One-step Protocol Research Articles

Functionalization of Gadolinium Chelates Silica Nanoparticle through Silane Chemistry for Simultaneous MRI/64Cu PET Imaging.

Multimodal nanoprobes are highly demanded for biomedical imaging applications to enhance the reliability of the diagnostic results. Among different types of nano-objects, ultrasmall silica gadolinium nanoparticle (SiGdNP) appears as a safe, effective, and versatile platform for this purpose. In this study, a new method to functionalize SiGdNP based on silane chemistry has been reported. Two types of chelating silanes (APTES-DOTAGA and APTES-NODAGA) have been synthesized and grafted on SiGdNP by a simple one-step protocol. This functionalization strategy requires no other reactants or catalyzers and does not compromise the ultrasmall size of the particles. NODAGA-functionalized particle has been labeled with 64Cu isotope and injected intravenously to mice bearing TS/A carcinoma tumor for biodistribution study to demonstrate its potential as a bimodal MRI/PET imaging agent. A fully integrated MRI/PET system was used to simultaneously monitor the distribution of the particle. The results showed that the functionalized particle maintained properties of a renal clearable NP which could rapidly escape through kidneys and had low retention in other organs, especially liver, even though its accumulation in the tumor was modest.

One-Step Multiplex Quantitative RT-PCR for the Simultaneous Detection of Viroids and Phytoplasmas.

A one-step multiplex quantitative reverse transcription polymerase chain reaction protocol is described, for the detection in pome trees of Pear blister canker viroid and Apple scar skin viroid, together with universal detection of phytoplasmas. Total nucleic acids extraction is performed according to a modified CTAB protocol and TaqMan MGB probes are used to surpass high genetic variability of viroids. The multiplex real-time assay is at least ten times more sensitive than conventional protocols and its features make it suitable for rapid and massive screening of pome fruit trees phytoplasmas and viroids in certification schemes and surveys.

Islet-Like Structures Generated In Vitro from Adult Human Liver Stem Cells Revert Hyperglycemia in Diabetic SCID Mice

A potential therapeutic strategy for diabetes is the transplantation of induced-insulin secreting cells. Based on the common embryonic origin of liver and pancreas, we studied the potential of adult human liver stem-like cells (HLSC) to generate in vitro insulin-producing 3D spheroid structures (HLSC-ILS). HLSC-ILS were generated by a one-step protocol based on charge dependent aggregation of HLSC induced by protamine. 3D aggregation promoted the spontaneous differentiation into cells expressing insulin and several key markers of pancreatic β cells. HLSC-ILS showed endocrine granules similar to those seen in human β cells. In static and dynamic in vitro conditions, such structures produced C-peptide after stimulation with high glucose. HLSC-ILS significantly reduced hyperglycemia and restored a normo-glycemic profile when implanted in streptozotocin-diabetic SCID mice. Diabetic mice expressed human C-peptide and very low or undetectable levels of murine C-peptide. Hyperglycemia and a diabetic profile were restored after HLSC-ISL explant. The gene expression profile of in vitro generated HLSC-ILS showed a differentiation from HLSC profile and an endocrine commitment with the enhanced expression of several markers of β cell differentiation. The comparative analysis of gene expression profiles after 2 and 4 weeks of in vivo implantation showed a further β-cell differentiation, with a genetic profile still immature but closer to that of human islets. In conclusion, protamine-induced spheroid aggregation of HLSC triggers a spontaneous differentiation to an endocrine phenotype. Although the in vitro differentiated HLSC-ILS were immature, they responded to high glucose with insulin secretion and in vivo reversed hyperglycemia in diabetic SCID mice.

Evaluation of a one-step sample preparation protocol for analysis of total serum testosterone by LC–MS/MS

In this study, a one-step method for liquid-liquid extraction has been compared against a two-step procedure for testosterone assays in terms of accuracy, specificity, recovery, lipid removal and baseline noise, using QCs and unknown samples. The difference in accuracy was less than 5% for adult sera, while it was less than 10% for prepubescent sera. To compare specificity, the ion ratio transition of 289 → 97 to 289 → 109 was monitored for all QCs and unknown samples; no interference in the testosterone peak was observed for any tested sample prepared by either the one-step or two-step procedure. The baseline comparison of LC–MS/MS chromatograms of samples indicated that samples prepared by the one-step procedure were of the same quality as those prepared by the two-step procedure; however, recovery in unaltered serum using the one-step procedure was approximately 15% greater across the low to high concentration range. Furthermore, recovery using the one-step procedure was more consistent between stripped and unstripped serum. Lipids were removed efficiently in the two-step procedure as verified by monitoring the typical phospholipid MRM transition of m/z 496 → 184. For every sample processed by the one-step procedure, a one-minute online column wash with 95% methanol was able to remove 95% of the bound lipids, thereby providing a column life-time approximately equivalent to that for the two-step procedure. The presented data indicate that the one-step procedure could replace the two-step procedure while maintaining accuracy, saving time, increasing recovery, and minimizing the potential for errors with the fewer steps required.

Self-assembly directed one-step synthesis of [4]radialene on Cu(100) surfaces

The synthetic challenges of radialenes have precluded their practical applications. Here, we report a one-step synthetic protocol of [4]radialene on a copper surface. High-resolution scanning tunneling microscopy measurements reveal that such catalytic reaction proceeds readily with high selectivity at the temperature below 120 K. First-principles calculations show that the reaction pathway is characterized by firstly the cooperative inter-molecular hydrogen tautomerization and then the C–C bond formation. The feasibility of such cyclotetramerization reaction can be interpreted by the surface effect of Cu(100), which firstly plays an important role in directing the molecular assembly and then serves as an active catalyst in the hydrogen tautomerization and C–C coupling processes. This work presents not only a novel strategy to the scant number of synthetic methods to produce [4]radialenes via a novel [1 + 1 + 1 + 1] reaction pathway, but also a successful example of C–C bond coupling reactions guided by the surface-induced C–H/π assembly.

Interrogating the Electrochemical Potential of Senna obtusifolia Mediated Biosynthesized Silver Nanoparticles

Hazards and toxicity associated with the physical and chemical synthesis protocol of metal nanoparticles has recently led researchers to seek alternative routes that is eco-friendly, cheap and fast; the green chemistry approach where plant extracts and microorganisms are used in the reduction of the metal salt is fast gaining popularity in the field of nanobiotechnology. However, controversy still trails acceptability; furthermore, electrochemical studies on these metal nanoparticles are limited. In this study, silver nitrate was reduced to its “nano silver form” through a one-step synthesis protocol using leaf extract of Senna obtusifolia . The usual microscopic and spectroscopic techniques such as UV-vis., FTIR, SEM, AFM and XRD were used to confirm the formation of silver nanoparticles. Electrochemical characterization using cyclic voltammetry and impedance spectroscopy further reveals the formation of silver nanoparticles via a redox process of Ag 1+ to Ag 0 with a formal potential value of 0.61 V vs. Ag|AgCl 3 M KCl.

Cryosurvival and pregnancy rates: One-step protocol for freezing-thawing Shangri-la Yak (Bos grunniens) Embryos.

The yak is one of the most important and economically useful animals for highlanders. The decline in the yak population requires effective measures for the conservation and multiplication of elite germplasm. A standardized protocol will simplify the freezing and warming of yak embryos in straw and facilitate embryo transfer. In this work, we investigated a one-step protocol that uses a stable basal medium, which comprised a warming medium (1.08M sucrose) and a freezing medium (EFS40). We also assessed the effects of the new transfer method on embryo survival. A total of 145 yak frozen embryos were thawed in a standard medium system. The one-step protocol led to a high recovery percentage (84.93) of yak embryos that survived vitrification and warming. The in vitro survival rates of these embryos significantly different from those of embryos frozen-thawed via the conventional method. The 95 embryos frozen-thawed via our one-step protocol were then implanted in selected recipients. Thirty-six singleton pregnancies were established. In conclusion, the proposed one-step method is a simple, safe, and standardized freezing-thawing protocol that ensures embryo survival and quality under field conditions. This study establishes new possibilities for the widespread use of embryo transfer in yaks.

Square Wave Voltammetric Detection of Nitrite on Platinum Electrode Modified with Moringa oleifera Mediated Biosynthesized Nickel Nanoparticles

In this study nickel chloride was reduced to its nanoform through a one-step synthesis protocol using the leaves extract of Moringa oleifera. Microscopic and spectroscopic techniques such as UV-vis., FTIR and SEM were used to confirm the formation of nickel nanoparticles. Electrochemical characterization using cyclic voltammetry and square wave voltammetry further reveals the electrochemical activity of synthesised nickel nanoparticles; as preliminary study on nitrite electro-oxidation shows that nickel nanoparticles can catalyse this process better than some other nanoparticles.

Acetate Anion Assisted Crystal Orientation Reconstruction in Organic–Inorganic Lead Halide Perovskite

The crystalline reconstruction along (112)/(200) planes rather than (110)/(002) planes of methylammonium lead iodide (MAPbI3) is realized via an alternative one-step fabrication protocol that does not use the antisolvent dripping process, where methylammonium acetate (MAAc) is incorporated into the precursor solution. It is disclosed that the MAAc additive causes the modulation of the initial orientation of PbI2 from (001) to (101) via formation of the intermediate adduct of PbI2·MAAc, and the sequential deconstruction-reconstruction procedures result in dense perovskite films composed of large grains along (112)/(200) planes, differing from the needle-like crystallites along (110)/(002) planes constructed without MAAc assistance. The resultant films present superior optoelectronic properties, and corresponding solar cells with efficiency as high as 18.91% have been achieved at the MAAc content of 11%. Furthermore, anisotropic properties have been observed with respect to different crystal orientation, wh...

One-Step Anionic Copolymerization Enables Formation of Linear Ultrahigh-Molecular-Weight Block Copolymer Films Featuring Vivid Structural Colors in the Bulk State.

Ultrahigh-molecular-weight (UHMW) tapered block copolymers (BCPs) consisting of polyisoprene- block-poly(4-methylstyrene) featuring overall molar masses in the range of 1101-2033 kg mol-1 ( Mw) are synthesized via a convenient one-step anionic copolymerization protocol. The obtained UHMW BCPs are investigated by differential scanning calorimetry, size exclusion chromatography, and 1H NMR spectroscopy. Microphase separation for the UHMW BCPs in the bulk state is investigated by transmission electron microscopy (TEM) measurements and scanning electron microscopy (SEM), revealing well-ordered lamellar and spherical domains with large domain sizes in the range of 100-200 nm. Excellent order and periodicity are observed for lamellar morphologies over large film areas of 90 × 120 μm. Because of this high order of the underlying domains and the different refractive indices of the block segments, vivid structural colors can be observed in the bulk state. Structural colors of BCP films are investigated by angle-dependent UV/vis measurements, revealing intensive reflection colors according to Bragg's law of diffraction. The optical characteristics are directly correlated to TEM and SEM results. Moreover, colored BCP films featuring spherical domains for one block segment with domain sizes of 97-122 nm revealed blue structural colors stemming from disordered particle scattering.

Circular permutation profiling by deep sequencing libraries created using transposon mutagenesis.

Deep mutational scanning has been used to create high-resolution DNA sequence maps that illustrate the functional consequences of large numbers of point mutations. However, this approach has not yet been applied to libraries of genes created by random circular permutation, an engineering strategy that is used to create open reading frames that express proteins with altered contact order. We describe a new method, termed circular permutation profiling with DNA sequencing (CPP-seq), which combines a one-step transposon mutagenesis protocol for creating libraries with a functional selection, deep sequencing and computational analysis to obtain unbiased insight into a protein’s tolerance to circular permutation. Application of this method to an adenylate kinase revealed that CPP-seq creates two types of vectors encoding each circularly permuted gene, which differ in their ability to express proteins. Functional selection of this library revealed that >65% of the sampled vectors that express proteins are enriched relative to those that cannot translate proteins. Mapping enriched sequences onto structure revealed that the mobile AMP binding and rigid core domains display greater tolerance to backbone fragmentation than the mobile lid domain, illustrating how CPP-seq can be used to relate a protein's biophysical characteristics to the retention of activity upon permutation.

Comparison of methylation methods for fatty acid analysis of milk fat

Three acid- and alkaline-catalysed transesterification methods were compared with the aim to validate a simple yet reliable protocol for fatty acid (FA) profiling of milk fat. While both the acid- and alkaline-catalysed methods were able to convert completely triglycerides and phospholipids into fatty acid methyl esters (FAMEs), the acid catalyst caused significant degradation of conjugated linoleic acid C18:2c9t11 at high temperature. Although a milder temperature can mitigate this negative impact, a long reaction time (2 h) is required to achieve full methylation. By contrast, despite being unable to methylate free fatty acids (FFA), the alkaline-catalysed transesterification yielded comparable results for all major FA due to the very low level of FFA in milk. The alkaline-catalysed methylation is benign for C18:2c9t11. We recommend here a simple one-step protocol based on 0.2 M methanolic KOH, a short reaction time (20 min) and a mild reaction temperature (50 °C) for milk FAME preparation.

Detecting potato viruses using direct reverse transcription quantitative PCR (DiRT-qPCR) without RNA purification: an alternative to DAS-ELISA

Virus screening is obligatory to avoid the spread of plant viruses regionally and globally. Double-antibody sandwich (DAS)-ELISA is the standard for screening potato viruses owing to its high-throughput potential, robustness, and cost–benefit ratio. However, low virus titers present in dormant potato tubers may not be reliably detected by using DAS-ELISA. Virus enrichment for reliable virus detection by DAS-ELISA assay is time-consuming and can be avoided by switching to more sensitive molecular biological techniques. Therefore, we developed a TaqMan® qPCR-based one-step protocol, termed direct reverse transcription quantitative PCR (DiRT-qPCR) for detection of RNA potato viruses PVY, PLRV and PVS without sophisticated nucleic acid purification and providing a high-throughput potential. Compared with DAS-ELISA, DiRT-qPCR showed up to a 100,000,000-fold higher sensitivity depending on the virus species. We also compared the qualitative results of standard DAS-ELISA used in seed potato certification, performed by sampling leaves of at least 4-weeks-old cultivated tuber eye cuttings, to the 1.5 h long DiRT-qPCR protocol on dormant tubers. The DiRT-qPCR protocol achieved an agreement with the DAS-ELISA procedure of 92.8%, 84.1% and 82.3% for the detection of PLRV, PVY, and PVS, respectively. The investigated different virus species show different multiplication behavior in secondary infected potato tuber eye cuttings, which is assumed to be a reason for the remaining qualitative differences in the outcome of the DiRT-qPCR and DAS-ELISA comparison. In our opinion, DiRT-qPCR protocol can be used as a reliable, work- and resource-saving alternative to DAS-ELISA in qualitative directed virus detection, particularly because no RNA purification is needed and dormant potato tubers can be directly used.

Simultaneous reprogramming and gene editing of human fibroblasts.

The utility of human induced pluripotent stem cells (iPSCs) is enhanced by an ability to precisely modify a chosen locus with minimal impact on the remaining genome. However, the derivation of gene-edited iPSCs typically involves multiple steps requiring lengthy culture periods and several clonal events. Here, we describe a one-step protocol for reliable generation of clonally derived gene-edited iPSC lines from human fibroblasts in the absence of drug selection or FACS enrichment. Using enhanced episomal-based reprogramming and CRISPR/Cas9 systems, gene-edited and passage-matched unmodified iPSC lines are obtained following a single electroporation of human fibroblasts. To minimize unwanted mutations within the target locus, we use a Cas9 variant that is associated with decreased nonhomologous end-joining (NHEJ) activity. This protocol outlines in detail how this streamlined approach can be used for both monoallelic and biallelic introduction of specific base changes or transgene cassettes in a manner that is efficient, rapid (∼6-8 weeks), and cost-effective.

An efficient synthesis of multisubstituted 4-nitrobuta-1,3-dien-1-amines and application in cyclisation reactions

The synthesis of multisubstituted 4-nitrobuta-1,3-dien-1-amines (nitrodienamines) from 3-aminocrotonates and nitroacetaldehyde potassium salt, has been performed in 45–89% yields. This one-step protocol works efficiently with a broad range of N-H and N-substituted 3-aminocrotonates and delivers both primary and secondary nitrodienamines. In addition, the possible variations of the substituents at the positions 2 and 3 of 4-nitrobuta-1,3-dien-1-amine have been shown. Generally, the yields of secondary 4-nitrobuta-1,3-dien-1-amines were lower than those of primary ones. The synthetic usefulness of obtained 4-nitrobuta-1,3-dien-1-amines has also been demonstrated by achieving the synthesis of multisubstituted 5-nitro-1,6-dihydropyridines in two-component cyclocondensation reactions of 4-nitrobuta-1,3-dien-1-amines with aromatic or aliphatic aldehydes. Lastly, diverse N-H and N-substituted 5-nitro-1,6-dihydropyridines have been obtained in 35–87% yields.

Facile preparation of highly pure KF-ZrF4 molten salt

The preparation of highly pure KF-ZrF4 (FKZr) molten salt, a potential secondary coolant in molten salt reactors, was realized simply by heating a mixture of (NH4)2ZrF6 and KF. X-ray diffraction analysis indicated that the FKZr molten salt was mainly composed of KZrF5 and K2ZrF6. The melting point of the prepared FKZr molten salt was 420–422 °C under these conditions. The contents of all metal impurities were lower than 20 ppm, and the content of oxygen was lower than 400 ppm. This one-step protocol avoids the need for a tedious procedure to prepare ZrF4 and for an additional purification process to remove oxide impurities, and is therefore a convenient, efficient and economic preparation method for high-purity FKZr molten salt.

Retrieval and cryopreservation of sperm in spermatophores from cadaveric Indian white shrimp, Fenneropenaeus indicus (H. Milne Edwards, 1837)

This study focused on the quality of sperm obtained from spermatophores of cadaveric shrimp after long-term storage. Spermatophores were collected using the stripping method, which has resulted in maximum sperm viability when this approach was previously used. Cryoprotectants toxicity assessment of samples was conducted using dimethyl sulfoxide (DMSO), methanol (MeOH), ethylene glycol (EG), glycerol (Gly), dimethyl acetamide (DMA) and propylene glycol (PG) at different concentrations (5%, 10% and 30% v/v), prepared in Ca-F saline. Based on the results from the cryoprotectant toxicity assay, DMSO and MeOH were used individually as well as in combination for the subsequent study. Samples along with cryoprotectants were subjected to slow and fast freezing protocols (i.e. −0.5, and −10 °C/min to a final temperature of −80 °C) and were subsequently stored in LN2 (196 °C). Similarly, vitrification was performed by plunging the samples directly in to LN2. Samples of control and cryopreserved spermatophores that were stored for 45 days had sperm viabilities of 91.4 ± 3.6% and 53.9 ± 4.7%, respectively. Further observations with HOST and DNA integrity analyses of the cryopreserved sperm, resulted in percentages of 45.6 ± 4.2%; 58.1 ± 1.7% compared to control values of 82.3 ± 4.8%; 94.3 ± 1.9%, respectively. Use of the one-step slow freezing protocol at the rate of -0.5 °C/min between 4 °C and −80 °C in LN2 with DMSO (5%) + MeOH (5%) was a desirable preservation strategy of spermatophores, compared to other freezing protocols. Unlike sperm viability, the HOST results affirm the fertility potential of the sperm that have the capacity to participate in the fertilization process. Thus, the results of this study demonstrate that long term storage of sperm in spermatophores of Fenneropenaeus indicus collected from cadaveric specimens can result in viable sperm after cryopreservation if extender (Ca-F saline) containing DMSO and MeOH are used.

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts.

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslips for electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examples of the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skin biopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a wide array of biomedical applications, including disease modeling, drug screening, and target validation.

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslips for electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examples of the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skin biopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a wide array of biomedical applications, including disease modeling, drug screening, and target validation.

Rapid one-step extraction method for the identification of molds using MALDI-TOF MS

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has revolutionized fungal identification. Previously, we developed a MALDI-TOF MS mold extraction procedure and comprehensive database. While MALDI-TOF MS has become routine in a few laboratories, it has not yet become widespread. A major obstacle is the lack of a simple, reproducible and uniform protein extraction procedure. In this study, we developed and validated a rapid one-step protein extraction protocol for filamentous fungi. Excised molds were placed into tubes containing zirconia-silica beads and extraction solution without washing or ethanol inactivation steps. Extraction solutions containing different ratios of acetonitrile and formic acid were evaluated. Samples were then processed using a PowerLyzer high power bead based homogenizer and supernatants spotted for MALDI-TOF MS. The rapid method was evaluated prospectively and in parallel to our current mold extraction protocol for 3 months. Analysis of 106 clinical mold isolates resulted in an improved performance and a decrease in extraction time by 30 minutes to a total of 5 minutes of hands-on time. Acceptable identification scores (≥ 2.00) were achieved for up to 63.0% of mold isolates by the rapid method compared with 52.8% of isolates by the current routine protocol. Score comparisons between duplicate spots showed higher reproducibility of the rapid method as compared to the routine method. The rapid extraction method allows efficient analysis of clinical mold isolates both in scheduled batch runs and on an in-demand basis while providing a simple starting platform for laboratories adopting MALDI-TOF MS for mold identification.