Lower cost alternatives for molecular diagnosis of COVID-19: conventional RT-PCR and SYBR Green-based RT-qPCR.

In March 2020, WHO declared a pandemic state due to SARS-CoV-2 having spread. TaqMan-based real-time RT-qPCR is currently the gold standard for COVID-19 diagnosis. However, it is a high-cost assay, inaccessible for the majority of laboratories around the world, making it difficult to diagnose on a large scale. The objective of this study was to standardize lower cost molecular methods for SARS-CoV-2 identification. E gene primers previously determined for TaqMan assays by Colman et al. (2020) were adapted in SYBR Green assay and RT-PCR conventional. The cross-reactivity test was performed with 17 positive samples for other respiratory viruses, and the sensibility test was performed with 8 dilutions (10 based) of SARS-CoV-2 isolated and 63 SARS-CoV-2-positive samples. The SYBR Green assays and conventional RT-PCR have not shown amplification of the 17 respiratory samples positives for other viruses. The SYBR Green-based assay was able to detect all 8 dilutions of the isolate. The conventional PCR detected until 107 dilution, both assays detected the majority of the 63 samples, 98.42% of positivity in SYBR Green, and 93% in conventional PCR. The average Ct variation between SYBR Green and TaqMan was 1.92 and the highest Ct detected by conventional PCR was 35.98. Both of the proposed assays are less sensitive than the current gold standard; however, our data shows a low sensibility variation, suggesting that these methods could be used by laboratories as a lower cost molecular method for SARS-CoV-2 diagnosis.

Realizing a high-rate sulfidogenic reactor driven by sulfur-reducing bacteria with organic substrate dosage minimization and cost-effectiveness maximization.

Biological sulfur reduction is an attractive sulfidogenic technology for the treatment of organics-deficient metal-laden wastewater, because it theoretically reduces the electron donor consumption by 75%, compared to sulfate reduction. However, reducing the external organic substrate dosage may lower the sulfur reduction rate. Supplying with a more biodegradable organic substrate could possibly enhance sulfidogenic activity but also increase the chemical cost. Therefore, the sulfide production performance of a sulfur-reducing bioreactor feeding with varied levels of organic supply, and different types of organic substrates were investigated. The results showed that high-rate sulfide production (12.30 mg S/L/h) in a sulfur-reducing bioreactor can be achieved at the minimal dosage of organic substrate as low as 39 mg C/L of organic carbon in the influent. Changing the type of organic substrate posed a significant effect on the sulfidogenic activity in the sulfur-reducing bioreactor. Sodium acetate was found to be the optimal substrate to achieve the highest sulfide production rate (28.20 mg S/L/h) by sulfur-reducing bacteria (S0RB), followed by ethanol, methanol, glycerol, pyruvic acid, acetic acid, glucose, sucrose, malic acid, sodium formate, formic acid, N-propanol, N-butanol, lactic acid, sodium lactate, propionic acid and sodium propionate (2.87 mg S/L/h as the lowest rate). However, the cost-effectiveness analysis showed that glucose was the most cost-effective organic substrate to realize the sulfur reduction process in high sulfide production rate (20.13 mg S/L/h) and low chemical cost (5.94 kg S/$). The utilization pathway of the different organic substrates in the sulfur-reducing bioreactor was also discussed.

Effectiveness and cost-effectiveness of daily all-over-body application of emollient during the first year of life for preventing atopic eczema in high-risk children (The BEEP trial): protocol for a randomised controlled trial.

BACKGROUND: Atopic eczema (AE) is a common skin problem that impairs quality of life and is associated with the development of other atopic diseases including asthma, food allergy and allergic rhinitis. AE treatment is a significant cost burden for health care providers. The purpose of the trial is to investigate whether daily application of emollients for the first year of life can prevent AE developing in high-risk infants (first-degree relative with asthma, AE or allergic rhinitis). METHODS: This is a protocol for a pragmatic, two-arm, randomised controlled, multicentre trial. Up to 1400 term infants at high risk of developing AE will be recruited through the community, primary and secondary care in England. Participating families will be randomised in a 1:1 ratio to receive general infant skin-care advice, or general skin-care advice plus emollients with advice to apply daily to the infant for the first year of life. Families will not be blinded to treatment allocation. The primary outcome will be a blinded assessment of AE at 24 months of age using the UK Working Party Diagnostic Criteria for Atopic Eczema. Secondary outcomes are other definitions of AE, time to AE onset, severity of AE (EASI and POEM), presence of other allergic diseases including food allergy, asthma and hay fever, allergic sensitisation, quality of life, cost-effectiveness and safety of the emollients. Subgroup analyses are planned for the primary outcome according to filaggrin genotype and the number of first-degree relatives with AE and other atopic diseases. Families will be followed up by online and postal questionnaire at 3, 6, 12 and 18 months with a face-to-face visit at 24 months. Long-term follow-up until 60 months will be via annual questionnaires. DISCUSSION: This trial will demonstrate whether skin-barrier enhancement through daily emollient for the first year of life can prevent AE from developing in high-risk infants. If effective, this simple and cheap intervention has the potential to result in significant cost savings for health care providers throughout the world by preventing AE and possibly other associated allergic diseases. TRIAL REGISTRATION: ISRCTN registry; ID: ISRCTN21528841 . Registered on 25 July 2014.

Pulsed Direct Current Electrospray: Enabling Systematic Analysis of Small Volume Sample by Boosting Sample Economy.

We had developed pulsed direct current electrospray ionization mass spectrometry (pulsed-dc-ESI-MS) for systematically profiling and determining components in small volume sample. Pulsed-dc-ESI utilized constant high voltage to induce the generation of single polarity pulsed electrospray remotely. This method had significantly boosted the sample economy, so as to obtain several minutes MS signal duration from merely picoliter volume sample. The elongated MS signal duration enable us to collect abundant MS(2) information on interested components in a small volume sample for systematical analysis. This method had been successfully applied for single cell metabolomics analysis. We had obtained 2-D profile of metabolites (including exact mass and MS(2) data) from single plant and mammalian cell, concerning 1034 components and 656 components for Allium cepa and HeLa cells, respectively. Further identification had found 162 compounds and 28 different modification groups of 141 saccharides in a single Allium cepa cell, indicating pulsed-dc-ESI a powerful tool for small volume sample systematical analysis.

[Economic assessment in health and environment from control of persistent organic pollutants in Colombia]. / Valoración económica en salud y medio ambiente del control de contaminantes orgánicos persistentes en Colombia.

Objective To estimate the economic benefits related to environment and health in the context of the implementation of the Stockholm Convention for the control of Persistent Organic Pollutants in the country. METHOD: The estimation was conducted based on two scenarios: non-compliance with the agreement and compliance with the Convention. Gross profit was derived from the difference in present value between the health and environmental costs that are assumed in each scenario. Results Gross profit by decreasing health costs arising from the implementation of the Convention was estimated at USD $ 511 and USD $ 501 million. By introducing variables such as management costs and agreement on potential benefits for access to international markets, the benefits to the country were estimated at between USD $1 631 and USD $ 3 118 million. Discussion Despite the economic benefits generated by lower expenditure on health for the Convention implementation, the costs associated with reducing pollutant emissions generated a negative balance, compensated only by the expectation of higher revenues for international market access. We consider this initial economic assessment an important contribution, but it should be reviewed to include valuation methodologies involving other social profitability variables and different scenarios for emerging technologies, new scientific knowledge about these pollutants, changes in legislation and / or changes in trade agreement conditions, among others.

The economies of synthesis.

In this tutorial review the economies of synthesis are analysed from both detailed and macroscopic perspectives, using case-studies from complex molecule synthesis. Atom, step, and redox economy are more than philosophical constructs, but rather guidelines, which enable the synthetic chemist to design and execute an efficient synthesis. Students entering the field of synthesis might find this tutorial helpful for understanding the subtle differences between these economic principles and also see real-world situations where such principles are put into practice.

Real-time quantitative PCR with SYBR Green I detection for estimating copy numbers of nine drug resistance candidate genes in Plasmodium falciparum.

BACKGROUND: Evaluating copy numbers of given genes in Plasmodium falciparum parasites is of major importance for laboratory-based studies or epidemiological surveys. For instance, pfmdr1 gene amplification has been associated with resistance to quinine derivatives and several genes involved in anti-oxidant defence may play an important role in resistance to antimalarial drugs, although their potential involvement has been overlooked. METHODS: The DeltaDeltaCt method of relative quantification using real-time quantitative PCR with SYBR Green I detection was adapted and optimized to estimate copy numbers of three genes previously indicated as putative candidates of resistance to quinolines and artemisinin derivatives: pfmdr1, pfatp6 (SERCA) and pftctp, and in six further genes involved in oxidative stress responses. RESULTS: Using carefully designed specific RT-qPCR oligonucleotides, the methods were optimized for each gene and validated by the accurate measure of previously known number of copies of the pfmdr1 gene in the laboratory reference strains P. falciparum 3D7 and Dd2. Subsequently, Standard Operating Procedures (SOPs) were developed to the remaining genes under study and successfully applied to DNA obtained from dried filter blood spots of field isolates of P. falciparum collected in São Tomé & Principe, West Africa. CONCLUSION: The SOPs reported here may be used as a high throughput tool to investigate the role of these drug resistance gene candidates in laboratory studies or large scale epidemiological surveys.

Comparative analysis of the DNA staining efficiencies of different fluorescent dyes in preparative agarose gel electrophoresis.

Ethidium bromide (EB) is a mutagen and toxin that is widely used in the laboratory for visualization of nucleic acids. Safer nucleic acid stains, such as SYBR Gold, SYBR Green, GoldView, GeneFinder, and GoldStar, have been developed. However, there has been no systematic comparative analysis of the staining efficiencies of these dyes. In the present study, SYBR Gold, SYBR Green I, GoldView and EB were compared. Although both SYBR Gold and SYBR Green alter electrophoretic mobility and thus DNA size estimates, they are cost-effective alternatives to EB. SYBR Gold was more sensitive than SYBR Green I at detecting short fragments, but 50-bp bands were clearly visible using either dye when visualized with a long integration time. SYBR Gold or SYBR Green I are sensitive and relatively safe alternatives to EB. In our laboratory, the SYBR Gold method is now used routinely by all members of our group with great consistency and success.

The commercial production of chemicals using pathway engineering.

Integration of metabolic pathway engineering and fermentation production technologies is necessary for the successful commercial production of chemicals. The 'toolbox' to do pathway engineering is ever expanding to enable mining of biodiversity, to maximize productivity, enhance carbon efficiency, improve product purity, expand product lines, and broaden markets. Functional genomics, proteomics, fluxomics, and physiomics are complementary to pathway engineering, and their successful applications are bound to multiply product turnover per cell, channel carbon efficiently, shrink the size of factories (i.e., reduce steel in the ground), and minimize product development cycle times to bring products to market.