Search

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare skin fragility disorder caused by mutations in COL7A1. RDEB is hallmarked by trauma-induced unremitting blistering, chronic wounds with inflammation, and progressive fibrosis, leading to severe disease complications. There is currently no cure for RDEB-associated fibrosis. Our previous studies and increasing evidence highlighted the profibrotic role of NOTCH pathway in different skin disorders, including RDEB. In this study, we further investigated the role of NOTCH signaling in RDEB pathogenesis and explored the effects of its inhibition by γ-secretase inhibitors DAPT and PF-03084014 (nirogacestat). Our analyses demonstrated that JAG1 and cleaved NOTCH1 are upregulated in primary RDEB fibroblasts (ie, RDEB-derived fibroblasts) compared with controls, and their protein levels are further increased by TGF-β1 stimulation. Functional assays unveiled the involvement of JAG1/NOTCH1 axis in RDEB fibrosis and demonstrated that its blockade counteracts a variety of fibrotic traits. In particular, RDEB-derived fibroblasts treated with PF-03084014 showed (i) a significant reduction of contractility, (ii) a diminished secretion of TGF-β1 and collagens, and (iii) the downregulation of several fibrotic proteins. Although less marked than PF-03084014-treated cells, RDEB-derived fibroblasts exhibited a reduction of fibrotic traits also upon DAPT treatment. This study provides potential therapeutic strategies to antagonize RDEB fibrosis onset and progression., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Five different diamines, namely 1,2-diaminopropane, N,N-diethylethylenediamine, piperazine, 1-methylpiperazine and N,N,N',N'-tetramethylethylenediamine, have been investigated as liquid sorbents for the direct capture of CO2 from air (DAC – Direct Air Capture) in both aqueous and non-aqueous solutions, using a 1:1 (volume scale) mixture of ethylene glycol and 1-propanol as an organic diluent. The capture performance of the 1.5 mol dm−3 diamine solutions was evaluated on the basis of the CO2 absorption efficiency from an atmospheric air stream, continuously measured by means of a gas chromatograph during 24-hour bench-scale absorption experiments, carried out in an absorption column maintained at 25°C. The carbonated species formed in solution upon the CO2 uptake and their relative amounts were evaluated by 13C NMR spectroscopy, a powerful non-invasive analytical technique that can provide valuable information on the reaction mechanisms. The correlations between the aerial CO2 absorption efficiency, the chemical structure of diamines and the carbonated species formed in their different solutions during capture were presented and discussed. Furthermore, the CO2 capture performance in DAC processes of the diamine solutions was compared to that of some of the more conventional alkanolamine solutions under the same operating conditions.

Objective: This study compared the protective effect of an experimental TiF 4 /Chitosan toothpaste with a commercial toothpaste on the prevention of erosive tooth wear (ETW) in situ., Methods: Fifteen subjects took part in this crossover and double-blind study, in which they wore a palatal appliance containing 4 bovine enamel and 4 dentin in 3 phases (5 days each). Half of the samples were subjected to erosive challenges (90 s in 0.1 % citric acid, pH 2.5, 4 times/day), and the other half to erosive plus abrasive challenges (15 s plus 45 s of contact, 2 times/day). The phases corresponded to the application of the different toothpastes: 1) TiF 4 (1400 ppm F - ) plus Chitosan, 2) Elmex®, Erosion Protection (1400 ppm F - , Chitosan), and 3) Placebo (negative control). Tooth wear was measured using contact profilometry (μm) and submitted to two-way RM ANOVA/Tukey test (p < 0.05)., Results: No significant differences were detected between the experimental and commercial toothpastes, regardless of the challenge on both tissues. Both significantly reduce ETW compared to negative control (p < 0.0006). Tooth wear was increased by brushing only on eroded enamel (p < 0.01), but not on dentin (p = 0.6085). TiF 4 /Chitosan [erosion 2.98 ± 1.12 μm vs. erosion and abrasion 3.12 ± 1.33 μm] and Elmex® toothpastes [erosion 2.35 ± 0.93 μm vs. erosion and abrasion 2.98 ± 1.0 μm] minimized the impact of brushing compared to placebo on enamel [erosion 4.62 ± 1.48 μm vs. erosion and abrasion 5.15 ± 1.50 μm]., Conclusions: TiF 4 plus chitosan toothpastes showed to be effective in minimizing the ETW as the commercial toothpaste is in situ., Clinical Relevance: The experimental toothpaste has similar effect against ETW compared to the commercial toothpaste. Considering the increased ETW prevalence worldwide, this result supports clinical trials and a possible application of this experimental anti-erosive toothpaste in the future., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Putrescine (Butane-1,4-diamine) has been regarded as a vital marker of spoiling protein-rich foods, especially meat and seafood. The detection of putrescine in food is considered a convenient and powerful method for evaluating the degree of spoilage of protein-rich foods. Herein, a novel rhodol-based fluorescent probe RSMA (formyl-rhodol Schiff base with methoxyaniline) was developed to detect putrescine. RSMA exhibited excellent linearity (R 2  = 0.9912) in the concentration range of 0-45 μM of putrescine with a detection limit as low as 0.45 μM. Although RSMA had moderate responses to some aliphatic diamines, the selectivity of RSMA for putrescine was one of the best reported in the literature so far. Moreover, RSMA was successfully fabricated to solid-state sensors for on-site detection of putrescine in shrimp, that demonstrated its application in monitoring food spoilage., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Poly(hexamethylenebicyanoguanide-hexamethylenediamine) hydrochloride (PHMB) is a biocide with a broad spectrum of antibacterial activity. Its use as a disinfectant and preservative in consumer products results in human exposure to PHMB. Toxicity studies on PHMB mainly focus on systemic toxicity or skin irritation; however, its effects on developmental neurotoxicity (DNT) and the underlying mechanisms are poorly understood. In this study, the DNT effects of PHMB were evaluated using IMR-32 and SH-SY5Y cell lines and zebrafish. In both cell lines, PHMB concentrations ≥ 10 µM reduced neurite outgrowth, and cytotoxicity was observed at concentrations up to 40 µM. PHMB regulated expression of neurodevelopmental genes and induced reactive oxygen species (ROS) production and mitochondrial dysfunction. Treatment with N-acetylcysteine reversed the toxic effects of PHMB. Toxicity tests on zebrafish embryos showed that PHMB reduced viability and heart rate and caused irregular hatching. PHMB concentrations of 1-4 µM reduced the width of the brain and spinal cord of transgenic zebrafish and attenuated myelination processes. Furthermore, PHMB modulated expression of neurodevelopmental genes in zebrafish and induced ROS accumulation. These results suggested that PHMB exerted DNT effects in vitro and in vivo through a ROS-dependent mechanism, highlighting the risk of PHMB exposure., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

With the diversification of people's demand for textile functions, the preparation of multifunctional fabrics is still a current research hotspot. In this study, the water-soluble epoxy compound N 1 , N 6 -bis(oxiran-2-ylmethyl) hexane-1,6-diamine (EH) was introduced into cellulose macromolecule blended fabrics (cotton/modal) by two-phase vaporization technique, resulting in excellent wrinkle, hydrophobicity, and certain UV protection effects. It could be observed by electron microscopy that EH formed a polymer film on the fiber surface. In addition, the results of EDS scans and fiber swelling rate tests showed that EH was uniformly distributed and formed a cross-linked structure in the amorphous zones inside the fibers. Compared with the control fabrics, the wrinkle recovery angle of the EH-treated fabric was increased by 39.7 %. The fabrics could reach a contact angle of 136.9°, providing excellent hydrophobic effect. In addition, the fabrics achieved certain UV protection effects (UPF of 50+). The EH-treated fabrics were less stabilized in strong acid and alkali conditions, but exhibited greater durability in other environments. In summary, the internal and external synergistic effects of EH in forming polymer films on the fibers surface and internal cross-linking structures provided a cleaner, simple, and feasible method for the preparation of multifunctional cellulose macromolecule fibers textiles., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Aminolysis is widely recognized as a valuable chemical route for depolymerizing polymeric materials containing ester, amide, or urethane functional groups, including polyurethane foams. Bio-based polyurethane foams, pristine and reinforced with 40 wt% of sustainable fillers, were depolymerized in the presence of bio-derived butane-1,4-diamine, BDA. A process comparison was made using fossil-derived ethane-1,2-diamine, EDA, by varying amine/polyurethane ratio (F/A, 1:1 and 1:0.6). The obtained depolymerized systems were analyzed by FTIR and NMR characterizations to understand the effect of both diamines on the degradation pathway. The use of bio-based BDA seemed to be more effective with respect to conventional EDA, owing to its stronger basicity (and thus higher nucleophilicity), corresponding to faster depolymerization rates. BDA-based depolymerized systems were then employed to prepare second-generation bio-based composite polyurethane foams by partial replacement of isocyanate components (20 wt%). The morphological, mechanical, and thermal conductivity properties of the second-generation polyurethane foams were evaluated. The best performances (σ10 %=71 ± 9 kPa, λ = 0.042 ± 0.015 W∙ m -1 ∙K -1 ) were attained by employing the lowest F/A ratio (1:0.6); this demonstrates their potential application in different sectors such as packaging or construction, fulfilling the paradigm of the circular economy., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Two dicationic water soluble mixed triamine/diamine copper (II) complexes, of general formula [Cu(dien)NN]Br2 (1–2) [dien = diethelenetriamine and NN is en = ethylenediamine or Me4en = N,N′,N,N′-tetramethylethylenediamine] were prepared under ultrasonic mode with a relatively high yield. These complexes were characterized by elemental microanalysis, UV visible IR spectroscopy, and thermal and electrochemical techniques. In addition, complex 2 structure was solved by X-ray single crystal and Hirshfeld surface analysis. The complex exhibits a distorted square pyramidal coordination environment around Cu(II) centre. The solvatochromism of the desired complexes was investigated in water and other suitable organic solvents. The results show that the Guttmann’s DN parameter values of the solvents have mainly contributed to the shift of the d–d absorption band towards the linear increase in the wavelength of the absorption maxima of the complexes. The complex 1 showed higher antibacterial activity against the studied microorganisms compared to complex 2. Both complexes revealed promising antibacterial activities.

The present study focuses on structural and chemical analyses of N-phenylmorpholine-4-carboxamide benzene-1,2-diamine (PMCBD) using quantum computational methods. The calculated bond angle, length, and dihedral angle between atoms were compared with measured values. The observed and stimulated FT-IR (Fourier Transform Infrared Spectroscopy) spectra parameters for vibrational wavenumbers and their associated PED (Potential Energy Distribution) values in percentage have been obtained from VEDA4 software. The electronic transitions of PMCBD were discussed by TD-SCF/DFT/B3LYP based on the 6-311++G(d,p) basis set with solvents such as chloroform, ethanol, and dimethyl sulfoxide (DMSO) and gas. Density functional computations were used to study the band energy between HOMO and LUMO using the B3LYP/6-311++G(d,p) level. Mulliken analysis and natural population analysis were used for a better understanding of charge levels on different atoms such as N, H and O. The natural bonding orbital (NBO) analysis proved helpful in studying molecular and bond strengths. (NBO). The ESP acquired data on the molecule's size, shape, charge density distribution, and chemical reactivity site. This was done by mapping electron density on the surface with electrostatic potential. Non-linear optical detection of PMCBD was also discussed. Aside from the electron localization function map, state densities are also mapped using Multiwfn software, a wave function analyzer., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Amine-terminated PANs were prepared in two steps. The first step includes free radical polymerization of acrylonitrile (AN) using initiator pair of ammonium persulfate and sodium thiosulfate as redox system. In the second step, the amino groups were introduced through the reaction of polyacrylonitrile with excess of different diamines (10-fold) including ethylenediamine (EDA), hexamethylenediamine (HMDA) and octamethylenediamine (OMDA), to yield PAN–EDA, PAN–HMDA and PAN–OMDA, respectively. Optimization of the amine-terminated PANs synthesis was carried out at different temperatures (30–90 °C) and different time intervals (4–24 h). In addition, the introduction of the amino group was followed by the piperidine test and recording of the FT-IR spectra. All polymers were characterized by, 1H NMR spectra, thermogravimetric analysis (TGA), and FT-IR spectra.

Two cyclodextrin-based nanosponges (CD-NSs) were synthesized using diamines with 6 and 12 methylene groups, CDHD6 and CDHD12, respectively, and used as adsorbents to remove 2,4-D from aqueous solutions. The physico-chemical characterization of the CD‒NSs demonstrated that, when using the linker with the longest chain length, the nanosponges show a more compact structure and higher thermal stability, probably due to hydrophobic interactions. SEM micrographs showed significant differences between the two nanosponges used. The adsorption of 2,4-D was assessed in terms of different parameters, including solid/liquid ratio, pH, kinetics and isotherms. Adsorption occurred preferentially at lower pH values and for short-chain crosslinked nanosponges; while the former is explained by the balance of acid-base characteristics of the adsorbent and adsorbate, the latter can be justified by the increase in the crosslinker-crosslinker interactions, predominantly hydrophobic, rather than adsorbent-adsorbate interactions. The maximum adsorption capacity at the equilibrium (q e ) was 20,903 mmol/kg, obtained using CDHD12 with an initial 2,4-D concentration of 2 mmol/L. An environmentally friendly strategy, based on alkali desorption, was developed to recycle and reuse the adsorbents. On the basis of the results obtained, cyclodextrin-based nanosponges appear promising materials for an economically feasible removal of phenoxy herbicides, to be used as potential adsorbents for the sustainable management of agricultural wastewaters., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Purpose: Scrub typhus (ST) is a zoonotic disease, caused by O. tsutsugamushi is a major cause of acute febrile illness (AFI) in India. There is a need to study the prevalence and risk factors in various regions of India., Methods: A study to estimate the prevalence and study the risk factors of ST in patients presenting with acute febrile illness (AFI) was performed. All patients underwent serology for IgM antibodies to Orientia tsutsugamushi (In Bios International Inc, Seattle, WA) as per the manufacturers' protocol. Following this, Polymerase Chain reaction (PCR) (real time SYBR green based targeting groEL gene and conventional PCR targeting 56 ​kDa type specific antigen gene) was performed from stored serum samples., Results: During the study period, 473 patients were admitted. Of these 56 (11.8%) patients were ST positive by IgM serology. The conventional PCR targeting 56 ​kDa type specific antigen gene of O. tsutsugamushi was positive in six patients while Ot groEL SYBR green based PCR) was positive in five. PCR was positive in patients who had demonstrated a higher OD value in ELISA. Conventional PCR positive amplicons were sent for Sanger sequencing and confirmed to be O. tsutsugamushi. The mean age of the patients was 49 ​± ​18.3 years and males constituted a higher number of patients (67.9%, n ​= ​38). The pathognomonic eschar was present in 7 (12.5%) patients. Phylogenetic analysis revealed that sequences clustered close to Kato-like Hualein-20 strain and Karp-like Linh DT strains. All patients were administered doxycycline in our study. Mortality was recorded in 8.9% of the patients., Conclusions: In patients presenting with acute febrile illness, ST should be considered as a differential diagnosis, especially in post-monsoon season. Along with serology, serum can also be used as sample for PCR in an intracellular bacterium like O. tsutsugamushi., (Copyright © 2022 Indian Association of Medical Microbiologists. Published by Elsevier B.V. All rights reserved.)

Corynebacterium glutamicum is well known as the amino acid-producing workhorse of fermentation industry, being used for multi-million-ton scale production of glutamate and lysine for more than 60 years. However, it is only recently that extensive research has focused on engineering it beyond the scope of amino acids. Meanwhile, a variety of corynebacterial strains allows access to alternative carbon sources and/or allows production of a wide range of industrially relevant compounds. Some of these efforts set new standards in terms of titers and productivities achieved whereas others represent a proof-of-principle. These achievements manifest the position of C. glutamicum as an important industrial microorganism with capabilities far beyond the traditional amino acid production. In this review we focus on the state of the art of metabolic engineering of C. glutamicum for utilization of alternative carbon sources, (e.g. coming from wastes and unprocessed sources), and construction of C. glutamicum strains for production of new products such as diamines, organic acids and alcohols.

Corynebacterium glutamicum is well known as the amino acid-producing workhorse of fermentation industry, being used for multi-million-ton scale production of glutamate and lysine for more than 60 years. However, it is only recently that extensive research has focused on engineering it beyond the scope of amino acids. Meanwhile, a variety of corynebacterial strains allows access to alternative carbon sources and/or allows production of a wide range of industrially relevant compounds. Some of these efforts set new standards in terms of titers and productivities achieved whereas others represent a proof-of-principle. These achievements manifest the position of C. glutamicum as an important industrial microorganism with capabilities far beyond the traditional amino acid production. In this review we focus on the state of the art of metabolic engineering of C. glutamicum for utilization of alternative carbon sources, (e.g. coming from wastes and unprocessed sources), and construction of C. glutamicum strains for production of new products such as diamines, organic acids and alcohols.

With the interest in radiometal-containing diagnostic and therapeutic pharmaceuticals increasing rapidly, appropriate ligands to coordinate completely and stably said radiometals is essential. Reported here are two novel, bis(amido)bis(oxinate)diamine ligands, H 2 amidohox (2,2'-(ethane-1,2-diylbis(((8-hydroxyquinolin-2-yl)methyl)azanediyl))diacetamide) and H 2 amidoC3hox (2,2'-(propane-1,3-diylbis(((8-hydroxyquinolin-2-yl)methyl)azanediyl))diacetamide), that combine two 8-hydroxyquinoline and amide donor groups and differ by one carbon in their 1,2-ethylenediamine vs. 1,3-diaminopropane backbones, respectively. Both ligands have been thoroughly studied via metal complexation, solution thermodynamics and radiolabeling with three radiometal ions: [ nat/64 Cu]Cu 2+ , [ nat/111 In]In 3+ , and [ nat/203 Pb]Pb 2+ . X-ray crystallography determined the structures of the hexacoordinated Cu 2+ -ligand complexes, indicating a better fit of Cu 2+ to the H 2 amidohox binding pocket. Concentration dependent radiolabeling with [ 64 Cu]Cu 2+ was successfully quantitative as low as 1 μM with H 2 amidohox and 10 μM with H 2 amidoC3hox within 5 min at room temperature. However, [ 64 Cu][Cu(amidohox)] maintained higher kinetic inertness against a superoxide dismutase enzyme-challenge assay and ligand challenges compared to the [ 64 Cu][Cu(amidoC3hox)] counterpart. Similarly, H 2 amidohox had significantly higher radiochemical conversion with both [ 111 In]In 3+ (97% at 1 μM) and [ 203 Pb]Pb 2+ (97% at 100 μM) under mild conditions compared to H 2 amidoC3hox (76% with [ 111 In]In 3+ at 1 μM and 0% with [ 203 Pb]Pb 2+ ). By studying non-radioactive and radioactive complexation with both ligands, a comprehensive understanding of the coordination differences between two- and three‑carbon diamine backbones is discussed. Overall, the ethylenediamine backbone of H 2 amidohox proves to be superior in rapid, mild radiolabeling and kinetic inertness towards competing ligands and proteins., (Copyright © 2022. Published by Elsevier Inc.)

The highly selective chemical reaction of carbon dioxide with organic amines is considered to a mature technology and a feasible initial path for carbon capture. In order to solve the disadvantages of high volatility, equipment corrosion and high energy consumption of traditional organic amines, amine alcohol "mixture based" solution has been developed and showed excellent carbon dioxide absorption capacity, which is due to the positive effect of intermolecular interaction in amine alcohol "mixture based" solution system on thermodynamic properties. However, the influencing factors of the intermolecular force in multicomponent solution system are complex, including the chemical, physical, structural effects. Therefore, it is necessary to comprehensively use a variety of characterization methods to systematically understand the form of intermolecular interaction in multicomponent solution system. This review systematically discusses the determination of intermolecular interactions in diamine-diol multicomponent solutions by three mainstream research methods, theoretical calculation method, spectral method, and thermodynamic method, aiming to provide a theoretical reference for the industrial production, the supplement to experimental data, and construction and understanding of theoretical models of multicomponent solution system., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Two multiplex SYBR Green based real-time PCR assays were standardized and evaluated to detect DNA from four canine haemoparasites (Babesia gibsoni, Babesia vogeli, Ehrlichia canis and Hepatozoon canis), along with internal controls from dogs from selected districts of Punjab state, India. Amplicons of 126 bp, 337 bp, 234 bp and 106 bp corresponding to B. gibsoni (18S rRNA gene), B. vogeli (18S rRNA gene), E. canis (virB9 gene), and H. canis (18S rRNA gene) were obtained, without any non-specific amplification. Microscopic evaluation of 200 blood samples from dogs revealed the prevalence of B. gibsoni, E. canis and H. canis as 1.5%, 1.5% and 1.0%, respectively, while with the multiplex real-time PCR assays the values for B. gibsoni, B. vogeli, E. canis, and H. canis were 8.0%, 1.5%, 3.5% and 23.5%, respectively, with concurrent infections of B. gibsoni and H. canis (3.5%); E. canis and H. canis (2.0%) and B. gibsoni, B. vogeli, E. canis, and H. canis (0.5%). The diagnostic sensitivity of the multiplex real-time PCR assays with respect to microscopy in the detection of B. gibsoni, E. canis and H. canis was 100% while the specificity for B. gibsoni, B. vogeli, E. canis, and H. canis was 93%, 100%, 98% and 77%, respectively, revealing the respective strength of agreement as ″fair″, ″slight″, ″moderate″ and ″slight″ by kappa value statistics, and the data were statistically significant, for detection of B. gibsoni and E. canis infections, by Fisher's exact test. The analytical sensitivity of the multiplex PCR assays in detection of DNAs was 8.59 × 10 5 and 9.9 × 10 6 copies for B. vogeli and E. canis, respectively, and 1.15 × 10 6 and 3.41 × 10 5 copies for B. gibsoni and H. canis, respectively. Assessment of risk factors viz. age, sex, breed, season and locations showed no significant association with the prevalence of these haemoparasites except for B. vogeli, E. canis and H. canis where significant associations were found for location, age and breed, respectively by multiplex real-time PCR assays., (Copyright © 2022. Published by Elsevier GmbH.)

A simple method for the determination of polyamines and their N-acetylated forms was developed using benzoyl chloride as derivatization reagent, and 1,6-diaminohexane as internal standard, followed by liquid-liquid extraction with ethyl acetate. The organic extract was injected in a gas chromatograph using a programmed temperature vaporizer and the determination and quantification was performed with a quadrupole mass spectrometer. There was no matrix effect with the proposed method, so internal calibration was used to quantify the corresponding derivatives. Good linear responses were obtained in the range from the limits of detection to 500 µg L -1 (50 µg L -1 for spermidine), with correlation coefficients varying from 0.9591 to 0.9968. The limits of quantification (S/N = 10) ranged 1.0 - 8.3 µg L -1 . Recoveries were found between 82 - 117%, showing the good accuracy of the proposed method. Intra- and inter-day precision assays, expressed as relative standard deviation (RSD) were evaluated at two different concentration levels (low and high), showing values in the range of 2.4 - 6.1% and 5.2 - 9.0% for repeatability and reproducibility, respectively (6.9 - 9.7% and 14.1 - 14.6% for spermidine). Successful determination of the studied polyamines and their N-acetylated forms was performed on the saliva of 17 volunteers., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2021 Elsevier B.V. All rights reserved.)

Phospholipase A 2 (PLA 2 ) may be a vital biomarker for the prediction and diagnosis of some diseases. Consequently, it is of great significance to quantitatively detect PLA 2 in biologic samples. Herein, on the basis of the principle of luminescence resonance energy transfer (LRET) between upconversion nanoparticles (UCNPs) and SYBR Green I (SG), we proposed a technology for the highly sensitive detection of PLA 2 amount. Therein, as an energy receptor, SG will be quantitatively loaded into liposomes firstly. Then, due to the hydrolysis of liposomes under the catalysis of PLA 2 , SG will be released and inserted into the double-stranded DNA (dsDNA) on the surface of UCNPs, which triggers the LRET because of the shortening of effective spatial distance between UCNPs and SG. Under exciting of NIR light, UCNPs emit luminescence at 476 nm, which makes SG emit fluorescence at 522 nm through LRET. Under optimal conditions, the emission intensity ratio (I 522 nm /I 476 nm ) increased linearly with the PLA 2 amount in the range of 20 U/L to 400 U/L, and the limit of detection (LOD) reached 15 U/L. Here, after comparing with the clinical standard method, it is found that the biosensor is expected to provide a convenient and sensitive assay for the detection of PLA 2 in actual serum samples. Furthermore, such biosensor can also be used to test the inhibitor of PLA 2 ., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Understanding reverse transcriptase (RT) activity is critical for designing fast one-step RT-PCRs. We report a stopped-flow assay that monitors SYBR Green I fluorescence to investigate RT activity in PCR conditions. We studied the influence of PCR conditions on RT activity and assessed the accuracy of cDNA synthesis predictions for one-step RT-PCR. Nucleotide incorporation increased from 26 to 89 s -1 between 1.5 and 6 mM MgCl 2 but was largely unaffected by changes in KCl. Conversely, increasing KCl from 15 to 75 mM increased apparent rate constants for RT-oligonucleotide binding (0.010-0.026 nM -1  s -1 ) and unbinding (0.2-1.5 s -1 ). All rate constants increased between 22 and 42 °C. When evaluated by PCR quantification cycle, cDNA predictions differed from experiments using RNase H+ RT (average 1.7 cycles) and RNase H- (average 4.5 cycles). Decreasing H+ RT concentrations 10 to 10 4 -fold from manufacturer recommendations improved cDNA predictions (average 0.8 cycles) and increased RT-PCR assay efficiency. RT activity assays and models can be used to aid assay design and improve the speed of RT-PCRs. RT type and concentration must be selected to promote rapid cDNA synthesis but minimize nonspecific amplification. We demonstrate 2-min one-step RT-PCR of a Zika virus target using reduced RT concentrations and extreme PCR., Competing Interests: Declaration of competing interest Related patents: US 9,932,634 and US 10,144,960 (CTW). Related published patent application: US20190002954A1 (CTW and JFQ). New aspects of this work are also the subject of a provisional patent application., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Introduction: Cryptosporidium is an intestinal parasite responsible for gastroenteritis. Conventional diagnosis of Cryptosporidium is made by microscopy. The most frequent molecular detection method for this parasite is polymerase chain reaction (PCR). The objective of the present study was to identify the novel DNA targets and development of PCR-based assays for the specific detection of two major human infecting species Cryptosporidium parvum and Cryptosporidium hominis., Methodology: Sensitive and specific SYBR green quantitative PCR (qPCR) and TaqMan qPCR assays were developed and validated at both diagnostic and analytical level using the new identified targets TU502HP-1 and TU502HP-2., Results: Assay validation results showed that the newly developed real-time PCR assays are 100% specific with a reliable limit of detection. Overall repeatability and reproducibility of these assays showed good quality results over intra- and inter-laboratory analysis., Conclusion: Novel target-based qPCR assays can be rapid an efficient tool for simultaneous detection of a C. parvum and C. hominis. These genes could also be utilized for the development of innovative DNA-based Point-of-Care test development., Competing Interests: None

We herein describe Hairpin probe-mediated Isothermal Amplification (HIAmp), a novel isothermal method to detect a target nucleic acid. This method employs a hairpin probe (HP) designed to be opened through binding to the target nucleic acid. Upon opening of the HP, the primer binds to the free stem of the opened HP followed by its extension by DNA polymerase, consequently displacing and recycling the target nucleic acid to open another HP and producing an intermediate product (IP) containing a nicking site. The IP then continuously produces a trigger probe (TP), which subsequently initiates the isothermal amplification cycles in two separate ways by binding to either the intact HP or the overhang region of the IP. Through the following well-designed interconnected pathways, a large amount of final double-stranded DNA products (FPs) is produced and a high fluorescent signal is generated from the duplex-specific fluorescent dye, SYBR Green I. By employing this isothermal strategy, target DNA was very sensitively detected down to 64 zmol with the capability to discriminate the target DNA against non-specific DNAs. This work would provide remarkable insight into the design of a new DNA network enabling isothermal amplification., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Plasmodium malariae mainly causes asymptomatic submicroscopic parasitemia in the endemic Amazon and non-endemic Atlantic Forest, where the number of cases and transmission of malaria through blood transfusion has increased. This study developed a P. malariae/P. brasilianum Real Time PCR (rtPCR) targeting the cytochrome b oxidase (cytb), a highly repetitive gene (20-150 copies/parasite) that should detect more cases than the 18S rRNA (4-8 copies/parasite) gene-based amplification systems. Cytb from human and non-human Plasmodium species (including P. brasilianum) aligned to the only 20 African P. malariae cytb sequences identified polymorphic regions within which we designed P. malariae species-specific primers. Non-human Plasmodium species, related parasites, anemia-causing microorganisms, normal human DNA and 47 blood bank donors samples that were truly negative to malaria accessed rtPCR specificity. Truly positive samples (n = 101) with species identification by semi-nested, nested or TaqMan PCR, and four samples from the Atlantic Forest that were suspected of malaria but three of them had negative genus TaqMan and 18S rRNA nested PCR. The cloned amplification product used in standard curves determined qPCR detection limit (0.5-1 parasite equivalent/μL). The 10 positive P. malariae samples among truly positives yielded positive rtPCR results and more importantly, rtPCR detected the four samples suspected of malaria from the Atlantic Forest. The rtPCR specificity was 100%, reproducibility 11.1% and repeatability 6.7%. In conclusion, the proposed rtPCR is fast, apparently more sensitive than all 18S rRNA amplification systems for detecting extremely low parasitemia. The rtPCR is also specific to P. malariae/P. brasilianum species. This new molecular tool could be applied to the detection of P. malariae/brasilianum infections with submicroscopic parasitemias in the context of epidemiological studies and blood bank safety programs., Competing Interests: Declaration of Competing Interest The authors have no conflict of interest to declare., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Haemonchus contortus is one of the most important gastrointestinal nematodes (GINs) infecting sheep, goats, and cattle worldwide. We developed a SYBR Green real-time PCR (qPCR) assay for detection and quantification of H. contortus by using specific primers based on a conserved region of the mitochondrial cytochrome oxidase subunit I (mt-COI) gene, and evaluated this technique in the detection of H. contortus infections in cattle in Central Anatolia Region of Turkey. The newly developed qPCR assay successfully discriminated H. contortus from other GIN species infecting cattle in the specificity evaluations, with a specific melt peak of 77.5 °C. Our results revealed the efficient amplification of the proposed target COI region within the range of plasmid copies, from 2 × 10 6 to 2 × 10 1 per μl, with 96.9 % efficiency, R² value of 0.999, and a slope of -3.398. Among the 920 cattle fecal samples from the field, 58 samples (6.3 %) were positive with qPCR assay, whereas 45 samples (4.9 %) were positive, as determined by larval culture, suggesting the utility of SYBR Green qPCR. Phylogenetic characterization of the partial COI gene of H. contortus isolates was also evaluated for 100 eggs and third stage larvae recovered from positive cattle faecal samples, which were verified with the qPCR assay prior to analyses. COI sequences were classified into three haplotypes (THC1 to THC3) with intraspecific nucleotide differences of 0.50 to 0.76 %. Phylogenetic analyses revealed that the haplotypes grouped with H. contortus isolates from several countries in a monophyletic cluster, with evidence of at least two main haplogroups. Overall, the SYBR Green qPCR assay was highly specific and sensitive, suggesting that it can be used for screening of H. contortus infections in livestock populations in epidemiological studies and the control of this important parasite., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Because STAT3 is a potent proto-oncogene, screening STAT3 gene has potential for use in tumor diagnosis, classification of subtypes, and molecular target therapy. Thus, in this study, using STAT3 gene as the model molecule, we developed a novel amplification strategy, ultrasensitive rolling circle amplification (THP-RCA) based on target-catalyzed hairpin structure-mediated padlock cyclization, for the ultrasensitive detection of human proto-oncogenes in a homogenous solution. In this system, HP1 was designed as the cyclization template and RCA reaction primer, while HP2 was the padlock probe. The two probes can fold into a hairpin structure via the self-hybridization and thus lock the signaling process in the absence of target species. The hybridization of HP2 with HP1 in an end-to-end fashion occurs with the help of target DNA. Subsequently, HP2 is cyclized by ligase on HP1 template. Interestingly, during the hybridization and enzymatic cyclization of HP2, the target DNA only serves as the catalytic probe and is not exhausted. The cyclized HP2 enables the rolling circle amplification, generating a long tandem single-stranded (ss) DNA product that is capable of hybridizing with considerable quantity of molecular beacons (MBs). As a result, the dramatically amplified fluorescence value is achieved for the ultrasensitive detection of the STAT3 gene. As a result, target DNA is able to be quantified down to 100 fM with a high specificity towards wild-type target DNA. Moreover, the sensing system is suitable for the target detection in human serum. The novel sensing strategy shows tremendous prospect for application in tumor diagnosis and clinical therapy guidance., (Copyright © 2019 Elsevier B.V. All rights reserved.)

An ultrasensitive strategy based on sandwich immunoassay coupled with isothermal exponential amplification reaction (IMEXPAR) is proposed for the determination of tumor protein Mucin 1 (MUC1). An immuno-PCR plate was prepared from modification of the primary MUC1-antibody (Ab 1 ) onto the inner-well of the PCR plate. A biotinylated secondary MUC1-antibody tagged with the biotinylated EXPAR primer (P-Ab 2 ) was prepared through biotin-streptavidin reaction. In the presence of target MUC1, sandwich-type combinations were specifically formed in the immuno-PCR plate. With further addition of amplification template, polymerase and nicking enzyme, EXPAR was specifically triggered, producing numerous primer replica in minutes, and greatly enhanced fluorescence of SYBR Green I. The proposed strategy has a good linear relationship with the logarithm of the MUC1 concentration ranging from 3 pM to 3 nM with a limit of detection of 1.63 pM (S/N = 3), which is two orders of magnitude lower than those of other methods. Owing to the specificity of immuno-reaction and EXPAR, the selectivity of the strategy is favorable, even if for the homologous protein. The proposed strategy was further applied for the MUC1 determination in human serum, and a satisfactory recovery range of 98.7%-105.3% was obtained. The strategy can be facilely extended to the ultrasensitive determination of various proteins., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Micrococcal nuclease (MNase) is a nonspecific endo-exonuclease that digests single-stranded/double-stranded DNA and RNA. The existence of MNase can serves as an important diagnostic biomarker of Staphylococcus aureus (S. aureus) infection. However, most of the substrates in MNase-based sensors are single-stranded DNA, which could also be digested by exonuclease I or S1 nuclease and interfere the MNase detection. In this work, we developed a highly selective fluorescent method for MNase detection using a specific dsDNA and nucleic acid dye SYBR Green I (SGI) as the indicator. After rational design, an AT-rich dsDNA with 3' protruding termini was screened as the high-specific substrate of MNase assay and efficient enhancer of SGI. The AT-rich dsDNA substrate can resist the digestion of other exonuclease and greatly enhance the fluorescence of SGI. This high-specific substrate-based probe can detect MNase in buffer as well as biological sample with highly selectivity. Moreover, this method was also applied to monitor the MNase secreted by S. aureus. Thus, the proposed MNase-based assay has a strong potential to identify S. aureus in food safety and microbial infection due to its excellent analytical sensitivity and high selectivity., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The chromatographic properties of three hyperbranched anion exchangers having various diamines in the external part of the functional layer are studied in order to reveal diamine influence on selectivity toward mono- and divalent organic acids. The obtained stationary phases have the same structure of the internal part of the functional layer formed by repeating 4 modification cycles including alkylation with 1,4-butanediol diglycidyl ether (1,4-BDDGE) and amination with methylamine (MA) and differ by the structure of diamine used in the 5 th modification cycle. For the first time several diamines (ethylenediamine, (2-aminoethyl)aminoethanol, and N,N'-bis(2-hydroxyethyl)ethylenediamine) are used for completing the last modification cycle in hyperbranching. The performance of three prepared anion exchangers is investigated using KOH and NaHCO 3 as eluents and discussed with respect to the differences in hydrophilicity of the external part of the functional layer showing its effect on the separation of organic acids., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Bombing accounts for the largest share of terrorist incidents worldwide. Most involve an improvised explosive device (IED): a bomb made from household items. Touch DNA may be left on parts of an IED during assembly. However, an IED conflagration degrades DNA, and there has never been a way to locate where touch DNA may remain. To solve this problem, we combined the use of fluorescent dye to locate latent DNA and direct PCR to improve STR profiles of DNA obtained from IEDs. Six fluorescent DNA-binding dyes were evaluated at various concentrations for the purpose of staining latent DNA. SYBR® Green I and Diamond™ Nucleic Acid dye were able to visualize touch DNA on IED substrates. Inhibition studies with extracted DNA and touch DNA using both dyes revealed that Diamond™ dye inhibited direct STR amplification, while SYBR® Green I did not. Stability studies at three temperatures showed optimum performance of SYBR® Green I up to 24 h after formulation. As such, only SYBR® Green I was further used to develop a "visualized-direct PCR" method. Using the conventional approach and the novel "visualized-direct PCR" approach in a single-blind investigation of mock IED evidence, the "visualized-direct PCR" approach had a 98.6% chance of obtaining more alleles (95% highest density interval (HDI): 0.7 to 10.0 alleles). A decrease in non-donor's alleles (mixed profiles) was also observed. The developed approach has the potential to revolutionize the process of STR typing from touch DNA., (Copyright © 2019 Elsevier B.V. All rights reserved.)

In this study, based on hybridization chain reaction (HCR) amplification and graphene oxide (GO), we developed a facile enzyme-free signal amplification strategy for sensitive detection of Staphylococcus aureus (S. aureus). Two hairpin probes (HP 1 and HP 2 ) labeled by fluorophore 6-carboxyfluorescein (FAM) are designed. The HP 1 and HP 2 can not only trigger to the HCR but also form a long nicked double strand DNA (dsDNA) with the target (16S rRNA). In the absence of target (16 s RNA), the free FAM-labeled HP1 and HP2 are adsorbed by the GO via π-π stacking, the fluorescence signal is quenched. In the presence of target (16 s RNA), the HCR is triggered and dsDNA complexes are generated. As a result, the fluorescence signal can be strongly amplified by the synergistic effect of FAM and the dsDNA dye SYBR Green I. Based on this mechanism, a fluorescence method is designed for the detection of 16S rRNA of S. aureus. Under the optimal conditions, it has low detection limit (50 pM) and a linear response in a concentration range of 50 pM to 100 nM for 16S rRNA. Furthermore, this method has also been successfully applied to the detection of S. aureus in milk sample with the detection limit of 4 × 10 2  CFU·mL -1 ., (Copyright © 2019 Elsevier B.V. All rights reserved.)

MicroRNAs (miRNAs) play an important role in the regulation of various biological processes and have been used as potential biomarkers for biomedical research and clinical diagnosis. Here, nicking-mediated rolling circle amplification (N-RCA)/symmetric isothermal circular strand-displacement amplification (S-SDA)-integrated combined cascade amplification (rs-CCA) was proposed for let-7a miRNA detection. Via introducing a palindromic fragment-integrated recognition sites for nicking endonuclease Nt.AlWI into the padlock probe, the hybridization of target miRNA can induce N-RCA and continuously generate the nicked fragments (NFs). Because the released NFs each have a palindromic sequence and daughter nicking site at the 3' end, they hybridize with each other, followed by alternately extension by polymerase and nicking by endonuclease. This leads to S-SDA effect, and the released single-stranded products in turn hybridize with NFs, accomplishing the rs-CCA process. When the Sybr Green I dyes intercalate into double-stranded DNA products, the amplified fluorescence signal is achieved. Thus, the target miRNA can be detected down to 5 pM. Importantly, the rs-CCA system is capable of distinguishing the single base difference between target miRNAs, indicating the high sequence specificity. Moreover, its potential application in disease diagnosis was demonstrated via detecting target miRNA in complex biological matrix and analyzing the total RNAs extracted from HeLa cells. As a proof-of-concept building, the impressive rs-CCA scheme is expected to provide a valuable insight into constructing powerful signal amplification strategies via sophisticated combination of biotechnologies available for nucleic acid manipulation and significantly benefit biomedical research and disease diagnostics., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Mercury ion (Hg 2+ ) is a heavy metal that can cause serious water pollution. With the accumulation of large quantities in lakes, rivers, freshwater and aquatic life, Hg 2+ can pass through the food chain, entering the human body and endangering health. Hg 2+ detection has therefore become important thereby attracting extensive interests. Currently, several DNA-based sensors have been used for Hg 2+ detection because they are not easy to degrade and are very stable. This paper summarizes the application of some DNA-based sensors based on fluorescence resonance energy transfer (FRET), analyzes their characteristic, and compares their sensitivity. Future perspectives and possible challenges in this area are also outlined., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A novel and highly sensitive method based on bio-barcode with rolling circle amplification (RCA) was developed for the detection of T-2 toxin. Gold nanoparticles (AuNPs) were modified with anti-T-2 monoclonal antibody and single-stranded thiol-oligonucleotides (SH-ssDNAs) and magnetic microparticles (MMPs) coated with T-2 antigen. The T-2 toxin competes with the antigen on MMPs for the anti-T-2 antibody on AuNPs. Then, the isolating complex system was separated by a magnetic field, and the DNA of the probes was released after washing in dithiothreitol solution. The barcode DNA via RCA and products were stained by SYBR Green I and then detected by fluorescence spectrophotometry. The optimized method was performed on oats, millet, flour, and other substances. This method exhibits a low limit of detection (0.26 pg mL -1 ) and linear range of 0.002-200 ng mL -1 . Moreover, the approach offers good recovery and relative standard deviations ranging from 88.65% to 10.04% and 0.6%-13.1%, respectively. In conclusion, this method exhibits potential for use as an ultrasensitive assay for the detection of a variety of small molecules in complex matrices., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In this study, a pair of primers were designed and synthesized for the gB gene of infectious laryngotracheitis virus (ILTV) (GenBank accession number: EU104985). The recombinant plasmid was constructed as the positive reference material, and a real-time fluorescence-based quantitative PCR (RFQ-PCR) method was established to detect ILTV using synergy brands (SYBR) Green I. This method could detect 3.34 × 103 copies/μL viral nucleic acid in the initial template, the sensitivity of this method was higher than that of the conventional PCR, and the coefficient of variation (CV) in the repeatability test by this method was 3.35%. At the same time, the method was used to detect 14 suspected pathological samples for clinical analysis, and the results showed that 10 positive samples were detected, and the standard S-shaped curve was amplified. It was concluded that the RFQ-PCR method established in this study was highly sensitive, specific and repeatable, it was suitable for early clinical detection and epidemiological investigation of ILTV, and was significant in effectively controlling the occurrence and transmission of ILTV.

A novel fluorescent method based on tetracycline-binding aptamers and the luminescence of SYBR Green I (SGI) was established for the sensitive and selective detection of tetracycline. Under natural conditions, the aptamers of tetracycline show the G-quadruplex spatial structures while SGI is nearly nonfluorescent in aqueous solution. After mixture with the G-quadruplex structured aptamers, SGI can recognize and intercalate into the aptamers, resulting in a strong fluorescence emission. When the target tetracycline was added into the solution, the specific recognition and high-affinity binding of aptamers with tetracycline will induce the conformational changes of aptamers from G-quadruplex structures to hairpin structures. Thereafter, SGI will be released from the aptamer molecules, leading to the fluorescence decline. The quantitative detection of tetracycline can be achieved by measuring the fluorescence change of the system. Under the optimum conditions, the linear range of tetracycline in the milk was from 5 to 25 μg/mL, and the detection limit was as low as 0.10 μg/mL. The recoveries of the spiked milk samples were in the range of 98.98%-104.67% with the relative standard deviations (RSDs) of 0.16%-0.67%, and the results were in agreement with those from HPLC. Therefore, the biosensor based on the specific recognition of aptamers and the fluorescence properties of SGI can detect the tetracycline in milk accurately, rapidly and specifically., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Avian leukosis virus subgroup K (ALV-K) is an emerging ALV tumor virus of chickens. We developed a SYBR green-based real-time polymerase chain reaction (PCR) assay for the rapid and economical detection of ALV-K in chicken flocks. The assay was specific for ALV-K and did not cross-react with other ALV subgroup or avian influenza virus, Newcastle disease virus, or Marek's Disease virus. The method was 100 times more sensitive than conventional PCR and 10 times more sensitive than the enzyme-linked immunosorbent assay (ELISA) for the P27 antigen. The assay was also more sensitive than conventional PCR in tests of 86 clinical plasma samples. DF-1 tissue culture cells infected with 1 TCID50 ALV-K particle were identified as negative using ELISA but tested positive with the real-time PCR method. The viral loads in organs and tissues in infected chickens were highest in kidney, lungs, and glandular stomach, and these results matched ELISA findings.