Design of Dyes Based on the Quinoline or Quinoxaline Skeleton towards Visible Light Photoinitiators.

Dyes based on quinoline and quinoxaline skeletons were designed for application as visible light photoinitiators. The obtained compounds absorb electromagnetic radiation on the border between ultraviolet and visible light, which allows the use of dental lamps as light sources during the initiation of the photopolymerization reaction. Their another desirable feature is the ability to create a long-lived excited state, which enables the chain reaction to proceed through the mechanism of intermolecular electron transfer. In two-component photoinitiating systems, in the presence of an electron donor or a hydrogen atom donor, the synthesized compounds show excellent abilities to photoinitiate the polymerization of acrylates. In control tests, the efficiency of photopolymerization using modified quinoline and quinoxaline derivatives is comparable to that obtained using a typical, commercial photoinitiator for dentistry, camphorquinone. Moreover, the use of the tested compounds requires a small amount of photoinitiator (only 0.04% by weight) to initiate the reaction. The research also showed a significant acceleration of the photopolymerization process and shortening of the reaction time. In practice, this means that the new two-component initiating systems can be used in much lower concentrations without slowing down the speed of obtaining polymer materials. It is worth emphasizing that these two features of the new initiating system allow for cost reduction by reducing financial outlays on both materials (photoinitiators) and electricity.

amer1 Regulates Zebrafish Craniofacial Development by Interacting with the Wnt/ß-Catenin Pathway.

Microtia-atresia is a rare type of congenital craniofacial malformation causing severe damage to the appearance and hearing ability of affected individuals. The genetic factors associated with microtia-atresia have not yet been determined. The AMER1 gene has been identified as potentially pathogenic for microtia-atresia in two twin families. An amer1 mosaic knockdown zebrafish model was constructed using CRISPR/Cas9. The phenotype and the development process of cranial neural crest cells of the knockdown zebrafish were examined. Components of the Wnt/ß-catenin pathway were examined by qPCR, Western blotting, and immunofluorescence assay. IWR-1-endo, a reversible inhibitor of the Wnt/ß-catenin pathway, was applied to rescue the abnormal phenotype. The present study showed that the development of mandibular cartilage in zebrafish was severely compromised by amer1 knockdown using CRISPR/Cas9. Specifically, amer1 knockdown was found to affect the proliferation and apoptosis of cranial neural crest cells, as well as their differentiation to chondrocytes. Mechanistically, amer1 exerted an antagonistic effect on the Wnt/ß-catenin pathway. The application of IWR-1-endo could partially rescue the abnormal phenotype. We demonstrated that amer1 was essential for the craniofacial development of zebrafish by interacting with the Wnt/ß-catenin pathway. These findings provide important insight into the role of amer1 in zebrafish mandibular development and the pathology of microtia-atresia caused by AMER1 gene mutations in humans.

Efficacy of montelukast for adenoid hypertrophy in paediatrics: A systematic review and meta-analysis.

INTRODUCTION: Leukotrienes play a significant role in the pathogenesis of adenoid hypertrophy (A.H.). Therefore, we aimed to analyse the role of montelukast, a leukotriene receptor antagonist, alone or in combination with mometasone, a potent local intranasal steroid, for the treatment of A.H. METHODS: Participants were children with A.H. were treated with montelukast alone or montelukast and mometasone furoate. The main outcome measures were effect of montelukast on clinical symptoms of A.H. A literature review was conducted using online search engines, Cochrane Library, PubMed, Web of Science and Scopus, for randomized clinical trials assessing children with A.H. treated with montelukast alone or montelukast and mometasone furoate. Seven randomized clinical trials (RCTs) were included with 742 children. RESULTS: Our study reveals that montelukast alone or in combination with intranasal mometasone furoate significantly improves clinical symptoms of adenoid hypertrophy such as snoring, sleeping disturbance, mouth breathing and A/N ratio. Montelukast was superior to placebo in decreasing snoring (SMD = -1.00, 95% CI [-1.52, -0.49]), sleep discomfort (SMD = -1.26, 95% CI [-1.60, -0.93]), A/N ratio (MD = -0.11, 95% CI [-0.14, -0.09]) and mouth breathing (SMD = -1.36, 95% CI [-1.70, -1.02]). No difference was detected between montelukast and mometasone versus mometasone alone in snoring (SMD = -0.21, 95%CI [-0.69, 0.27]); however, the combination group was superior to the mometasone alone in mouth breathing (SMD = -0.46, 95% CI [-0.73, -0.19]). CONCLUSIONS: The limitation of studies included a small sample size, with an overall low to medium quality. Thus, further larger, higher-quality RCTs are recommended to provide more substantial evidence.

Cyanine Masking: A Strategy to Test Functional Group Effects on Antibody Conjugate Targeting.

Conjugates of small molecules and antibodies are broadly employed diagnostic and therapeutic agents. Appending a small molecule to an antibody often significantly impacts the properties of the resulting conjugate. Here, we detail a systematic study investigating the effect of various functional groups on the properties of antibody-fluorophore conjugates. This was done through the preparation and analysis of a series of masked heptamethine cyanines (CyMasks)-bearing amides with varied functional groups. These were designed to exhibit a broad range of physical properties, and include hydrophobic (-NMe2), pegylated (NH-PEG-8 or NH-PEG-24), cationic (NH-(CH2)2NMe3+), anionic (NH-(CH2)2SO3-), and zwitterionic (N-(CH2)2NMe3+)-(CH2)3SO3-) variants. The CyMask series was appended to monoclonal antibodies (mAbs) and analyzed for the effects on tumor targeting, clearance, and non-specific organ uptake. Among the series, zwitterionic and pegylated dye conjugates had the highest tumor-to-background ratio (TBR) and a low liver-to-background ratio. By contrast, the cationic and zwitterionic probes had high tumor signal and high TBR, although the latter also exhibited an elevated liver-to-background ratio (LBR). Overall, these studies provide a strategy to test the functional group effects and suggest that zwitterionic substituents possess an optimal combination of high tumor signal, TBR, and low LBR. These results suggest an appealing strategy to mask hydrophobic payloads, with the potential to improve the properties of bioconjugates in vivo.

Encapsulation of Trimethine Cyanine in Cucurbit[8]uril: Solution versus Solid-State Inclusion Behavior.

Inclusion of polymethine cyanine dyes in the cavity of macrocyclic receptors is an effective strategy to alter their absorption and emission behavior in aqueous solution. In this paper, the effect of the host-guest interaction between cucurbit[8]uril (CB[8]) and a model trimethine indocyanine (Cy3) on dye spectral properties and aggregation in water is investigated. Solution studies, performed by a combination of spectroscopic and calorimetric techniques, indicate that the addition of CB[8] disrupts Cy3 aggregates, leading to the formation of a 1 : 1 host-guest complex with an association constant of 1.5×106  M-1 . At concentrations suitable for NMR experiments, the slow formation of a supramolecular polymer was observed, followed by precipitation. Single crystals X-ray structure elucidation confirmed the formation of a polymer with 1 : 1 stoichiometry in the solid state.

One-step SYBR green-based real-time RT-PCR assay for detection of foot-and-mouth disease virus circulating in India.

Rapid, sensitive, and reliable laboratory detection of foot-and-mouth disease virus (FMDV) infection is essential for containing and controlling virus infection in any geographical area. In this report a SYBR green-based 3Dpol-specific one-step real-time RT-PCR (rRT-PCR) assay was developed for the pan-serotype detection of FMDV in India. The detection limit of the SYBR green-based rRT-PCR was 10-2 TCID50/50 µl, which is 10 times more sensitive than the traditional agarose gel electrophoresis-based RT-multiplex PCR (RT-mPCR). The standard curve exhibited a linear range across 8-log10 units of viral RNA dilution. The reproducibility and specificity of this assay were reasonably high suggesting that the 3Dpol-specific SYBR green rRT-PCR could detect FMDV genome specifically and with little run-to-run variation. The new 3Dpol-specific SYBR green rRT-PCR assay was evaluated alongside the established RT-mPCR using the archived FMDV isolates and clinical field samples from suspected FMD outbreaks. A perfect concordance was observed between the new rRT-PCR and the traditional RT-mPCR on viral RNA in the archived FMDV cell culture isolates tested. Furthermore, 73% of FMDV-suspected clinical samples were detected positive through the 3Dpol-specific SYBR green rRT-PCR, while the detection rate through the traditional RT-mPCR was 57%. Therefore, the SYBR green-based 3Dpol-specific one-step rRT-PCR could be considered as a valuable assay with higher diagnostic sensitivity to complement the routine assays that are being used for FMD virus diagnosis in India.

What Is the Effect of Pre-Emptive Oral Montelukast on Postoperative Pain Following Bimaxillary Orthognathic Surgery? A Triple-Blind Randomized Clinical Trial.

PURPOSE: The aim of this study was to assess the effect of preoperative administration of oral montelukast on the amount of postoperative pain following bimaxillary orthognathic surgery. METHODS AND MATERIALS: All healthy skeletal class III deformity candidates for bimaxillary orthognathic surgery were included in this triple-blind randomized clinical trial. The subjects were randomly divided into placebo and montelukast groups. One hour before the surgery, a 10 mL of apple juice was given to each and every patient; however, a 10 mg tablet of montelukast was dissolved in the juice for the intervention group. All operations were performed by the same surgical team, under the same general anesthesia protocols. The outcome variable was the amount of postoperative pain (1-, 3-, 6-, 12-, 18-, and 24-hour intervals) which was measured during the first 24 hours using a Visual Analog Scale. For statistical analysis, the significance level was set at 0.05 using SPSS 23. RESULTS: A total of 60 consecutive patients, comprising 31 females (51.7%) and 29 males (48.3%) with an average age of 25.2 ± 2.2 were recruited. The average surgical duration was 193 ± 28.0 minutes. In general, pain intensity exhibited an increasing trend from the first hour postoperatively, reaching its peak in the 12th hour and decreasing thereafter. Nevertheless, the average amount of pain was significantly higher in the placebo group compared with the montelukast group, in all the studied time intervals (P < .05). The number of patients who required postoperative opioid analgesics was significantly higher in the placebo group compared to the montelukast group (P = .024). Moreover, the duration of surgery had a direct and significant effect on the postoperative pain intensity (P < .001). CONCLUSIONS: It might be concluded that preoperative administration of montelukast is effective in reducing postoperative pain following bimaxillary orthognathic surgery. Further studies are necessary for more relevancy.

Application of Green Chiral Chromatography in Enantioseparation of Newly Synthesized Racemic Marinoepoxides.

Enantioseparation of the newly synthesized series of novel quinoline-2(1H)-one epoxide structures rac-6a-c and rac-8a-c, named marinoepoxides, is described. Marinoepoxide rac-6a, the key intermediate in the total synthesis of natural products marinoaziridines A and B, as well as their structural analogues, was synthesized by addition of the achiral ylide generated in situ from the sulfonium salt 5 or 7, to the carbon-oxygen double bond of the corresponding quinoline-2(1H)-one-4-carbaldehyde 4a-c in good yield. Separation of enantiomers of (±)-2,3,3-trisubstituted marinoepoxides rac-6a-c and (±)-trans-2,3-disubstituted marinoepoxides rac-8a-c was studied using two immobilized polysaccharide type chiral stationary phases (CSPs); tris-(3,5-dichlorophenylcarbamoyl)cellulose stationary phase (CHIRAL ART Cellulose-SC) and tris-(3,5-dimethylphenylcarbamoyl)amylose stationary phase (CHIRAL ART Amylose-SA). Enantioseparation conditions were explored by high-performance liquid chromatography (HPLC) using dimethyl carbonate/alcohol mixtures and n-hexane/ethanol (80/20, v/v) as mobile phase, and by supercritical fluid chromatography (SFC) using CO2/alcohol mixtures as mobile phase. In all examined racemates, enantioseparation was successfully achieved, but its efficiency largely depended on the structure of chiral selector and type/composition of the mobile phase.

Parasitological and immunological evaluation of a quinoline derivative salt incorporated into a polymeric micelle formulation against Leishmania infantum infection.

Leishmaniasis is a parasitic disease caused by Leishmania protozoa, which presents a large spectrum of clinical manifestations. In the present study, a quinoline derivative salt named N-(2-((7-chloroquinolin-4-yl)amino)ethyl)-N-(prop-2-yn-1-yl)prop-2-yn-1-aminium chloride or QDS3 was in vitro and in vivo tested against L. infantum by means of its incorporation in Poloxamer 407-based polymeric micelles (QDS3/M). The in vitro antileishmanial activity of QDS3 and QDS3/M was investigated in L. infantum promastigotes, axenic amastigotes and infected macrophages. BALB/c mice were infected with L. infantum, and parasitological parameters were evaluated 1 and 15 days post-treatment by determining the parasite load by a limiting dilution assay, besides a quantitative PCR (qPCR) method. Immunological response was assessed based on production of cellular cytokines, as well as by quantification of nitrite levels and specific antibodies. In vitro results showed that QDS3 free or in micelles presented effective antileishmanial action against both parasite stages, being more effective in amastigotes. In vivo data showed that treatment using QDS3 or QDS3/M reduced the parasite load in the livers, spleens, draining lymph nodes (dLN) and bone marrows of the treated animals, 1 and 15 days after treatment, when compared to values found in the control groups. Additionally, treated mice developed a polarized Th1-type immune response, with higher levels of IL-12, IFN-γ, GM-CSF and nitrite, besides high production of specific IgG2a antibodies, when compared to the controls. Parasitological and immunological data obtained using the micellar composition were better than the others. In conclusion, QDS3, mainly when applied in a delivery adjuvant system, could be considered for future studies as therapeutic candidate against VL.

Transition-Metal-Free Synthesis of Functionalized Quinolines by Direct Conversion of ß-O-4 Model Compounds.

Direct production of heterocyclic aromatic compounds from lignin ß-O-4 models remains a huge challenge due to the incompatible catalysis for aryl ether bonds cleavage and heterocyclic ring formation. Herein, the first example of quinoline synthesis from ß-O-4 model compounds by a one-pot cascade reaction is reported in yields up to 89 %. The reaction pathway involves selective cleavage of C-O bonds, dehydrogenation, aldol condensation, C-N bond formation along with heterocyclic aromatic ring construction. The control experiments suggest that both imine and chalcone were identified as the key intermediates, and the rate determining step as well as the preferred pathway were experimentally clarified and supported by density functional theory (DFT) calculations. Based on this protocol, the conversion of ß-O-4 polymer delivered 56 wt % yield of quinoline derivative in three steps. This transformation provides a potential petroleum-independent choice for heterocyclic aromatic chemicals.

New copolyhydrazides with anthrazoline fragments in the main chain: synthesis and optical properties.

Several new 2,8-diphenylpyrido[3,2-g]quinoline-4,6-dicarbohydrazides were synthesized and copolyhydrazides based on them were obtained. Molecular weight, thermal, stress-strain and optical properties were investigated. It was shown that all polymer films exhibit significant luminescence in the 450-650 nm region, the profile and intensity of which depended on the nature and position of substituents in the phenylene fragment.

Sterilizing Immunity against SARS-CoV-2 Infection in Mice by a Single-Shot and Lipid Amphiphile Imidazoquinoline TLR7/8 Agonist-Adjuvanted Recombinant Spike Protein Vaccine*.

The search for vaccines that protect from severe morbidity and mortality because of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 (COVID-19) is a race against the clock and the virus. Here we describe an amphiphilic imidazoquinoline (IMDQ-PEG-CHOL) TLR7/8 adjuvant, consisting of an imidazoquinoline conjugated to the chain end of a cholesterol-poly(ethylene glycol) macromolecular amphiphile. It is water-soluble and exhibits massive translocation to lymph nodes upon local administration through binding to albumin, affording localized innate immune activation and reduction in systemic inflammation. The adjuvanticity of IMDQ-PEG-CHOL was validated in a licensed vaccine setting (quadrivalent influenza vaccine) and an experimental trimeric recombinant SARS-CoV-2 spike protein vaccine, showing robust IgG2a and IgG1 antibody titers in mice that could neutralize viral infection in vitro and in vivo in a mouse model.

Development and in vitro evaluation of effervescent floating matrix tablet of neritinib: An anticancer drug.

Neratinib is a potent anticancer drug, used for the treatment of breast cancer. It is poorly soluble at higher pH, which tends to minimize the therapeutic effects in the lower GIT leads to its poor bioavailability. An attempt has been made to prepare and develop a novel gastro-retentive system of neratinib to improve the drug bioavailability in the GIT by enhancing the gastric retention time. The floating matrix tablets were prepared by various proportions of carbopol 940, micro-crystalline cellulose (MCC) and ethyl cellulose (EC), sodium bicarbonate (NaHCO3) as gas forming agent, by direct compression. The formulation mixture was assessed for pre and post compression test, lag time, in-vitro floating, FTIR, water uptake/swelling index, in vitro and kinetic release studies. The findings revealed that, the parameters of compression (pre and post) were within USP limits. The floating tablets swelled well and floated for more than 24h, with less than 120 seconds of buoyancy lag time. The optimized formulation F3 showed sustained release up to 12h; a non-Fickian mechanism. Therefore, all the results and findings have shown that developed neratinib floating matrix system is a promising approach as a drug delivery system and application in the treatment of breast cancer.

Potent and Prolonged Innate Immune Activation by Enzyme-Responsive Imidazoquinoline TLR7/8 Agonist Prodrug Vesicles.

Synthetic immune-stimulatory drugs such as agonists of the Toll-like receptors (TLR) 7/8 are potent activators of antigen-presenting cells (APCs), however, they also induce severe side effects due to leakage from the site of injection into systemic circulation. Here, we report on the design and synthesis of an amphiphilic polymer-prodrug conjugate of an imidazoquinoline TLR7/8 agonist that in aqueous medium forms vesicular structures of 200 nm. The conjugate contains an endosomal enzyme-responsive linker enabling degradation of the vesicles and release of the TLR7/8 agonist in native form after endocytosis, which results in high in vitro TLR agonist activity. In a mouse model, locally administered vesicles provoke significantly more potent and long-lasting immune stimulation in terms of interferon expression at the injection site and in draining lymphoid tissue compared to a nonamphiphilic control and the native TLR agonist. Moreover, the vesicles induce robust activation of dendritic cells in the draining lymph node in vivo.

Patients with adenoid cystic carcinomas of the salivary glands treated with lenvatinib: Activity and quality of life.

BACKGROUND: The treatment of patients with recurrent and/or metastatic (R/M) salivary gland adenoid cystic carcinoma (ACC) remains an unmet need. METHODS: Patients with R/M disease with a history of clinical or symptomatic disease progression within 6 months and a maximum of 1 previous line of chemotherapy or a multiple kinase inhibitor received oral lenvatinib at a dose of 24 mg/day. The primary endpoint was the objective response rate; secondary endpoints included quality of life (QOL) (according to the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire-Core 30 Items [EORTC QLQ-C30] and the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire-Core Module Head and Neck Module [EORTC QLQ-H&N35]), progression-free survival and overall survival, duration of response, and toxicities. RESULTS: Twenty-eight patients with R/M ACC were enrolled. Among 26 evaluable patients, 3 partial responses (11.5%) were reported. Target lesion reductions between 23% to 28% were observed in 4 of 20 patients with stable disease. Treatment-related adverse events were frequent (all grades, 96%; grade≥3 in 50% of cases according to version 4.03 of the National Cancer Institute Common Terminology Criteria for Adverse Events). The dose of lenvatinib was reduced in 24 patients, whereas in 21 patients the dose was reduced within the first 12 weeks and 4 patients maintained the full dose throughout treatment. The QOL deteriorated between baseline and 6 months with regard to Fatigue and Dry Mouth. There was no evidence of changes in Swallowing and Physical Functioning. At a median follow-up of 29 months, 2 patients remained on treatment, 10 patients were off protocol for disease progression and were alive with disease, and 14 patients had died of disease progression. The median overall survival, progression-free survival, and duration of response were 27 months, 9.1 months, and 3.1 months, respectively. CONCLUSIONS: Lenvatinib appears to have modest activity in ACC. Toxicities are common but manageable and QOL was found to deteriorate in some domains.

Deep Imaging Analysis in VISUAL Reveals the Role of YABBY Genes in Vascular Stem Cell Fate Determination.

Stem cells undergo cell division and differentiation to ensure organized tissue development. Because plant cells are immobile, plant stem cells ought to decide their cell fate prior to differentiation, to locate specialized cells in the correct position. In this study, based on a chemical screen, we isolated a novel secondary cell wall indicator BF-170, which binds to lignin and can be used to image in vitro and in situ xylem development. Use of BF-170 to observe the vascular differentiation pattern in the in vitro vascular cell induction system, VISUAL, revealed that adaxial mesophyll cells of cotyledons predominantly generate ectopic xylem cells. Moreover, phloem cells are abundantly produced on the abaxial layer, suggesting the involvement of leaf adaxial-abaxial polarity in determining vascular cell fate. Analysis of abaxial polarity mutants highlighted the role of YAB3, an abaxial cell fate regulator, in suppressing xylem and promoting phloem differentiation on the abaxial domains in VISUAL. Furthermore, YABBY family genes affected in vivo vascular development during the secondary growth. Our results denoted the possibility that such mediators of spatial information contribute to correctly determine the cell fate of vascular stem cells, to conserve the vascular pattern of land plants.

In vivo delivery of an exogenous molecule into murine T lymphocytes using a lymphatic drug delivery system combined with sonoporation.

Physical delivery of exogenous molecules into lymphocytes is extremely challenging because conventional methods have notable limitations. Here, we evaluated the potential use of acoustic liposomes (ALs) and sonoporation to deliver exogenous molecules into lymphocytes within a lymph node (LN). MXH10/Mo-lpr/lpr (MXH10/Mo/lpr) mice, which show systemic LN swelling, were used as the model system. After direct injection into the subiliac LN, a solution containing both ALs and TOTO-3 fluorophores (molecular weight: 1355) was able to reach the downstream proper axillary LN (PALN) via the lymphatic vessels (LVs). This led to the accumulation of a high concentration of TOTO-3 fluorophores and ALs in the lymphatic sinuses of the PALN, where a large number of lymphocytes were densely packed. Exposure of the PALN to >1.93 W/cm2 of 970-kHz ultrasound allowed the solution to extravasate into the parenchyma and reach the large number of lymphocytes in the sinuses. Flow cytometric analysis showed that TOTO-3 molecules were delivered into 0.49 ± 0.23% of CD8+7AAD- cytotoxic T lymphocytes. Furthermore, there was no evidence of tissue damage. Thus, direct administration of drugs into LVs combined with sonoporation can improve the delivery of exogenous molecules into primary lymphocytes. This technique could become a novel approach to immunotherapy.

Fluorescent contact lens for continuous non-invasive measurements of sodium and chloride ion concentrations in tears.

Rapid and non-invasive measurement of hydration status is medically important because even mild levels of dehydration can have a significant impact on physical and cognitive performance. Despite the potential value of determining whole-body hydration based on the electrolytes found in tears, very few tests are available. An area of intense interest is the development of a contact lens which could measure ion concentrations in tears, specifically that of sodium (Na+) and chloride (Cl-) ions, the dominant electrolytes in blood plasma and tears. Here, we describe a method to make fluorescent contact lenses which allow determination of Na+ and Cl- ion concentrations in tears. Fluorophores known to be sensitive to Na+ and Cl- were derivatized to bind non-covalently to two commercially-available silicone hydrogel (SiHG) contact lenses-the Biofinity (Comfilcon A) or MyDay (Stenfilcon A) lenses. The sodium- and chloride-sensitive fluorophores displayed spectral changes in the physiological range for Na+ and Cl- ions in tears. The lenses for both Na+ and Cl- ions were completely reversible. The sodium responses were not sensitive to protein interference including human lysozyme, human serum albumin and mucin type 2. The chloride sensitivity was similar with both lenses, but the sodium-sensitive range was different in the Biofinity and MyDay lenses. We also fabricated a lens with both the Na+ and Cl- probes in a single MyDay lens resulting in a contact lens that independently measured Na+ and Cl- concentrations without physical separation of the fluorophores. Our findings indicated that a sodium and chloride-sensitive contact lens (NaCl-lens) could be used for rapid non-invasive detection of whole-body hydration, as well as associated diseases or other infections.

The extended recovery ring-stage survival assay provides a superior association with patient clearance half-life and increases throughput.

BACKGROUND: Tracking and understanding artemisinin resistance is key for preventing global setbacks in malaria eradication efforts. The ring-stage survival assay (RSA) is the current gold standard for in vitro artemisinin resistance phenotyping. However, the RSA has several drawbacks: it is relatively low throughput, has high variance due to microscopy readout, and correlates poorly with the current benchmark for in vivo resistance, patient clearance half-life post-artemisinin treatment. Here a modified RSA is presented, the extended Recovery Ring-stage Survival Assay (eRRSA), using 15 cloned patient isolates from Southeast Asia with a range of patient clearance half-lives, including parasite isolates with and without kelch13 mutations. METHODS: Plasmodium falciparum cultures were synchronized with single layer Percoll during the schizont stage of the intraerythrocytic development cycle. Cultures were left to reinvade to early ring-stage and parasitaemia was quantified using flow cytometry. Cultures were diluted to 2% haematocrit and 0.5% parasitaemia in a 96-well plate to start the assay, allowing for increased throughput and decreased variability between biological replicates. Parasites were treated with 700 nM of dihydroartemisinin or 0.02% dimethyl sulfoxide (DMSO) for 6 h, washed three times in drug-free media, and incubated for 66 or 114 h, when samples were collected and frozen for PCR amplification. A SYBR Green-based quantitative PCR method was used to quantify the fold-change between treated and untreated samples. RESULTS: 15 cloned patient isolates from Southeast Asia with a range of patient clearance half-lives were assayed using the eRRSA. Due to the large number of pyknotic and dying parasites at 66 h post-exposure (72 h sample), parasites were grown for an additional cell cycle (114 h post-exposure, 120 h sample), which drastically improved correlation with patient clearance half-life compared to the 66 h post-exposure sample. A Spearman correlation of - 0.8393 between fold change and patient clearance half-life was identified in these 15 isolates from Southeast Asia, which is the strongest correlation reported to date. CONCLUSIONS: eRRSA drastically increases the efficiency and accuracy of in vitro artemisinin resistance phenotyping compared to the traditional RSA, which paves the way for extensive in vitro phenotyping of hundreds of artemisinin resistant parasites.

Development of a novel SYBR green I-based quantitative RT-PCR assay for Senecavirus A detection in clinical samples of pigs.

Porcine vesicular disease caused by Senecavirus A (SVA) is a newly emerging disease in many countries. Based on clinical signs only, it is very challenging to distinguish SVA infection from other similar diseases, such as foot and mouth disease, swine vesicular disease, and vesicular stomatitis. Therefore, it is crucial to establish a detection assay for the clinical diagnosis of SVA infection. In this study, a pair of specific primers were designed based on the highly conserved L/VP4 gene sequence of SVA. The established SYBR green I-based quantitative reverse transcription polymerase chain reaction (qRT-PCR) method was used to detect SVA nucleic acids in clinical samples. The limit of detection SVA nucleic acids by qRT-PCR was 6.4 × 101 copies/µL, which was significantly more sensitive than that by gel electrophoresis of 6.4 × 103 copes/µL. This assay was specific and had no cross-reaction with other seven swine viruses. Using SYBR green I-based qRT-PCR, the SVA positive rates in experimental animal samples and field samples were 67.60% (96/142) and 80% (24/30) respectively. The results demonstrate that SYBR green I-based qRT-PCR is a rapid and specific method for the clinical diagnosis and epidemiological investigation of related vesicular diseases caused by SVA.