Dilution Fold Research Articles

Real-time detection and analysis of foodborne pathogens via machine learning based fiber-optic Raman sensor

Food safety is a critical concern worldwide, and in recent years, it has become a growing public concern in the United States due to mass production and multi-channel distribution of high-nutrient and fresh-cut foods that have increased the prevalence and diversity of foodborne pathogens (e.g., Escherichia coli, Salmonella, Listeria, etc.). Traditional methods for the detection of these pathogens rely heavily on low-efficiency techniques, which may expose the public to contaminated foods that have not been tested or identified. Therefore, rapid, simple, sensitive, and inexpensive detection methods are urgently needed for food safety investigations with higher efficiency and accuracy. One of the promising methods for the detection of foodborne pathogens is Raman spectroscopy, which is a molecular-level analytical tool with the advantages of high selectivity and sensitivity and simple operation at a relatively low cost. In this study, a novel fiber-optic-based portable Raman probe was developed and used for real-time detection of a panel of pathogen-specific molecular fingerprint volatile organic compounds (VOCs). Furthermore, machine learning (ML) algorithms were applied to assist in the extraction of molecular information from the raw Raman spectra, resulting in high accuracy prediction of complex VOC mixtures, even at high dilution folds (100x). The developed ML-assisted Raman probe has immense potential for rapid on-site detection of complex chemical mixtures in food safety and beyond. This innovative approach achieves high accuracy in comprehensively sorting mixed chemicals with varying concentrations and provides several advantages over previous studies, including speed, portability, non-contact operation, and precise classification of foodborne pathogen VOCs.

Discovery and validation of bladder cancer related excreted nucleosides biomarkers by dilution approach in cell culture supernatant and urine using UHPLC-MS/MS

The exploration of nucleoside changes in human biofluids has profound potential for cancer diagnosis. Herein, we developed a rapid methodology to quantify 17 nucleosides by UHPLC-MS/MS. Five pairs of isomers were successfully separated within 8 min. The ME was mostly eliminated by sample dilution folds of 1000 for urine and 40 for CCS. The optimized method was firstly applied to screen potential nucleoside biomarkers in CCS by comprising bladder cancer cell lines (5637 and T24) and normal human bladder cell line SV-HUC-1 together with student's t-test and OPLS-DA. Nucleosides with significant differences in the supernatant of urine samples were also uncovered comparing BCa with the non-tumor group, as well as a comparison of BCa recurrence group with the non-recurrence group. By intersecting the differential nucleosides in CCS and urine supernatant, and then further confirmed using validation sets, the combination of m3C and m1A with AUC of 0.775 was considered as a potential biomarker for bladder cancer diagnosis. A panel of m3C, m1A, m1G, and m22G was defined as potential biomarkers for bladder cancer prognosis with an AUC of 0.819. Above all, this method provided a new perspective for diagnosis and recurrence monitoring of bladder cancer. SignificanceThe exploration of nucleoside changes in body fluids has profound potential for the diagnosis and elucidation of the pathogenesis of cancer. In this study, we developed a rapid methodology for the simultaneous quantitative determination of 17 nucleosides in the supernatant of cells and urine samples using UHPLC-MS/MS to discover and validate bladder cancer related excreted nucleoside biomarkers. The results of this paper provide a new strategy for diagnosis and postoperative recurrence monitoring of bladder cancer and provide theoretical support for the exploration of its pathogenesis.

A Novel Methodology for Detecting Variations in Cell Surface Antigens Using Cell-Tearing by Optical Tweezers.

The quantitative analysis of cell surface antigens has attracted increasing attention due to the antigenic variation recognition that can facilitate early diagnoses. This paper presents a novel methodology based on the optical “cell-tearing” and the especially proposed “dilution regulations” to detect variations in cell surface antigens. The cell attaches to the corresponding antibody-coated slide surface. Then, the cell-binding firmness between a single cell and the functionalized surface is assayed by optically tearing using gradually reduced laser powers incorporated with serial antibody dilutions. Groups B and B3 of red blood cells (RBCs) were selected as the experiment subject. The results indicate that a higher dilution called for lower power to tear off the cell binding. According to the proposed relative-quantitative analysis theory, antigenic variation can be intuitively estimated by comparing the maximum allowable dilution folds. The estimation result shows good consistency with the finding in the literature. This study suggests a novel methodology for examining the variation in cell surface antigens, expected to be widely capable with potential sensor applications not only in biochemistry and biophysics, but also in the micro-/nano- engineering field.

Assessment of Emulsification Potentials and Bio- Diversities of Bacterial Isolates from Bonny Loading Jetty Crude Oil Polluted Soil, Bonny Island, Rivers State.

Surfactant producing microorganisms are indispensable in utility; In the present study, contaminated homogenized soil samples from Bonny Island River showed the following physicochemical properties: pH (5.67), Chloride ion (Cl-) (321±0.2 mg/g),Potassium ion (K+) (5.26±0.01mg/g), Magnesium ion(Mg2+) (2.19±0.05 mg/g), Iron II ion(Fe2+) (38.73±0.13ppm), Calcium ion (Ca2+) (4.12±0.1mg/g), Copper II ion (Cu2+) (3.2±0.01ppm), Lead II ion (Pb2+) (0.21±0.02 ppm), Phosphate ion (PO3) (6.24±0.1 mg/g), Total petroleum hydrocarbons(TPH 364±0.05 Mg/g), Total organic carbon  (TOC 56.5±0.05 mg/g) and Total organic matter contents (TOM) (63.45±0.2) .Prior to the emulsification screening carried out; microbial isolation and total heterotrophic counting (CFU/g) carried out showed a total of twelve microbial distinct isolates from the polluted soil with a microbial population of 2.5 x 108 and 2.1 x 108 CFU/g of the soil. Dilution folds of 10-1 and 10-2 from the Bushnell-Hass media showed microbial population of 0.33 x 104 and 0.78 x 104CFU/g respectively. Klebsiella sp. showed emulsification indices of43.3%, 51.2%, 50 and 48.9%with diesel and coconut oil, kerosene and crude oil respectively. Pseudomonas sp. showed emulsification indices of 40, 43, 54 and 45.6% with diesel, coconut oil, kerosene and crude oil respectively. Bacillus sp. showed emulsification indices of 46, 45, 32 and 37% with diesel, coconut oil, kerosene and crude oil respectively. The present study have shown the great potential of utilization of the bacterial consortium in oil recovering from various damp oil-wells and in cost efficient bioremediation process.

Safety and immunogenicity of Nanocovax, a SARS-CoV-2 recombinant spike protein vaccine: Interim results of a double-blind, randomised controlled phase 1 and 2 trial

SummaryBackgroundNanocovax is a recombinant severe acute respiratory syndrome coronavirus 2 subunit vaccine composed of full-length prefusion stabilized recombinant SARS-CoV-2 spike glycoproteins (S-2P) and aluminium hydroxide adjuvant.MethodsWe conducted a dose-escalation, open label trial (phase 1) and a randomized, double-blind, placebo-controlled trial (phase 2) to evaluate the safety and immunogenicity of the Nanocovax vaccine (in 25 mcg, 50 mcg, and 75 mcg doses, aluminium hydroxide adjuvanted (0·5 mg/dose) in 2-dose regime, 28 days apart (ClinicalTrials.gov number, NCT04683484). In phase 1, 60 participants received two intramuscular injection of the vaccine following dose-escalation procedure. The primary outcomes were reactogenicity and laboratory tests to evaluate the vaccine safety. In phase 2, 560 healthy adults received either vaccine doses similar in phase 1 (25 or 50 or 75 mcg S antigen in 0·5 mg aluminium per dose) or adjuvant (0·5 mg aluminium) in a ratio of 2:2:2:1. One primary outcome was the vaccine safety, including solicited adverse events for 7 day and unsolicited adverse events for 28 days after each injection as well as serious adverse event or adverse events of special interest throughout the study period. Another primary outcome was anti-S IgG antibody response (Index unit/ml). Secondary outcomes were surrogate virus neutralisation (inhibition percentage), wild-type SARS-CoV-2 neutralisation (dilution fold), and T-cell responses by intracellular staining for interferon gamma (IFNg). Anti-S IgG and neutralising antibody levels were compared with convalescent serum samples from symptomatic Covid-19 patients.FindingsFor phase 1 study, no serious adverse events were observed for all 60 participants. Most adverse events were grade 1 and disappeared shortly after injection. For phase 2 study, after randomisation, 480 participants were assigned to receive the vaccine with adjuvant, and 80 participants were assigned to receive the placebo (adjuvant only). Reactogenicity was absent or mild in the majority of participants and of short duration (mean ≤3 days). Unsolicited adverse events were mild in most participants. There were no serious adverse events related to Nanocovax. Regarding the immunogenicity, Nanocovax induced robust anti-S antibody responses. In general, there humoral responses were similar among vaccine groups which reached their peaks at day 42 and declined afterward. At day 42, IgG levels of vaccine groups were 60·48 [CI95%: 51·12–71·55], 49·11 [41·26–58·46], 57·18 [48·4-67·5] compared to 7·10 [6·32-13·92] of convalescent samples. IgG levels reported here can be converted to WHO international standard binding antibody unit (BAU/ml) by multiplying them to a conversion factor of 21·8. Neutralising antibody titre of vaccine groups at day 42 were 89·2 [52·2–152·3], 80·0 [50·8–125.9] and 95·1 [63·1–143·6], compared to 55·1 [33·4-91·0] of the convalescent group.InterpretationUp to day 90, Nanocovax was found to be safe, well tolerated, and induced robust immune responses.FundingThis work was funded by the Coalition for Epidemic Preparedness Innovations (CEPI), the Ministry of Science and Technology of Vietnam, and Nanogen Pharmaceutical Biotechnology JSC.

Effects of Platostoma palustre ethanolic extracts and commercial herbal tea on the cell viability of colorectal cancer cells

Platostoma palustre jelly is a traditional food. Platostoma palustre has been used as folk medicine and is effective against heat-shock, hypertension and diabetes. Therefore, the aim of this study was to determine the effects of ethanolic extracts and commercial herbal tea of Platostoma palustre in inhibiting colorectal cancer cell viability. The ethanolic extracts of Platostoma palustre by using 90% ethanol for extraction. In this study, 2-fold serial dilution of 100 mg/mL Platostoma palustre extracts were applied. On other hand, the same dilution fold was also performed for 100% commercial herbal tea with Platostoma palustre. Additionally, CT-26 and HT-29 colorectal cancer cell lines were also used in this study. After co-culturing for 24 hours, the cell viability of CT-26 and HT-29 colorectal cancer cell lines were performed by using 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. According to these data, the 1.56-100 mg/mL Platostoma palustre extracts possessed the significant inhibition effects of CT-26 colorectal cancer cell viability. The 3.13-100% commercial herbal tea with Platostoma palustre possessed the significant inhibition effects of CT-26 colorectal cancer cell viability. The 6.25-100 mg/mL Platostoma palustre extracts possessed the significant inhibition effects of HT-29 colorectal cancer cell viability. The 25-100% commercial herbal tea with Platostoma palustre possessed the significant inhibition effects of HT-29 colorectal cancer cell viability. However, the 0.39-3.13 mg/mL Platostoma palustre extracts possessed the significant promoting effects of HT-29 colorectal cancer cell viability. The 0.39-12.5% commercial herbal tea with Platostoma palustre also possessed the significant promoting effects of HT-29 colorectal cancer cell viability. Comparison of CT-26 and HT-29 cell lines was on the cell viability after Platostoma palustre ethanolic extracts and commercial herbal tea treatments, CT-26 cell line was better sensitive than HT-29 cell line on the inhibition of cell viability after treatment of Platostoma palustre ethanolic extracts and the commercial herbal tea. Taken these results together, Platostoma palustre ethanolic extracts and commercial herbal tea may have a potential for inhibiting the growth of colorectal cancer cells.

Ultrasensitive and High Reproducible Detection of Urinary Metabolites Using the Tip–Contact Extraction Method Coupled with Negative LDI-MS

More and more evidence has proved that urinary metabolites can instantly reflect disease state. Therefore, ultra-sensitive and reproducible detection of urinary metabolites in a high-throughput way is urgently desirable for clinical diagnosis. Matrix-free laser desorption/ionization mass spectrometry (LDI-MS) is a high-throughput platform for metabolites detection, but it is encountered by severe interference from numerous salts in urine samples, because the crystallized urine salt on dried samples could result in poor reproducibility in LDI-MS detection. The present work proposed a tip-contact extraction (TCE) technique to eliminate interference from the urine salt. Vertical silicon nanowire arrays decorated with the fluorinated ethylene propylene film (FEP@VSiNWs) could effectively extract metabolites from the urine sample dropping on its surface. High salt tolerance was observed in the subsequent LDI-MS detection of the metabolites extracted on the tip of FEP@VSiNWs even in the presence of 1 M urea. Stable and reproducible mass spectra for non-target metabolic analysis were obtained in real urine samples with different dilution folds. Urinary metabolites collected from bladder cancer (BC) patients were reliably profiled by the TCE method coupled with negative LDI-MS. Based on this platform, potential metabolic biomarkers that can distinguish BC patients and normal controls were uncovered.

Hydrogen production by sequential dark and photofermentation using wet biomass hydrolysate of Spirulina platensis: Response surface methodological approach

Microalgae and cyanobacteria can be used as a potential biomass to produce hydrogen from stored glycogen and starch through fermentation and photofermentation. In this study, the potential of algal biomass i.e. Spirulina platensis hydrolysate as a substrate for sequential fermentative (I-stage) and photo-fermentative (II-stage) biohydrogen production was evaluated. Response Surface Methodology (RSM) was employed to find the optimum photofermentation conditions. From the preliminary optimization experiments, it was found that the significantly affecting factors for H2 production were pH, dilution fold (D.F.) of fermentate and Fe(II) sulfate concentration during photofermentation (second stage). In the present study, 1% (w/v) Spirulina platensis hydrolyzate produced 23.06 ± 3.63 mmol of H2 with yield of 1.92 ± 0.20 mmol H2/g COD reduced. In the second stage experiment 1510 ± 35 mL/l hydrogen was produced using inoculum volume-20.0% (v/v) and inoculum age-48 h of co-culture of Rhodobacter sphaeroides NMBL-01 and Bacillus firmus NMBL-03 under conditions pH-5.95, D.F. of dark fermentate-20.30 folds, Fe(II) sulfate concentration-0.412 μM, temperature-32±2 °C and light intensity-2.5 klux.

Dairy Manure Wastewater Remediation Using Non-airtight Digestion Pretreatment Followed by Microalgae Cultivation.

The non-airtight digestion technology is emerging to be applied in the acidogenic phase for two-stage methane production. However, in this study, it was used to pretreat screened dairy manure (SDM) in order to provide microalgae cultivation with a substrate that might be more suitable for nutrient reduction, especially phosphorus. SDM was firstly underwent non-airtight digestion applying different dilution folds, i.e., blank (no dilution), 5-fold, 10-fold, and 15-fold. Total solids (TS), total dissolved solids (TDS), and chemical oxygen demand (COD) of the SDM were mostly reduced when there was no dilution applied. Five-fold dilution is the most beneficial one for ammonia reduction. Total phosphorus (TP) was reduced the most efficiently in the blank SDM. After the non-airtight digestion, 5-fold diluted original SDM, 5-fold diluted digested original SDM, and digested 5-fold diluted SDM were used to grow microalgae for 8days. Microalgae grown in 5-fold diluted digested original SDM and digested 5-fold diluted SDM had better removal efficiencies in COD and NH4-N. From the monitoring of pH and TP during the 8-day culture period, it is found that pHs were peaked on the 4th day for microalgae grown in 5-fold diluted digested original SDM and digested 5-fold diluted SDM, corresponding to the maximal TP removal. Non-airtight digestion of SDM could help achieve better nutrient removal by microalgal cultivation in a shorter time span.

Allelopathic effects of water extracts from different parts of foxtail millet straw on three kinds of weeds

We examined the allelopathic effect of extracts from different parts of foxtail millet straw with different concentrations (undiluted water extracts, 10-fold, 50-fold, and 100-fold dilution) on three different kinds of malignant weeds (Amaranthus retroflexus, Chenopodium album, and Setaria viridis) by water extract. In this experiment, we measured biological indicators for seed germination period and potted seedling physiological parameters. The results showed that water extract of foxtail millet leaves and stems had significant allelopathic effects on the three species of weeds, with the effects of different concentrations being different. Undiluted water extract inhibited the growth, while dilution (10-fold, 50-fold, 100-fold dilution) promoted the growth. Under the treatment of undiluted water extract of leaf and stem, the germination rate of three weeds decreased 63.9%, 37.3% and 41.7%, respectively, while root length was only 27.8%, 37.8% and 18.4% of the control. The bud length was only 34.5%, 27.7% and 17.6% of the control. The net photosynthetic rate accounted for 66.6%, 89.9% and 88.2% of the control. The transpiration rate accounted for 69.0%, 87.5% and 56.1% of the control, while the synthesis allelopathic index of the three weeds were -0.699, -0.716 and -0.795 by undiluted water extract, respectively. Results implied strong allelopathic inhibition. The allelopathy promoting effect of dilution increased first and then decreased with the increases of dilution folds. Among which, 50-fold dilution had the strongest promoting effect with the germination rate, root length and bud length of the three weeds being significantly different from those of the control, with the synthesis allelopathic index being 0.261, 0.217, and 0.165, respectively. A large number of associated weeds grow in foxtail millet field which is related to the leaching of allelopathic substances in straw.

Optimisation of Almond Milk Producing Using Response Surface Method

The purpose of this study was to find the optimal production’s condition of almond milk. By this purpose it was determined dilution fold (3–7) and dilution temperature (25–80 0C) as producing parameter by pro - tests. Totally 13 milkwas produced. Under these conditions, effects of dilution fold and dilution temperature were investigated on the chemical compositions, energy and sensory properties of producted almond milks. According to results, dry matters of almond milks were determined as averagely 12.77%; ash 0.43%, protein 3.21%; fat 6.85%; carbohydrate 2.44%. Results of energy values were changed between 67 cal.100-1 mL and 103 cal.100-1 mL. Produced almond milk at 71.2 oC dilution temperature and 3 fold dilution, it has been the maximum desirability as 94%.

Almond Milk: Preparation, Chemical, Fatty Acids, Mineral, Total Phenolic Compositions and Antioxidant Activity

The aim of this study was producing a new alternative milk for person who is vegetarian, lactose intolerance sick, cardio-vascular diseased, diabetic or for old people. The dilution fold (3–7) and dilution temperature (25–80 0C) as producing parameter by pro – tests were carried out. Totally 13 milks were produced. In these conditions, effects of dilution fold and dilution temperature on the proximate, fatty acid, mineral, total phenolic compositions and antioxidant activity of produced milks were investigated. Keywords: Almond milk, Antioxidant activity, Fatty acids composition, Mineral composition, Total phenolic compositions DOI: 10.7176/JSTR/6-08-11

Microalgae Cultivation Using Screened Liquid Dairy Manure Applying Different Folds of Dilution: Nutrient Reduction Analysis with Emphasis on Phosphorus Removal.

A number of dairies in southern Idaho employed stationary inclined screens to separate large solid particles out of liquid dairy manure. In this way, the total solid content of the liquid dairy manure can drop about 20%. Solids in dairy wastewater cause high turbidities, which could block the incident light, a key factor in the microalgae cultivation process using wastewaters as culture media. In this study, screened liquid dairy manure was used as the microalgae Chlorella vulgaris culture media. The aim was to optimize the dilution folds for the best growth of Chlorella vulgaris and nutrients' reduction with a special focus on phosphorus removal and recovery. Four folds of dilution, designated as 5*, 10*, 15*, 20*, were applied to the liquid dairy manure to alleviate hindrance of the high turbidity together with the high ammonium. Microalgal cultivation removed a significant amount of turbidity and major nutrients. For differently diluted liquid dairy manures, although the initial turbidities varied a lot, the final removal rates were not significantly different, falling in the range of 88.11-91.73%. Chemical oxygen demand (COD) in the 5-fold diluted liquid dairy manure dropped from 6700 to 1200mg/L, corresponding to a removal rate of 79.81%. For the 10-fold, 15-fold, and 20-fold diluted manures, Chlorella removed around 67-69% of the initial CODs. Total Kjeldahl nitrogen (TKN) was removed at rates ranging from 70.84 to 73.99% from the four differently diluted liquid dairy manures without significant differences. NH4-N was removed most efficiently by 88.92% from the 20-fold diluted liquid dairy manure, and the least at 68.65% from the 5-fold diluted one. Although the original total phosphorus (TP) concentrations were distinctive for each group, the TP removal rates stayed in the range of 52.16 to 65.22%. Scanning electron microscopy (SEM) and energy-dispersive spectrometry (EDS) analysis of the precipitates harvested from the microalgal cultivation suggested possible phosphate precipitate forms. The chelation of Ca or Mg cations by dissolved organic matter (DOM) under alkaline conditions caused by microalgae cultivation could explain the unsatisfactory phosphorus removals observed in this study.

Effects of alginic acid on phosphorus and potassium absorption in radish under cadmium contamination

In order to study the effects of alginic acid at different dilution folds on phosphorus and potassium absorption of radish under cadmium contamination, foliar application of radish seedlings was carried out using alginic acid solutions with different dilution folds. The results showed that the alginic acid solution at 600, 900, 1200, and 1500 dilution folds increased the potassium content of radish, and increased the content of available phosphorus and available potassium in the soil. The solution of lower dilution folds (600 and 900) increased phosphorus content of radish, while the solution with higher dilution folds (1200 and 1500) only increased phosphorus content in radish roots and root tubers. It showed that foliar application of alginic acid could alleviate the harm of cadmium stress to radish to a certain extent, and increase the absorption of phosphorus and potassium nutrients in radish. The best effect was achieved when the dilution fold of alginic acid solution was 600.

Effects of alginic acid on radish growth and osmotic adjustment substance content under cadmium stress

To study the effects of alginic acid on the growth of vegetables, a pot experiment was conducted to study the effects of spraying alginic acid at different dilution folds on the growth and osmotic adjustment substance content of radish under cadmium stress condition. The results showed that the biomass, leaf length and leaf width of radish with 600, 900, 1200 and 1500 dilution folds alginic acid were higher than those of the control, while the soluble protein content, relative conductivity and malondialdehyde content of the radish were lower than those of the control. When the dilution factor of alginic acid is 600 dilution folds, the biomass, leaf length and leaf width of radish’s roots, root tubers, leaves and edible parts all reach the maximum. Increased by 19.39%, 35.27%, 19.74%, 29.44%, 22.11% and 14.01% compared with the control, respectively; the soluble protein content, relative conductivity and MDA content of radish reached the lowest, which were 38.76%, 29.86% and 35.61% lower than the control, respectively. Sprayed diluted alginic acid can promote the growth of radish, and the best dilution factor is 600 dilution folds.

Two novel protein chips for the detection of antibodies against porcine parvovirus

BackgroundPPV is one of the most important pathogens causing porcine reproductive disorder. It has been shown in clinical cases to be a commonly mixed infection with other important swine diseases which can aggravate the severity of the disease and bring serious economic losses to the pig industry. Serological methods, such as hemagglutination inhibition assays (HAI), serum neutralization (SN), and the modified direct complement-fixation (MDCF) test were utilized earlier, whereas the enzyme-linked immunosorbent assay (ELISA) is the most frequently applied assay to detect PPV-specific antibodies.ResultsWe establish the visible protein chip and the cyanine dye 3 (Cy3)-labeled protein chip to detect the clinical serum from pigs. In this study, the recombinant protein VP2 of PPV was expressed in E.coli, purified with nickel magnetic beads, and then printed onto epoxy-coated glass slides for preparation of the protein chip. After a series of experiments, the conditions of antigen protein concentration, incubation time of primary antibody or secondary antibody, and optimal serum dilution fold were optimized, resulting in a successful visible protein chip and Cy3-labeled protein chip. The results showed that the positive serum, diluted up to 6000-fold, can be detected by the visible protein chip, and the positive serum, diluted up to 12,800-fold, can be detected by the Cy3-labeled protein chip, suggesting the high sensitivity of these protein chips. Moreover, the positive detection ratio, sensitivity, and specificity of these two kinds of protein chips were higher than those of commercial ELISA antibody detection kits.ConclusionOverall, these two protein chips can be used to rapidly diagnose clinical samples with high throughput.

A direct PCR–based SNP marker–assisted selection system (D-MAS) for different crops

As more and more re-sequencing genome data in crops were released, SNPs (single nucleotide polymorphisms) are easily achieved for genotyping and show the highest abundance among all kinds of molecular markers. However, high-throughput SNP genotyping methods, such as KASP (Kompetitive Allele Specific PCR), TaqMan, or ARMS (amplification refractory mutation system), are always labor on DNA extraction and depending on expensive equipment. Therefore, most breeders are undergoing the bottleneck of lacking an easy, cost-saving, and stable genotyping system. Here, we report a direct PCR–based medium-throughput SNP marker–assisted selection (D-MAS) system suits available major crops including rice, wheat, maize, and rapeseed. The D-MAS system, which reduces the time of manual operation and result analysis dramatically, contains (1) seedling breeding in greenhouse; (2) high-throughput DNA extraction by alkaline lysis; and (3) gel-free SNP marker detection with 384-well by PARMS (penta-primer amplification refractory mutation) or KASP genotyping system. The stability of alkaline lysis DNA was validated by flexible dilution fold and long storing time under low temperatures. The alkaline lysis DNA from four F2 populations of rice, wheat, maize, and rapeseed showed equal efficiency in SNP calling relative to the ones from cetyl trimethylammonium bromide (CTAB) method. With the alkaline lysis DNA, PAMRS showed denser genotype cluster than KASP. Furthermore, the D-MAS system was adaptive with rice old leaves. The throughput and efficiency of the D-MAS system were validated in the hybrid rice seed purity test with a Xian-Geng-specific SNP marker which is also recommended to select ipa1-2d gene in the rice molecular breeding. In conclusion, we proposed a direct PCR–based SNP calling pipeline, which could be a simple, cheap, and robust standard operation procedure (SOP) of molecular breeding for different crops and get extensive use in most laboratory.

Determination of major and minor elements in Mexican red wines by microwave-induced plasma optical emission spectrometry, evaluating different calibration methods and exploring potential of the obtained data in the assessment of wine provenance

In this work, the determination of major and trace elements in Mexican wines was carried out by microwave-induced plasma optical emission spectrometry (MIP OES), setting sample dilution as the pretreatment procedure. The elements were grouped according with the required dilution fold: 1:100 for K; 1:50 for Na, Mg, Ca; 1:10 for Rb, Sr, Mn, Fe, Al and 1:4 for Cu, Cr. Due to troublesome background noise, different for each sample, matrix-matching with individual wines was necessary and three quantification methods were explored: classic standard additions (SA), standard dilution analysis (SDA) and multi-energy calibration (MEC). In order to avoid extrapolation in assessing the actual concentration of internal standard at each dilution point and to allow for longer integration time during signal acquisition, SDA was modified (nominated MSDA) and was recommended for the determination of minor elements whereas for major elements the suitable method was MEC. The results obtained by MSDA and MEC were in good agreement with those provided by SA; however, background correction was not required and both methods offer more simplicity as compared to SA. The concentration ranges found in the Mexican wines were as follows: 866–4896 mg L−1 K, 32.7–326 mg L−1 Na, 56.6–164 mg L−1 Mg, 63.9–121 mg L−1 Ca, 0.56–1.90 mg L−1 Sr, 0.35–6.31 mg L−1 Rb, 0.21–2.11 mg L−1 Fe, 0.69–1.72 mg L−1 Mn, 0.28–0.78 mg L−1 Al, 0.10–0.42 mg L−1 Cu, and Cr was not detected. Principal component analysis (PCA) allowed for separation of wines according with the winery regions: Valle de Guadalupe, Guanajuato, Aguascalientes. Furthermore, PCA revealed association of Rb, K, Ca, Sr with wines produced in Guanajuato and association of Na, Cu and Mn with those produced in Valle de Guadalupe; wines from Aguascalientes contained relatively high concentration of Fe and Al, although tight clustering of variables was not observed in this case. To establish reliable discrimination model, more samples from these and possibly from other winery regions in Mexico need to be analyzed.

Formulation and In-vitro Characterization of Self Nano-emulsifying Drug Delivery System (SNEDDS) for enhanced Solubility of Candesartan Cilexetil

Candesartan cilexetil is the prodrug of candesartan, a selective angiotensin II receptor antagonist, used in the management of hypertension and heart failure. It is characterized by poor aqueous solubility resulting in low bioavailability. Self-nanoemulsifying drug delivery systems (SNEDDS) loaded with candesartan cilexetil were successfully developed to overcome such obstacles. Preliminary screening was carried out to select proper oil, surfactant and co-surfactant combination for successful SNEDDS formulation. Ternary and pseudo ternary diagrams were constructed to optimize the system. Capryol 90® as an oil showed high emulsification ability. The nature of obtained dispersions was found to be nanoemulsions. Six formulations were visually inspected and evaluated for stability, robustness to dilution, effect of drug loading on the nano-emulsion boundary, viscosity, content uniformity, their mean droplet size, polydispersity index (PDI), zeta potential in addition to in vitro drug release study. Three formulations showed a very short emulsification time of less than 3minutes. The emulsification efficiency was significantly superior at pH6.8. All selected formulations showed mean droplet size (124.25 to 460.15nm), very low PDI values (0.311–0.562) and zeta potential (30.5–44.45 mV). Formulation F16 (10% w/w Capryol 90®, 30% w/w Tween80, 60% w/w Transcutol®) was selected for further characterization. Droplet size of F16 showed robustness to different dilution folds with different media. These results indicate that, SNEDDS could be promising delivery systems with a rapid onset of action and prolonged therapeutic effect of candesartan cilexetil.

Development and in vitro/in vivo performance of self-nanoemulsifying drug delivery systems loaded with candesartan cilexetil

Candesartan cilexetil is widely used in the management of hypertension and heart failure. The drug delivery encounters obstacles of poor aqueous solubility, efflux by intestinal P-glycoprotein and vulnerability to enzymatic degradation in small intestine. Self-nanoemulsifying drug delivery systems (SNEDDS) loaded with candesartan cilexetil were successfully developed to overcome such obstacles. Preliminary screening was carried out to select proper surfactant, co-surfactant and oil combination for successful SNEDDS formulation. All screened excipients were reported for their P-glycoprotein and cytochrome P450 3A4 (CYP3A4) modulation activity. Ternary and pseudo ternary diagrams were constructed to optimize the system. Peppermint oil and clove oil showed a high emulsification ability. The nature of obtained dispersions was identified to be nanoemulsions. Twenty-four formulations were evaluated for stability, robustness to dilution and self-emulsification efficiency. All formulations showed a very short emulsification time of <2min. The emulsification efficiency was significantly superior at pH6.8, at which the largest self-emulsifying region was also observed. Eight formulations were selected for further characterization according to cloud point measurement; mean droplet size, poly dispersity index (PDI) and zeta potential determination in addition to in vitro drug release study. All selected formulations showed very high cloud points (70–90°C), ultrafine mean droplet size (12±1.4 to 24.5±2.13nm), very low PDI values (0.015–0.1305) and almost a complete drug release after 12h. Formulation F15 (Peppermint oil 55% w/w: Cremophor RH40 25% w/w: Labrasol 20% w/w) was selected for further characterization. Its droplet size showed robustness to different dilution folds with different media and its TEM photograph showed spherical particles without any apparent aggregation even after 24h. Formulation F15 successfully controlled the systolic blood pressure of hypertensive rats for 24h with the maximum effect was observed after 2h. These results indicate that, SNEDDS could be promising delivery systems with a rapid onset of action and prolonged therapeutic effect of candesartan cilexetil.