%) was obtained for both targets in non-infected animals. Conclusion: Chitosan microparticles is innovative method and could be used to capture and concentrate T.cruzi DNA in urine (Tr-DNA).The detection of Tr-DNA could be used for diagnosis of Chagas disease.

Semi-quantitative Digital Analysis for Human Papillomavirus Detection from Environmental Specimens

Background: Recently, human papillomavirus (HPV) deoxyribonucleic acid (DNA) has been detected in urban wastewater, indicating that the virus can reach the sewer and, eventually, other water environments. This study aimed to develop a semi-quantitative assay for HPV DNA detection from environmental specimens using the PCR gel electrophoresis method. Method: This was an experimental descriptive qualitative study conducted from July to November 2019 in a standard molecular laboratory and non-laboratory administration room without air conditioner. Three brands of PCR reagents and different annealing temperatures were compared to identify the best condition for conventional PCR of plasmid DNA containing the HPV L1 gene. The semi-quantitative data were obtained from densitometry digital analysis using an imaging software. The optimized protocol was then applied on DNA serial dilutions to seek for the lower limit of detection (LLOD) value and the linear range of the assay. To evaluate the robustness of the assay, the protocol was further applied to spiked specimens of wastewater. Finally, several wastewater samples were tested for the presence of HPV DNA using this protocol. Results: A broad linear range and HPV L1 gene detection ability were observed with an LLOD of less than 2pg plasmid DNA in field condition. Although the assay successfully detected HPV DNA from several spiked wastewater samples, certain wastewater could interfere with the assay and gave false negative result. Conclusion: A semi-quantitative conventional PCR method to detect HPVDNA from environmental samples has been established and proven to be robust in field condition with non-optimum cold chain.