A Lattice Model on the Rate of DNA Hybridization

We develop a lattice model on the rate of hybridization of the complementary single-stranded DNAs (c-ssDNAs). Upon translational diffusion mediated collisions, c-ssDNAs interpenetrate each other to form correct (cc), incorrect (icc) and trap-correct contacts (tcc) inside the reaction volume. Correct contacts are those with exact registry matches which leads to nucleation and zipping. Incorrect contacts are the mismatch contacts which are less stable compared to tcc which can occur in the repetitive c-ssDNAs. Although tcc possess registry match within the repeating sequences, they are incorrect contacts in the view of the whole c-ssDNAs. The nucleation rate (kN) is directly proportional to the collision rate and the average number of correct-contacts (<ncc>) formed when both the c-ssDNAs interpenetrate each other. Detailed lattice model simulations suggest that 〈n_cc 〉∝L⁄V where L is the length of c-ssDNAs and V is the reaction volume. Further numerical analysis revealed the scaling for the average radius of gyration of c-ssDNAs (Rg) with their length as R_g∝√L. Since the reaction space will be approximately a sphere with radius equals to 2Rg and V∝L^(3⁄2), one obtains k_N∝1/√L. When c-ssDNAs are nonrepetitive, then the overall renaturation rate becomes as k_R∝k_N L and one finally obtains k_R∝√L in line with the experimental observations. When c-ssDNAs are repetitive with a complexity of c, then earlier models suggested the scaling k_R∝√L/c which breaks down at c = L. This clearly suggested the existence of at least two different pathways of renaturation in case of repetitive c-ssDNAs viz. via incorrect contacts and trap correct contacts. The trap correct contacts can lead to the formation of partial duplexes which can keep the complementary strands in the close vicinity for a prolonged timescale. This is essential for the extended 1D slithering, inchworm movements and internal displacement mechanisms which can accelerate the searching for the correct contacts. Clearly, the extent of slithering dynamics will be inversely proportional to the complexity. When the complexity is close to the length of c-ssDNAs, then the pathway via incorrect contacts will dominate. When the complexity is much lesser than the length of c-ssDNA, then pathway via trap correct contacts would be the dominating one.

Saving Resources: SARS-CoV-2 Diagnostics by Real-Time RT-PCR Using Reduced Reaction Volumes

Since the beginning of 2020, the betacoronavirus SARS-CoV-2 is causing a global pandemic of an acute respiratory disease termed COVID-19. The diagnostics of the novel disease is primarily based on direct virus detection by RT-PCR; however, the availability of test kits may become a major bottleneck, when millions of tests are performed per week. To increase the flexibility of SARS-CoV-2 diagnostics, three real-time RT-PCR assays listed on the homepage of the World Health Organization were selected and investigated regarding their compatibility with three different RT-PCR kits. Furthermore, the reaction volume of the PCR chemistry was reduced up to half of the original protocol to make the individual reactions more cost- and resource-effective. When testing dilution series of culture-grown virus, nearly identical quantification cycle values (Cq) were obtained for all RT-PCR assay/chemistry combinations. Regarding the SARS-CoV-2 detection in clinical samples, agreeing results were obtained for all combinations for virus negative specimens and swabs containing high to medium viral genome loads. In cases of very low SARS-CoV-2 genome loads (Cq > 36), inconsistent results were observed, with some test runs scoring negative and some positive. However, no preference of a specific target within the viral genome (E, RdRp, or N) or of a certain chemistry was seen. In summary, a reduction of the reaction volume and the type of PCR chemistry did not influence the PCR sensitivity.

Iron-Nickel Alloy with Starfish-like Shape and Its Unique Magnetic Properties: Effect of Reaction Volume and Metal Concentration on the Synthesized Alloy

Iron-nickel alloy is an example of bimetallic nanostructures magnetic alloy, which receives intensive and significant attention in recent years due to its desirable superior ferromagnetic and mechanical characteristics. In this work, a unique starfish-like shape of an iron-nickel alloy with unique magnetic properties was presented using a simple, effective, high purity, and low-cost chemical reduction. There is no report on the synthesis of such novel shape without complex precursors and/or surfactants that increase production costs and introduce impurities, so far. The synthesis of five magnetic iron-nickel alloys with varying iron to nickel molar ratios (10–50% Fe) was undertaken by simultaneously reducing Fe(II) and Ni(II) solution using hydrazine hydrate as a reducing agent in strong alkaline media for 15 min at 95–98 °C. The effect of reaction volume and total metal concentration on the properties of the synthesized alloys was studied. Alloy morphology, chemical composition, crystal structure, thermal stability, and magnetic properties of synthesized iron-nickel alloys were characterized by means of SEM, TEM, EDX, XRD, DSC and VSM. ImageJ software was used to calculate the size of the synthesized alloys. A deviation from Vegard’s law was recorded for iron molar ration higher than 30%., in which superstructure phase of FeNi3 was formed and the presence of defects in it, as well as the dimensional effects of nanocrystals. The saturation magnetization (Ms), coercivity (Hc), retentivity (Mr), and squareness are strongly affected by the molar ratio of iron and nickel and reaction volume as well as the total metal concentration.

Low-cost alternative biodiesel production apparatus based on household food blender for continuous biodiesel production for small communities

Fatty acid methyl esters (FAMEs) are sustainable biofuel that can alleviate high oil costs and environmental impacts of petroleum-based fuel. A modified 1200 W high-efficiency food blender was employed for continuous transesterification of various refined vegetable oils and waste cooking oil (WCO) using sodium hydroxide as a homogeneous catalyst. The following factors have been investigated on their effects on FAME yield: baffles, reaction volume, total reactant flow rate, methanol-oil molar ratio, catalyst concentration and reaction temperature. Results indicated that the optimal conditions were: 2000 mL reaction volume, 50 mL/min total flow rate, 1% and 1.25% catalyst concentration for refined palm oil and WCO, respectively, 6:1 methanol-to-oil molar ratio and 62–63 °C, obtaining yield efficiency over 96.5% FAME yield of 21.14 × 10–4 g/J (for palm oil) and 19.39 × 10–4 g/J (for WCO). All the properties of produced FAMEs meet the EN 14214 and ASTM D6751 standards. The modified household food blender could be a practical and low-cost alternative biodiesel production apparatus for continuous biodiesel production for small communities in remote areas.

Low-Cost Alternative Biodiesel Production Apparatus Based on Household Food Blender for Continuous Biodiesel Production for Small Communities

Fatty acid methyl esters (FAMEs) are sustainable biofuel that can alleviate high oil cost and environmental impacts of petroleum-based fuel. A modified 1,200 W high efficiency fruit blender was employed for continuous transesterification of various refined vegetable oils and waste cooking oil (WCO) using sodium hydroxide as a homogeneous catalyst. The following factors have been investigated on their effects on FAME yield: baffles, reaction volume, total reactant flow rate, methanol-oil molar ratio, catalyst concentration and reaction temperature. Results indicated that the optimal conditions were: 2,000 mL reaction volume, 50 mL/min total flow rate, 1% and 1.25% catalyst concentration for refined palm oil and WCO, respectively, 6:1 methanol-to-oil molar ratio and 62 - 63oC, obtaining yield efficiency over 96.5% FAME yield of 21.14 ´ 10-4 g.J-1 (for palm oil) and 19.39 ´ 10-4 g.J-1 (for WCO). All the properties of produced FAMEs meet the EN 14214 and ASTM D6751 standards. The modified household fruit blender could be a practical and low-cost alternative biodiesel production apparatus for continuous biodiesel production for small communities in remote areas.

A Novel Approach to the Bioluminescent Detection of the SARS-CoV-2 ORF1ab Gene by Coupling Isothermal RNA Reverse Transcription Amplification with a Digital PCR Approach

The COVID-19 pandemic caused by the SARS-CoV-2 virus, which first emerged in December 2019, represents an ongoing global public health emergency. Here, we developed an improved and highly sensitive approach to SARS-CoV-2 detection via coupling bioluminescence in real-time (BART) and reverse-transcriptase loop-mediated amplification (RT-LAMP) protocols (RT-LAMP-BART) and was also compatible with a digital LAMP system (Rainsuit), which did not allow for real-time quantification but did, nonetheless, facilitate absolute quantification with a comparable detection limit of 104 copies/mL. Through improving RNA availability in samples to ensure the target RNA present in reaction, we additionally developed a simulated digital RT-LAMP approach using this same principle to enlarge the overall reaction volume and to achieve real-time detection with a limit of detection of 10 copies/mL, and with further improvements in the overall dynamic range of this assay system being achieved through additional optimization.

90Y-DOTA-nimotuzumab: Synthesis of a Promising β⁻ radiopharmaceutical

Background: Nimotuzumab is a humanized anti-epidermal growth factor receptor (EGFR) monoclonal antibody, nowadays used for tumour immunochemotherapy. This study aimed to label the conjugate DOTA-nimotuzumab with yttrium-90, in order to provide a β- emitting radioimmunoconjugate (90Y-DOTA-nimotuzumab) potentially useful to assess the feasibility of a new radio-guided surgery approach. Methods: The synthesis of 90Y-DOTA-nimotuzumab was performed in two days. Nimotuzumab was conjugated with a 50 fold excess of DOTA and then labelled with 90Y3+. The 90Y-DOTA-nimotuzumab preparation was optimized considering several parameters such as pH, temperature and reaction volume. Moreover, the 90Y-DOTA-nimotuzumab stability was evaluated in human plasma. Results: The radioimmunoconjugate 90Y-DOTA-nimotuzumab was obtained with a radiochemical purity greater than 96%, and showed a good stability at 20°C as well as at 37°C in human plasma. Conclusions: The optimized conditions for a mild and easy preparation of 90Y-DOTA-nimotuzumab joined to a promising stability under physiological conditions suggest to propose this radioimmunoconjugate as a potential diagnostic radiopharmaceutical for β- radio-guided surgery.

Validation of Half-reaction Volumes of the Promega PowerPlex® Forensic Amplification Kits (PowerPlex® 18D Systems, PowerPlex ® 21System, PowerPlex® Fusion System and PowerPlex® Y23 System) in STR Analysis

DNA amplification is known to be the most expensive step during forensic DNA analysis. This study evaluated the half-reaction amplification protocol (12.5 µL PCR product) using DNA amplification kits from Promega PowerPlex® (PowerPlex® 18D System, PowerPlex ®21System, PowerPlex® Fusion System and PowerPlex® Y23 System), which might aid in reducing sample analysis cost by half and allow the analysis of more samples. A sensitivity study (15 samples) along with testing of various blood stain samples (n=100) that were submitted to the Medico-Legal Directorate laboratory for DNA testing was accomplished to compare the DNA profiles resulting from half-reaction volume procedure to those with full-reaction volume procedure, using three differed methods along with standard protocol to evaluate the effect of half reaction volume with some variables. Results demonstrated the use of half-reaction amplification protocol preceded by washing step for all aforementioned DNA amplification kits gave a robust and reliable amplification result that aid to increase the number of samples analyzed and decreased the test cost for each kit without compromising the quality of 3DNA profiles obtained.