Electrochemical aptasensor detection of electron transfer flavoprotein subunit beta for leptospirosis diagnosis.

Electron transfer flavoprotein subunit beta (ETFB) of Leptospira interrogans is a biomarker for diagnosing leptospiral infection. Thus, the ETFB-specific nuclease-resistant RNA aptamer ETFB3-63 was developed and used in an electrochemical aptasensor to assay ETFB. Although the majority of reported biosensors detect various genes and antibodies of L. interrogans, this is the first attempt to construct an electrochemical biosensor to detect ETFB protein for the diagnosis of leptospiral infection. The ETFB protein can be detected without any extraction phase. In this assay, a single-stranded DNA probe complementary to the ETFB3-63 sequence was immobilized on a screen-printed carbon electrode (SPCE). The aptamer was then incubated and hybridized with the antisense probe on the SPCE. In the presence of ETFB, the aptamer dissociates from the aptamer/probe complex on the SPCE to bind with the protein. Methylene blue was then added to intercalate with the remaining hybridized aptamers, and its signal was measured using differential pulse voltammetry. The signal arising from the intercalated methylene blue decreased with increasing concentration of ETFB, showing a linear response in the range of 50-500 nM of ETFB and 10 to 109 leptospira cells per mL, respectively. The aptasensor signal was also specific to L. interrogans but not to 12 related bacteria tested. In addition, the aptasensor showed similar performance in detecting ETFB spiked in human serum to that in buffer, indicating that proteins in the serum do not interfere with the assay. Therefore, this assay has great potential to develop into a point-of-care electrochemical device that is accurate, cost-effective, and user-friendly for leptospirosis diagnosis.

Study of siRNA Delivery via Polymeric Nanoparticles in Combination with Angiogenesis Inhibitor for The Treatment of AFP-Related Liver Cancer.

Angiogenesis inhibitor drugs have been explored as important pharmacological agents for cancer therapy, including hepatocellular carcinoma. These agents have several drawbacks, such as drug resistance, nonspecific toxicity, and systemic side effects. Therefore, combination therapy of the drug and small interfering RNA could be a promising option to achieve high therapeutic efficacy while allowing a lower systemic dose. Therefore, we studied adding an alpha-fetoprotein siRNA (AFP-siRNA) incorporated on polymeric nanoparticles (NPs) along with angiogenesis inhibitor drugs. The AFP siRNA-loaded NPs were successfully synthesized at an average size of 242.00 ± 2.54 nm. Combination treatment of AFP-siRNA NPs and a low dose of sunitinib produced a synergistic effect in decreasing cell viability in an in vitro hepatocellular carcinoma (HCC) model. AFP-siRNA NPs together with sorafenib or sunitinib greatly inhibited cell proliferation, showing only 39.29 ± 2.72 and 44.04 ± 3.05% cell viability, respectively. Moreover, quantitative reverse transcription PCR (qRT-PCR) demonstrated that AFP-siRNA incorporated with NPs could significantly silence AFP-mRNA expression compared to unloaded NPs. Interestingly, the expression level of AFP-mRNA was further decreased to 28.53 ± 5.10% when sunitinib was added. Therefore, this finding was considered a new promising candidate for HCC treatment in reducing cell proliferation and enhancing therapeutic outcomes.

The Ability of Nuclease-Resistant RNA Aptamer against Streptococcus suis Serotype 2, Strain P1/7 to Reduce Biofilm Formation In Vitro.

Streptococcus suis, a Gram-positive bacterium, is an important swine and human pathogen, with serotype 2 being the most prevalent strain found worldwide. Deafness, meningitis, and death (in severe cases) are observed in S. suis-infected cases. Development of the ligands that can bind to S. suis with high affinity and specificity could be beneficial for the diagnosis and treatment of S. suis infection. Herein, the nuclease-resistant RNA aptamers based on 2'-fluoropyrimidine modification against S. suis serotype 2, strain P1/7, were established using the cell- Systematic Evolution of Ligands by Exponential enrichment (SELEX) technique. One of the aptamers, R8-su12, could bind to the S. suis target strain as well as other S. suis serotypes, i.e., 1, 1/2, 9, and 14, but not to other bacteria tested, i.e., S. pneumoniae ATCC 49619, Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853. Moreover, the R8-su12 RNA aptamer was also capable of inhibiting the biofilm formation of the S. suis target strain, making it potentially useful for the study of biofilm formation and the treatment of S. suis infection in humans and pigs in the future.

Targeted Nanoparticles for the Binding of Injured Vascular Endothelium after Percutaneous Coronary Intervention.

Percutaneous coronary intervention (PCI) is a common procedure for the management of coronary artery obstruction. However, it usually causes vascular wall injury leading to restenosis that limits the long-term success of the PCI endeavor. The ultimate objective of this study was to develop the targeting nanoparticles (NPs) that were destined for the injured subendothelium and attract endothelial progenitor cells (EPCs) to the damaged location for endothelium regeneration. Biodegradable poly(lactic-co-glycolic acid) (PLGA) NPs were conjugated with double targeting moieties, which are glycoprotein Ib alpha chain (GPIbα) and human single-chain antibody variable fragment (HuscFv) specific to the cluster of differentiation 34 (CD34). GPIb is a platelet receptor that interacts with the von Willebrand factor (vWF), highly deposited on the damaged subendothelial surface, while CD34 is a surface marker of EPCs. A candidate anti-CD34 HuscFv was successfully constructed using a phage display biopanning technique. The HuscFv could be purified and showed binding affinity to the CD34-positive cells. The GPIb-conjugated NPs (GPIb-NPs) could target vWF and prevent platelet adherence to vWF in vitro. Furthermore, the HuscFv-conjugated NPs (HuscFv-NPs) could capture CD34-positive cells. The bispecific NPs have high potential to locate at the damaged subendothelial surface and capture EPCs for accelerating the vessel repair.

Biological properties of reverse ankyrin engineered for dimer construction to enhance HIV-1 capsid interaction.

BACKGROUND: Assembly and budding in the late-stage of human immunodeficiency virus type 1 (HIV-1) production rely on Gag protein polymerization at the inner leaflet of the plasma membrane. We previously generated a monomeric ankyrin repeat protein (Ank1D4) that specifically interacts with capsid protein (CAp24) of HIV-1, however this protein had modest binding affinity. OBJECTIVE: This study aimed to improve the avidity of Ank1D4 by generating two Ank1D4 dimers: (Ank1D4NC-NC) and its inverted form (Ank1D4NC-CN), with each domain connected by a flexible (G4S)4 linker peptide. METHODS: Binding properties of monomeric and dimeric Ank1D4 was performed by capture enzyme-linked immunosorbent assay (ELISA). Sandwich ELISA was used to examine bifunctional module of dimeric Ank1D4. Ank1D4NC-NC and Ank1D4NC-CN were evaluated using bio-layer interferometry (BLI), compared to monomeric Ank1D4. RESULTS: Similar binding surfaces were observed in both dimers which was comparable with monomeric Ank1D4. The interaction of Ank1D4NC-CN with CAp24 was significantly greater than that of Ank1D4NC-NC and Ank1D4 by capture ELISA. Ank1D4NC-CN also exhibited bifunctionality using a sandwich ELISA. The KD of Ank1D4NC-CN, Ank1D4NC-NC and monomeric Ank1D4 was 3.5 nM, 53.7 nM, and 126.2 nM, respectively using bio-layer interferometry analysis. CONCLUSIONS: This study provides a strategy for increasing Ank1D4 avidity through the construction of novel inverted dimers with a flexible linker. Ank1D4NC-CN may provide an alternative treatment strategy for inhibiting HIV-1 replication.

Systemic absorption of epinephrine compared between the intranasal and intramuscular routes of administration in healthy adults.

BACKGROUND: Epinephrine 5 mg administered via the intranasal (IN) route was shown to be bioequivalent to epinephrine 0.3 mg administered via the intramuscular (IM) route in our preliminary study. OBJECTIVE: To investigate the pharmacokinetics and pharmacodynamics of IN and IM epinephrine absorption in a larger group of healthy adults (n = 12). METHODS: Each subject was administered IN saline, IN epinephrine (5 mg), and IM epinephrine (0.3 mg) on 3 separate days. Plasma epinephrine levels were determined using liquid chromatography-tandem mass spectrometry. RESULTS: IN epinephrine administration showed significant systemic absorption compared to IN saline control with the areas under the curve (AUC0-180 min) of 4.4 (4.9) ± 4.0 and 0.2 (0.5) ± 0.3 ng.min/mL, respectively; the values are mean (median) ± standard deviation. IN epinephrine absorption was about 0.5-fold that of IM epinephrine (AUC0-180 min 10.0 (9.2) ± 8.6 ng.min/mL), but the difference was not statistically significant (p = 0.16). The mean peak epinephrine concentration and the time to reach it were also not significantly different between the IN and IM routes. The corresponding values were 120 pg/mL and 41 min for IN, and 209 pg/mL and 41 min for IM, respectively. CONCLUSIONS: The systemic absorption of IN epinephrine 5 mg was significantly different from the control IN saline and about 0.5-fold that of IM epinephrine 0.3 mg. Although epinephrine administration via the less invasive IN route is safe and feasible, further investigations are necessary to achieve an adequate and consistent systemic absorption comparable to that of the conventional IM injection.

PLGA nanoparticles containing α-fetoprotein siRNA induce apoptosis and enhance the cytotoxic effects of doxorubicin in human liver cancer cell line.

Hepatocellular carcinoma (HCC) is one of the most common cancers and is a leading cause of death. Delivery of therapeutic molecules, e.g., siRNA, to HCC cells could potentially be an alternative treatment for HCC. In this study, the siRNA targeting α-fetoprotein (AFP) mRNA was found to specifically induce apoptosis and significant cell death in HepG2 cells. It also enhanced the cytotoxic effects of doxorubicin by about two-fold, making it the candidate therapeutic molecule for HCC treatment. To deliver the siRNAs into HCC cells, the AFP siRNAs were loaded into the nanoparticles based on poly (lactic-co-glycolic) acid (PLGA). These nanoparticles induced apoptosis in HepG2 cells and synergistically increased the cytotoxicity of doxorubicin. In summary, the delivery of the AFP siRNA-loaded PLGA nanoparticles in combination with doxorubicin could be a very promising approach for the treatment of HCC.

Curcuminoids supplementation ameliorates iron overload, oxidative stress, hypercoagulability, and inflammation in non-transfusion-dependent ß-thalassemia/Hb E patients.

Curcuminoids, polyphenol compounds in turmeric, possess several pharmacological properties including antioxidant, iron-chelating, and anti-inflammatory activities. Effects of curcuminoids in thalassemia patients have been explored in a limited number of studies using different doses of curcuminoids. The present study aims to evaluate the effects of 24-week curcuminoids supplementation at the dosage of 500 and 1000 mg/day on iron overload, oxidative stress, hypercoagulability, and inflammation in non-transfused ß-thalassemia/Hb E patients. In general, both curcuminoids dosages significantly lowered the levels of oxidative stress, hypercoagulability, and inflammatory markers in the patients. In contrast, reductions in iron parameter levels were more remarkable in the 1000 mg/day group. Subgroup analysis revealed that a marker of hypercoagulability was significantly decreased only in patients with baseline ferritin ≤ 1000 ng/ml independently of curcuminoids dosage. Moreover, the alleviation of iron loading parameters was more remarkable in patients with baseline ferritin > 1000 ng/ml who receive 1000 mg/day curcuminoids. On the other hand, the responses of oxidative stress markers were higher with 500 mg/day curcuminoids regardless of baseline ferritin levels. Our study suggests that baseline ferritin levels should be considered in the supplementation of curcuminoids and the appropriate curcuminoids dosage might differ according to the required therapeutic effect. Thai Clinical Trials Registry (TCTR): TCTR20200731003; July 31, 2020 "retrospectively registered".

Prevalence of and factors associated with thiamin deficiency in obese Thai children.

BACKGROUND AND OBJECTIVES: Obesity is a state that results from excessive energy consumption, and obese people often have micronutrient deficiencies. The objective of this study was to investigate the prevalence of and factors associated with thiamin deficiency in obese Thai children. METHODS AND STUDY DESIGN: This cross-sectional study was conducted at Faculty of Medicine Siriraj Hospital, Mahidol University during 2014 to 2017. Children aged 7-15 years old with exogenous obesity were recruited. Symptoms and signs of thiamin deficiency were evaluated. Erythrocyte transketolase activity was measured by thiamin pyrophosphate effect (TPPE), with ≥15% indicating thiamin deficiency. Dietary consumption from a 5-day food diary and food frequency questionnaire was calculated by INMUCAL software. Other medical complications of obesity were also evaluated. RESULTS: One hundred and twenty-four subjects (81 males and 43 females) were enrolled, with a mean age of 10.9 years. Fifty-two subjects had abnormal TPPE for an overall prevalence of thiamin deficiency of 42%. Manifestations of thiamin deficiency included numbness, weakness, and calf muscle cramping. TPPE test results were correlated with at least one symptom or a sign of thiamin deficiency (p<0.01). The thiamin-deficient group tended to have higher proportion of morbid obesity and larger waist circumferences than thiamin-sufficient group. The thiamindeficient group tended to consume less thiamin in relation to energy intake than the thiamin-sufficient group (p=0.057). Items of foods consumed were statistically indistinguishable between groups. CONCLUSIONS: The results of this study revealed a 42% prevalence of thiamin deficiency among obese Thai children, and most of those cases were subclinical.

Drug repurposing of minocycline against dengue virus infection.

Dengue virus infection is one of the most common arthropod-borne viral diseases. A complex interplay between host and viral factors contributes to the severity of infection. The antiviral effects of three antibiotics, lomefloxacin, netilmicin, and minocycline, were examined in this study, and minocycline was found to be a promising drug. This antiviral effect was confirmed in all four serotypes of the virus. The effects of minocycline at various stages of the viral life cycle, such as during viral RNA synthesis, intracellular envelope protein expression, and the production of infectious virions, were examined and found to be significantly reduced by minocycline treatment. Minocycline also modulated host factors, including the phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2). The transcription of antiviral genes, including 2'-5'-oligoadenylate synthetase 1 (OAS1), 2'-5'-oligoadenylate synthetase 3 (OAS3), and interferon α (IFNA), was upregulated by minocycline treatment. Therefore, the antiviral activity of minocycline may have a potential clinical use against Dengue virus infection.

A preliminary study of intranasal epinephrine administration as a potential route for anaphylaxis treatment.

BACKGROUND: The intranasal (IN) administration of epinephrine could be an alternative route for anaphylaxis treatment. Although IN epinephrine absorption has been demonstrated in animals, such data in humans are still lacking. OBJECTIVE: To study the pharmacokinetics of IN epinephrine absorption in humans. METHODS: Each healthy adult (n = 5) was administered IN saline, IN epinephrine at various doses (i.e., 0.3, 0.6, 1.25, 2.5 and 5 mg), and intramuscular (IM) epinephrine at 0.3 mg. Plasma epinephrine levels at baseline and various time points up to 120 minutes after administration were determined using high-performance liquid chromatography with electrochemical detection. RESULTS: Significant systemic absorption of epinephrine via IN route was observed only at the dose of 5 mg, and the absorption thereof was comparable to that of IM epinephrine; the average area-under-curve (AUC) values at 0-120 minutes for IN saline, IM epinephrine, and 5 mg IN epinephrine were 0.3, 18.3, and 19.4 ng.min/mL, respectively. In addition, the peak epinephrine concentrations and the time to reach them were also not significantly different between IM and 5-mg IN epinephrine; the corresponding values (mean ± SD) were 309 ± 88 pg/mL and 67 ± 43 min for IM epinephrine, and 386 ± 152 pg/mL and 70 ± 17 min for 5 mg IN epinephrine. CONCLUSION: This preliminary study showed that epinephrine can be significantly absorbed via the IN route in humans. However, it requires a higher IN dose (5 mg) than the usual IM dose (0.3 mg) to achieve comparable systemic epinephrine absorption.

Quantifying fecal and plasma short-chain fatty acids in healthy Thai individuals.

Short-chain fatty acids (SCFAs) are involved in important physiological processes such as gut health and immune response, and changes in SCFA levels can be indicative of disease. Despite the importance of SCFAs in human health and disease, reference values for fecal and plasma SCFA concentrations in healthy individuals are scarce. To address this gap in current knowledge, we developed a simple and reliable derivatization-free GC-TOFMS method for quantifying fecal and plasma SCFAs in healthy individuals. We targeted six linear- and seven branched-SCFAs, obtaining method recoveries of 73-88% and 83-134% in fecal and plasma matrices, respectively. The developed methods are simpler, faster, and more sensitive than previously published methods and are well suited for large-scale studies. Analysis of samples from 157 medically confirmed healthy individuals showed that the total SCFAs in the feces and plasma were 34.1 ± 15.3 µmol/g and 60.0 ± 45.9 µM, respectively. In fecal samples, acetic acid (Ace), propionic acid (Pro), and butanoic acid (But) were all significant, collectively accounting for 89% of the total SCFAs, whereas the only major SCFA in plasma samples was Ace, constituting of 93% of the total plasma SCFAs. There were no statistically significant differences in the total fecal and plasma SCFA concentrations between sexes or among age groups. The data revealed, however, a positive correlation for several nutrients, such as carbohydrate, fat, iron from vegetables, and water, to most of the targeted SCFAs. This is the first large-scale study to report SCFA reference intervals in the plasma and feces of healthy individuals, and thereby delivers valuable data for microbiome, metabolomics, and biomarker research.

Differential plasma proteome profiles of mild versus severe ß-thalassemia/Hb E.

The severity of thalassemia is currently classified based on clinical manifestations and multiple tests. In the present study, we performed a plasma proteome analysis to identify differentially expressed proteins compared between normal subjects and patients with mild and severe forms of ß-thalassemia/hemoglobin E (Hb E). Plasma samples were collected from patients with mild (n = 8) and severe (n = 12) forms as well as healthy normal individuals (n = 12). Clinical chemistry revealed that several parameters, i.e., hematological indices, oxidative stress markers, antioxidant enzymes, and erythropoietic activity, had significant differences among these three groups. After removal of seven major abundant proteins, the plasma proteome profiles were compared using two-dimensional gel electrophoresis. Spot matching, quantitative intensity analysis, and statistics revealed differential levels of 32 and 9 proteins when comparing normal vs. patients and mild vs. severe forms, respectively. These proteins were successfully identified by quadrupole time-of-flight mass spectrometry and/or tandem mass spectrometry. The decreased level of ADP-ribosylation factor guanine nucleotide-exchange factor 2 in ß-thalassemia/Hb E patients compared to healthy individuals and the decreased level of endothelin-converting enzyme 2 in severe form compared to the mild form of the disease were validated by Western blot analysis. Our data provide a number of proteins that may lead to better understanding of the pathophysiology of thalassemia or for novel biomarkers which can be used to simply differentiate mild and severe forms of ß-thalassemia/Hb E without any need for multiple tests.

Inhibition of p38MAPK and CD137 signaling reduce dengue virus-induced TNF-α secretion and apoptosis.

BACKGROUND: Hepatic injury in dengue virus (DENV) infection is authenticated by hepatomegaly and an upsurge in transaminase levels. DENV replicates in hepatocytes and causes hepatocyte apoptosis both in vitro and in vivo. Understanding the molecular mechanisms of DENV-induced hepatic injury could facilitate the development of alternate chemotherapeutic agents and improved therapies. FINDINGS: The p38 mitogen-activated protein kinase (MAPK) participates in both apoptosis-related signaling and pro- inflammatory cytokine production. The role of p38 MAPK in DENV-infected HepG2 cells was examined using RNA interference. The results showed that DENV infection activated p38 MAPK and induced apoptosis. The p38 MAPK activation and TNF-α production were controlled by p38 MAPK and CD137 signaling in DENV-infected HepG2 cells as activated p38 MAPK, TNF-α and apoptosis were significantly decreased in p38 MAPK and CD137 depleted DENV-infected HepG2 cells. Addition of exogenous TNF-α to p38 MAPK depleted DENV-infected HepG2 cells restored DENV-induced apoptosis in HepG2 cells. CONCLUSION: DENV induces CD137 signaling to enhance apoptosis by increasing TNF-α production via activation of p38 MAPK.

Epigallocatechin Gallate Potentiates the Anticancer Effect of AFP-siRNA-Loaded Polymeric Nanoparticles on Hepatocellular Carcinoma Cells.

To develop a potential cancer treatment, we formulated a novel drug delivery platform made of poly(lactic-co-glycolic) acid (PLGA) and used a combination of an emerging siRNA technology and an extracted natural substance called catechins. The synthesized materials were characterized to determine their properties, including morphology, hydrodynamic size, charge, particle stability, and drug release profile. The therapeutic effect of AFP-siRNA and epigallocatechin gallate (EGCG) was revealed to have remarkable cytotoxicity towards HepG2 when in soluble formulation. Notably, the killing effect was enhanced by the co-treatment of AFP-siRNA-loaded PLGA and EGCG. Cell viability significantly dropped to 59.73 ± 6.95% after treatment with 12.50 µg/mL of EGCG and AFP-siRNA-PLGA. Meanwhile, 80% of viable cells were observed after treatment with monotherapy. The reduction in the survival of cells is a clear indication of the complementary action of both active EGCG and AFP-siRNA-loaded PLGA. The corresponding cell death was involved in apoptosis, as evidenced by the increased caspase-3/7 activity. The combined treatment exhibited a 2.5-fold increase in caspase-3/7 activity. Moreover, the nanoparticles were internalized by HepG2 in a time-dependent manner, indicating the appropriate use of PLGA as a carrier. Accordingly, a combined system is an effective therapeutic strategy.

Differential expression of sprouty genes in hepatocellular carcinoma.

BACKGROUND AND OBJECTIVES: Sprouty (Spry) proteins are important modulators of the RTK/Ras/MAPK pathway, overactivation of which is associated with hepatocellular carcinoma (HCC). Thus far, the roles of Sprouty in HCC is still unclear. METHODS: The expressions of SPRY1, SPRY2, SPRY3, and SPRY4, at the mRNA levels were determined by quantitative RT-PCR in paired HCC and non-tumor liver tissues from 31 patients. RESULTS: The expression levels of SPRY1, SPRY2, and SPRY4 in tumor tissues were significantly different from those in non-tumor tissues with the average log fold change values of 0.15, -0.34, and -0.37, respectively; however, that of SPRY3 was not significantly different. SPRY1 expression was also found to be significantly up-regulated in the cases without underlying cirrhosis compared with those with cirrhosis (log fold change of 0.35 and -0.02, respectively, P < 0.05), whereas SPRY2 expression was significantly lower in the cases with advanced HCC (log fold change of -0.12 and -0.52 in early and advanced stages, respectively, P < 0.05) and in those with angiolymphatic invasion (log fold change of -0.47 and -0.16 in the presence and absence thereof, P < 0.05). CONCLUSION: This study demonstrates that Sprouty genes are differentially expressed in HCC and might provide some insight into their roles in HCC carcinogenesis.

Selection of RNA aptamers that bind HIV-1 LTR DNA duplexes: strand invaders.

RNA that can specifically bind to double-stranded DNA is of interest because it might be used as a means to regulate transcription of the target genes. To explore possible interactions between RNA and duplex DNA, we selected for RNA aptamers that can bind to the long terminal repeats (LTRs) of human immunodeficiency virus type 1 DNA. The selected aptamers were classified into four major groups based on the consensus sequences, which were found to locate in the non-stem regions of the predicted RNA secondary structures, consistent with roles in target binding. Analysis of the aptamer consensus sequences suggested that the conserved segments could form duplexes via Watson-Crick base-pairing with preferred sequences in one strand of the DNA, assuming the aptamer invaded the duplex. The aptamer binding sites on the LTR were experimentally determined to be located preferentially at these sites near the termini of double-stranded target DNA, despite selection schemes that were designed to minimize preferences for termini. The results presented here show that aptamer RNAs can be selected in vitro that strand-invade at preferred DNA duplex sequences to form stable complexes.

The helper oligonucleotides enable detection of folded single-stranded DNA by lateral flow immunoassay after HCR signal amplification.

A combination of Hybridization Chain Reaction (HCR) and Lateral Flow Immunoassay (LFIA) is an attractive strategy for a simple signal amplification DNA/RNA detection. The present study aimed to report a strategy used to solve a problem encountered when the target DNA contained folded secondary structure during HCR, enabling HCR hairpin probes to easily access the target site. The 24-nt conserved sequence within 3'-UTR, present only in dengue virus genome but not in other species, is an ideal target to use as a probe binding site for pan-dengue virus detection. Thus, the 105-nt target containing the 24-nt target sequence was chosen as a target with secondary structures. The 24-nucleotide (nt) synthetic target DNA successfully induced HCR reaction within 5 min at room temperature. However, the HCR detection of the 105-nt synthetic target DNA with secondary structures was problematic. The probe hybridization was prevented by the secondary structures of the target, resulting in a failure to generate HCR product. To solve this problem, two helper oligonucleotides (helper1 and helper2) were designed to linearize the folded structure of the 105-nt target through strand-displacement mechanism, allowing the HCR hairpin probes to easily access the target site. The HCR product with the labeled helper oligonucleotides and the labeled probes were successfully detected by LFIA. With this strategy, the combination of the helper-enhanced HCR and LFIA exhibited a limit of detection (LOD) in a nanomolar range of the 105-nt DENV synthetic target DNA. Our study demonstrated that signal amplification by the combination of HCR and LFIA could successfully detect the target DNA with secondary structure, but not target RNA with secondary structure. In summary, this work provided a proof of concept of two main issues including probe hybridization enhancement by helper oligonucleotide for the target with complicated secondary structure and the advantage of a combination of labeled helper and HCR probes design for LFIA to overcome the false positive result from HCR probe leakage. Our findings on the use of helper oligonucleotides may be beneficial for the development of other isothermal amplification, since the secondary structure of the target is one of the major obstacles among hybridization-based methods.

The Effect of high temperature on the stability of basal insulin in a pen: a randomized controlled, crossover, equivalence trial.

INTRODUCTION: Insulin is an essential medicine in the management of diabetes. When stored at high temperatures(HTs), its efficacy could rapidly decline. Therefore, appropriate storage of in-use insulin is necessary to achieve its maximum therapeutic effects. However, the ambient temperature in tropical countries is normally relatively high. This study aimed to compare the efficacies of basal insulin in a pen previously kept at 37°C for 21 days and basal insulin in a refrigerated pen (2°C-8°C). Continuous glucose monitoring (CGM) was used to evaluate daily mean glucose levels (MGLs). RESEARCH DESIGN AND METHODS: This randomized controlled, crossover, equivalence trial recruited adults with type 2 diabetes mellitus and glycated hemoglobin levels <8% who had used insulin glargine for >3 months. Subjects were randomized for sequential use of refrigerated basal insulin followed by basal insulin kept at HT, with a 2-week washout between phases. The HT insulin pens were stored in a 37°C incubator for 21 days before use, while the refrigerated insulin pens were stored at 2°C-8°C. Study patients received 7-day CGM. The primary outcome was the difference in the groups' MGLs. The secondary outcome parameters were glucose variability represented by the standard deviation (SD), mean amplitude of glycemic excursion (MAGE), and percentage of time in range (TIR). The remaining quantity of insulin was evaluated by ultrahigh-performance liquid chromatography (UHPLC) assay. RESULTS: Forty patients completed the study. The MGLwas 158.7±30.5 mg/dL and 157.0±40.9 mg/dL in the HT and refrigerated insulin pen groups, respectively (p=0.72). The groups had no significant differences in MAGE7day, SD, percentage of TIR, carryover period, or treatment effects (all p>0.05). There was also no significant difference in the remaining quantity of insulin evaluated by UHPLC (p=0.97). CONCLUSIONS: HT basal insulin pens retain their potency and have biological activity comparable to that of refrigerated pens.Trial registration number TCTR20210611002.

Patient-ready syringes containing 25 mg/mL methotrexate can be kept at temperature ranging from 4 °C to 37 °C for up to 12 weeks for use in psoriatic and rheumatologic conditions.

BACKGROUND: Methotrexate (MTX) is a mainstay drug in the treatment of psoriatic and rheumatologic conditions. Subcutaneous MTX has become a feasible treatment alternative with the development of prefilled syringes or autoinjectors containing MTX solution that can be self-administered by the patient at home. However, MTX prefilled auto-injector pens are still not available in some countries. OBJECTIVE: This study aimed to investigate the stability and sterility of 25 mg/mL MTX solution in a disposable plastic syringe over a 12-week period under light protection at temperatures of 4 °C, 25 °C, and 37 °C. METHODS: This study was conducted during November 2019 to February 2020 at the Faculty of Medicine Siriraj Hospital, Mahidol University. Stability was evaluated using ultra-high-performance liquid chromatography technique, and sterility was assessed by cultures for bacterial and fungal contamination. RESULTS: Our results revealed that patient-ready syringes containing 25 mg/mL MTX solution can be prepared in advance and kept for up to 12 weeks under light protection, and they can be kept at temperatures ranging from 4 to 37 °C. CONCLUSION: This system for delivering MTX to patients that are refractory to or intolerant of oral MTX via a self-administered pre-filled syringe is both efficient and easy to implement in care settings where commercially alternatives are not yet available.