Reagent Combination Research Articles

Field-friendly MUNNG® optima simple test kit for quick qualitative assessment of iodine and iron presence in double-fortified salt.

Data from several efficacy studies and a long-term effectiveness study have encouraged the governments to adopt a policy of providing double-fortified salt (DFS) in the Mid-Day Meal (MDM) programs in government schools across India. These envisaged food security events are likely to boost the manufacturing of DFS in a big way. Thus, it becomes pertinent to come up with a robust monitoring system involving community and field workers for quality checks. It is imperative to equip these field workers with simple testing kits (STKs) capable of qualitative detection of iron and iodine in DFS. As the consumer acceptance of foods is based on several factors including sensory characteristics, performance, convenience, cost, nutrition, and product image, a variety of iron compounds are in use for fortification. However, it becomes challenging to provide a kit that can overcome the chemical masking of iodine detection by iron compounds. We aimed at (1) the development of a field-friendly STK for quick qualitative assessment of iodine and various forms of iron present in DFS, (2) to check its validity under field conditions. We put in place reagents combined using known chemical reactions and balanced use of oxidants to overcome the problems of encapsulation and to maximize the use, by enabling reagent combination to react with all forms of iron. The kit reagents successfully detect iodine as well as three commonly used iron fortificants in DFS. Published field trials confirmed the specificity and sensitivity of the developed kit. The simplicity and use of the kit by a field worker can be seen in the enclosed video. The combination of improvised kit reagents allows early detection of iron and iodine in DFS. Iron is detected in a variety of iron-containing fortifications. The provision of diluted H2O2 ensures the presence of oxygen-free radicals that enhances iodine release captured by concentrated KI making iodine detection an easy task.

Machine Learning C-N Couplings: Obstacles for a General-Purpose Reaction Yield Prediction.

Pd-catalyzed C-N couplings are commonplace in academia and industry. Despite their significance, finding suitable reaction conditions leading to a high yield, for instance, remains a challenging and time-consuming task which usually requires screening over many sets of conditions. To help select promising reaction conditions in the vast space of reagent combinations, machine learning is an emerging technique with a lot of promise. In this work, we assess whether the reaction yield of C-N couplings can be predicted from databases of chemical reactions. We test the generalizability of models both on challenging data splits and on a dedicated experimental test set. We find that, provided the chemical space represented by the training set is not left, the models perform well. However, the applicability domain is quickly left even for simple reactions of the same type, as, for instance, present in our plate test set. The results show that yield prediction for new reactions is possible from the algorithmic side but in practice is hindered by the available data. Most importantly, more data that cover the diversity in reagents are needed for a general-purpose prediction of reaction yields. Our findings also expose a challenge to this field in that it appears to be extremely deceiving to judge models based on literature data with test sets which are split off the same literature data, even when challenging splits are considered.

Conceptual advances in nucleophilic organophosphine-promoted transformations.

Catalysis by trivalent nucleophilic organophosphines has emerged as an essential tool in organic synthesis. Several new organic transformations promoted by phosphines substantiate and complement the existing synthetic chemistry tools. Mere design of the substrate and reagent combinations has introduced new modes of reactivity patterns, which are otherwise difficult to achieve. These design considerations have led to the rapid build-up of complex molecular entities and laid a solid foundation to synthesise bioactive natural products and pharmaceuticals. This article presents an overview of some of the conceptual advances, including our contributions to nucleophilic organophosphine chemistry. The scope, limitations, mechanistic insights, and applications of these metal-free transformations are discussed elaborately.

STUDYING THE TECHNOLOGICAL POSSIBILITY OF OBTAINING SILICON DIOXIDE AND SEPARATION OF METALS FROM TECHNOGENIC ORE FORMATIONS

A combined pyro-hydrometallurgical technological method for obtaining silicon dioxide of. technical purpose and metal concentrates from technogenic ore formations resistant to chemical reagents is shown. The smelting of the raw materials prepared for the processing of technogenic formations is carried out by combined reagents, after which the melt formed is leached with an aqueous solution of hydrochloric acid under gel-like conditio¬ns¬. The reduced silicic acid is leached with a weak aqueous solution of hydrochlo¬r¬i¬c acid in order to remove impurities, and to be converted into silicon dioxide, it is subjected to heat treatment in atmospheric air. As a result of the process, the metals contained in technogenic formations are divided into solid and liquid concentrates.

A Combination of Physical and Chemical Treatments Is More Effective in The Preparation of Acellular Uterine Scaffolds.

Decellularized uterine scaffold, as a new achievement in tissue engineering, enables recellularization and regeneration of uterine tissues and supports pregnancy in a fashion comparable to the intact uterus. The acellular methods are methods preferred in many respects due to their similarity to normal tissue, so it is necessary to try to introduce an acellularization protocol with minimum disadvantages and maximum advantages. Therefore, this study aimed to compare different protocols to achieve the optimal uterus decellularization method for future in vitro and in vivo bioengineering experiments. In this experimental study, rat uteri were decellularized by four different protocols (P) using sodium dodecyl sulfate (SDS), with different doses and time incubations (P1 and P2), SDS/Triton-X100 sequentially (P3), and a combination of physical (freeze/thaw) and chemical reagents (SDS/Triton X-100). The scaffolds were examined by histopathological staining, DNA quantification, MTT assay, blood compatibility assay, FESEM, and mechanical studies. Histology assessment showed that only in P4, cell residues were completely removed. Masson's trichrome staining demonstrated that in P3, collagen fibers were decreased; however, no damage was observed in the collagen bundles using other protocols. In indirect MTT assays, cell viabilities achieved by all used protocols were significantly higher than the native samples. The percentage of red blood cell (RBC) hemolysis in the presence of prepared scaffolds from all 4 protocols was less than 2%. The mechanical properties of none of the obtained scaffolds were significantly different from the native sample except for P3. Uteri decellularized with a combination of physical and chemical treatments (P4) was the most favorable treatment in our study with the complete removal of cell residue, preservation of the three-dimensional structure, complete removal of detergents, and preservation of the mechanical property of the scaffolds.

Substrate specific closed-loop optimization of carbohydrate protective group chemistry using Bayesian optimization and transfer learning.

A new way of performing reaction optimization within carbohydrate chemistry is presented. This is done by performing closed-loop optimization of regioselective benzoylation of unprotected glycosides using Bayesian optimization. Both 6-O-monobenzoylations and 3,6-O-dibenzoylations of three different monosaccharides are optimized. A novel transfer learning approach, where data from previous optimizations of different substrates is used to speed up the optimizations, has also been developed. The optimal conditions found by the Bayesian optimization algorithm provide new insight into substrate specificity, as the conditions found are significantly different. In most cases, the optimal conditions include Et3N and benzoic anhydride, a new reagent combination for these reactions, discovered by the algorithm, demonstrating the power of this concept to widen the chemical space. Further, the developed procedures include ambient conditions and short reaction times.

Role of the reducing agent in tungsten extraction by chlorination with chlorine from an ore enriched by physical means

The effect of three reducing agents (sulphur dioxide, pyrite and carbon) on tungsten extraction from a scheelite-wolframite concentrate with chlorine gas was assessed. A low-grade ore (0.9% w/w WO3) was collected in the San Martín department, San Luis Province, Argentina, and subsequently enriched by physical means (65.73% w/w WO3). The minerals identified and their reactivities with chlorine were: scheelite (reactive), wolframite (reactive), pyrite (reactive), quartz (inert) and feldspars (inert). The metal recovered as WO2Cl2, with the different reagent combinations, was in increasing order: Cl2 + N2 < Cl2 + SO2 < pyrite + Cl2 + N2 < pyrite +Cl2 + SO2 < carbon + Cl2 + N2. It was proved that, according to its content, the pyrite initially present in the ore improves chlorination performance. At the same time, the non-volatile reaction products formed - CaCl2 and/or CaSO4 - and the manner of removing the nascent (desorbed) oxygen change depending on the reducing agent tested.

A Concentration Method for HIV Drug Resistance Testing in Low-Level Viremia Samples.

Drug resistance testing in HIV-1 low-level viremia (LLV) samples is challenging yet critical. Our study is aimed at assessing the performance of lentivirus concentration reagent (LCR) in combination with a validated Sanger sequencing (SS) for monitoring drug resistance mutations (DRMs) in LLV samples. A series of clinical samples were diluted and amplified for genotypic resistance testing (GRT) to prove the performance of the LCR. The Stanford HIV-1 drug resistance database (HIVdb version 8.9) was used to analyze the mutations. HIV-1 subtypes and CRFs were determined using the COMET online tool. The overall success rate of genotyping was compared with ultracentrifugation combined with SS. Furthermore, the success rates at varied VL of the two concentration methods were evaluated, and the DRMs of diluted samples were compared with those undiluted samples. When LCR was used, the overall success rate was 90% (72/80) in the PR and RT regions and 60% (48/80) in the IN region. In addition, when HIV RNA was 1000 copies/ml, 400 copies/ml, 200 copies/ml, and 100 copies/ml, the success rates of PR and RT regions were 100%, 100%, 95%, and 65%, respectively, while the success rates of IN region were 85%, 60%, 45%, and 50%, respectively. We found that the sample DR-387A2 missed the E138A mutation, and mutations in other samples were consistent with undiluted samples using LCR. LCR will support monitoring DRMs in HIV-1 patients with LLV and can be an effective alternative for small- and medium-sized laboratories that cannot afford an ultracentrifuge.

Synergistic detoxification by combined reagents and safe filling utilization of cyanide tailings

Cyanide tailings are the major hazardous wastes generated in the production process of the gold industry, which not only contain highly toxic cyanide, but also contain heavy metals with recycling value and other substances suitable for building materials or filling. These tailings are in urgent need of purification treatment and safe utilization. In this study, the impacts of treatment methods, types and combinations of reagents on decyanation effect were researched. Gold in cyanide tailings was recovered by flotation, and flotation tailings were used for filling after identifying the properties of solid waste. Results are as follows: (1) INCO method and 5 reagents (sodium sulfite, sodium persulfate, copper sulfate, ferrous sulfate and zinc sulfate) were selected for synergistic decyanation treatment, and cyanide concents in slurry and leaching solution were decreased to the minimum. (2) The gold recovery rate of the tailings through flotation was increased by 27.8% than without detoxification. (3) Flotation tailings were identified as general industrial solid wastes by leaching toxicity and toxic substance content analysis. (4) As filling aggregate, under the conditions of slurry concentration of 63% and cement-sand ratio of 1:6, the strength filling body of flotation tailings reached 1.32 Mpa after 28 days of maintenance. (5) This process and combined reagents were applied to engineering. The cyanide content in the leaching solution and the flotation recovery rate of gold were kept below 0.2 mg/L and above 60% respectively, and the strength of the filling body was stable to meet the requirements of underground filling.

Sorafenib inhibits doxorubicin-induced PD-L1 upregulation to improve immunosuppressive microenvironment in Osteosarcoma.

Although undergoing conventional chemotherapy significantly improves the prognosis of Osteosarcoma, chemoresistance and failure of therapy is still a significant challenge. Furthermore, conventional chemotherapy, like doxorubicin, would upregulate the expression of programmed death-ligand 1 (PD-L1) which caused an immunosuppressive microenvironment and unsatisfied treatment result in Osteosarcoma. Thus, it is urgent to explore a strategy to overcome this disadvantage. Human Osteosarcoma cell line MG63 and mouse Osteosarcoma cell line K7 were included in this study. Subcutaneous tumor model was used by injection of K7 cells in BALB/C mice to test the effect of doxorubicin and sorafenib on tumor growth. PD-L1 expression was tested in vitro (flow cytometry, western blot and PCR) and in vivo (flow cytometry and immunohistochemistry). Proportion of immune cells (CD4, CD8, Tregs, and cytotoxic T lymphocytes) in vivo was analyzed with flow cytometry. Combination of sorafenib and doxorubicin inhibited tumor growth significantly in vivo. Doxorubicin increased PD-L1 expression in vitro and in vivo, while sorafenib inhibited doxorubicin-induced PD-L1 upregulation in vitro and in vivo. Proportion of interferon-γ-secreting CD8 + T lymphocytes in tumor tissue was increased significantly when sorafenib was combined with doxorubicin, while proportion of CD4, CD8, and Tregs was not significantly changed. Extracellularsignal-regulatedkinases (ERK) pathway could be one of the key mechanisms by which doxorubicin induced upregulation of PD-L1 in Osteosarcoma cells. Combination of sorafenib and conventional chemotherapeutic reagents is a potent strategy to improve treatment effectiveness by modulating tumor microenvironment in Osteosarcoma through increasing proportion of cytotoxic T lymphocytes.

Accuracy of determination of free light chains (Kappa and Lambda) in plasma and serum by Swedish laboratories as monitored by external quality assessment

BackgroundFree light chain (FLC) measurements are important in diagnosing monoclonal gammopathies. As FLC are heterogeneous, different reagents and instruments for measuring FLC concentrations may give diverging results that affect assessment of patients with monoclonal gammopathies. Here we investigated agreement between different FLC methods using data from the Swedish external quality assurance (EQA) programme. MethodsThe two main FLC assays, N Latex FLC (Siemens) and Serum Freelite (The Binding Site), using four nephelometric or turbidimetric instrument platforms, were compared. Results from 27 EQA rounds distributed to 11–16 Swedish hospital laboratories during 2015–2020 were investigated. ResultsThe kappa (κ) FLC measurements deviated significantly over time, but when only nephelometry was used, deviation from the mean was lower (median ranges: −5% to 13 %). The CV was significantly higher for the Freelite assay (mean CV = 8.7) than for the N latex assay (mean CV = 5.7) (p < 0.0001). The coefficient of determination between all combinations of reagents and instrument platforms used was generally good (r2 = 0.76–0.87), and the correlation slope acceptable (0.81–1.2). For lambda (λ) FLC measurements, no concordance between combinations of instruments and reagents is apparent, deviating between −40 % to + 48 % from the mean. The CV was significantly higher for the combination with nephelometry and the Freelite assay (CV mean = 13.9 %) than nephelometry and the N latex assay (CV mean = 9.9 %) (p <0.001). The coefficient of determination varied between combinations of reagents and instrument platforms (r2 = 0.59–0.89) and the slope ranged between 0.48 and 1.5. Significant differences between the two reagents used were sometimes noted. ConclusionsImprecision in λFLC affects the κFLC/λFLC ratio. This may be important in clinical assessment of patients, especially differentiating between monoclonal and polyclonal gammopathies.

SSNIP-seq: A simple and rapid method for isolation of single-sperm nucleic acid for high-throughput sequencing.

We developed a simple and reliable method for the isolation of haploid nuclei from fresh and frozen testes. The described protocol uses readily available reagents in combination with flow cytometry to separate haploid and diploid nuclei. The protocol can be completed within 1 hour and the resulting individual haploid nuclei have intact morphology. The isolated nuclei are suitable for library preparation for high-throughput DNA and RNA sequencing using bulk or single nuclei. The protocol was optimised with mouse testes and we anticipate that it can be applied for the isolation of mature sperm from other mammals including humans.

Enantioselective Cyclopropanation Catalyzed by Gold(I)-Carbene Complexes.

The formation of polysubstituted cyclopropane derivatives in the gold(I)-catalyzed reaction of olefins and propargylic esters is a potentially useful transformation to generate diversity, therefore any method in which its stereoselectivity could be controlled is of significant interest. We prepared and tested a series of chiral gold(I)-carbene complexes as a catalyst in this transformation. With a systematic optimization of the reaction conditions, we were able to achieve high enantioselectivity in the test reaction while the cis:trans selectivity of the transformation was independent of the catalyst. Using the optimized conditions, we reacted a series of various olefins and acetylene derivatives to find that, although the reactions proceeded smoothly and the products were usually isolated in good yield and with good to exclusive cis selectivity, the observed enantioselectivity varied greatly and was sometimes moderate at best. We were unable to establish any structure-property relationship, which suggests that for any given reagent combination, one has to identify individually the best catalyst.

A Summary of Autophagy Mechanisms in Cancer Cells

Autophagy is a well-known vital process in cells and plays a significant role in biological evolution, the immune system, and cell death. It can be effective in fatal disorders, such as nervous system degeneration, autoimmune diseases, and cancer. Autophagy has a dual role; on the one hand, it increases cell survival, and on the other hand, it causes cell death in advanced stages although no agreement has yet been accomplished on the role of autophagy in cellular processes. There is evidence that autophagic signaling regulation is inversely related to oncogenic signaling. Numerous commonly activated oncogenes (class I PtdIns3K, Akt, TOR, Bcl-2) inhibit autophagy, while commonly mutated or epigenetically silenced tumor suppressor genes (p53, PTEN, TSC1/TSC2) promote autophagy. Autophagy promotes cancer progression by supplying sufficient nutrients that enable cancer cell growth. FIP200, a related- autophagy protein, interacts with ATG 13 and induces autophagy. Increased autophagy causes the interaction of Becklin 118 with HER2, resulting in an increase in tumorigenesis. In order to make complete use of the autophagic properties in cancer treatment, further studies on its role in disease in the different biologics fields are essential. Cancer stem cells (CSCs) can regenerate, cause cancer, and enhance resistance to treatment, metastasis, and recurrence. Autophagy moderates stressful conditions and promotes resistance to anticancer therapy. In addition, autophagy regulates the ability of radiation in CSCs and leads to failure in anticancer therapies. Hence, autophagy is a potential therapeutic target for metastasis resistance and anticancer therapy recurrence. Regulation of autophagy using autophagy modulators alone does not improve the therapeutic effects of anticancer reagents. In contrast, it has supplied nutrients for cancer cells. Consequently, clinical trials aiming for autophagy through a combination of autophagy modulations and anticancer reagents are crucial to consider autophagy as a potentially effective therapeutic strategy in anticancer therapy.

Analytical Performance of Different Laboratory Methods for Measuring Susoctocog-Alfa.

Recombinant porcine factor VIII (rpFVIII) is indicated for treating bleeding episodes in acquired haemophilia A, but there are few data regarding laboratory methods to adequately monitor treatment. This study involving three Italian laboratories aimed to evaluate the analytical performance of different assays for measuring rpFVIII. Five spiked rpFVIII samples (0.5–1.5 IU/mL) were analysed on three days, in triplicate, with eleven combinations of reagents (Werfen, Boston, MA, USA: SynthasIL and SynthaFax for one-stage assay, Chromogenix Coamatic FVIII for chromogenic assay), FVIII depleted plasmas (with or without von Willebrand factor—VWF) and calibrators (HemosIL human calibrator plasma, porcine calibrator diluted in FVIII deficient plasma with or without VWF). The assays were performed on ACL TOP analysers (Werfen, Boston, MA, USA). Intra- and inter-assay and inter-laboratory Coefficient of Variation (CV%) were calculated together with percentage of recovery (% recovery) on the expected value. The results showed that the reagent combinations reaching satisfactory analytical performance are: SynthasIL/human calibrator/deficient plasma+VWF (total recovery 99.4%, inter-laboratory CV 4.04%), SynthasIL/porcine calibrator/deficient plasma+VWF (total recovery 111%, inter-laboratory CV 2.75%) and Chromogenic/ porcine calibrator/deficient plasma+VWF (total recovery 96.6%, inter-laboratory CV 8.32%). This study highlights that the use of porcine standard (when available) and FVIII deficient plasma with VWF should be recommended.

Selective flotation of galena from sphalerite using a combination of KMnO4 and carboxylated chitosan

An effective reagent combination plays a crucial role in the flotation separation of galena and sphalerite. In this work, potassium permanganate (KMnO4) was used to amplify the property difference between galena and sphalerite, carboxylated chitosan (CCS) was used to selectively depress sphalerite. The mechanism of CCS in the separation of galena and sphalerite was investigated by microcalorimetric measurements, X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared (FTIR) spectroscopy. The results indicated that the rate of sphalerite oxidation by KMnO4 is significantly faster than galena, and more metal oxides will be produced on the surface of sphalerite. The metal oxides provided active sites for CCS to adsorb on the surface of sphalerite via chemisorption, and the adsorption of CCS hindered the further adsorption of potassium butylxanthate (PBX). Meanwhile, the slower oxidation rate of galena means that fewer metal oxides are produced. In addition, CCS is mainly adsorbed on the surface of galena through physical adsorption, which is easy to fall off, so the surface of galena can further adsorb PBX. Thus, flotation separation of galena and sphalerite is achieved.

Microfluidic Printing-Based Method for the Multifactorial Study of Cell-Free Protein Networks.

Protein networks can be assembled in vitro for basic biochemistry research, drug screening, and the creation of artificial cells. Two standard methodologies are used: manual pipetting and pipetting robots. Manual pipetting has limited throughput in the number of input reagents and the combination of reagents in a single sample. While pipetting robots are evident in improving pipetting efficiency and saving hands-on time, their liquid handling volume usually ranges from a few to hundreds of microliters. Microfluidic methods have been developed to minimize the reagent consumption and speed up screening but are challenging in multifactorial protein studies due to their reliance on complex structures and labeling dyes. Here, we engineered a new impact-printing-based methodology to generate printed microdroplet arrays containing water-in-oil droplets. The printed droplet volume was linearly proportional (R2 = 0.9999) to the single droplet number, and each single droplet volume was around 59.2 nL (coefficient of variation = 93.8%). Our new methodology enables the study of protein networks in both membrane-unbound and -bound states, without and with anchor lipids DGS-NTA(Ni), respectively. The methodology is demonstrated using a subnetwork of mitogen-activated protein kinase (MAPK). It takes less than 10 min to prepare 100 different droplet-based reactions, using <1 μL reaction volume at each reaction site. We validate the kinase (ATPase) activity of MEK1 (R4F)* and ERK2 WT individually and together under different concentrations, without and with the selective membrane attachment. Our new methodology provides a reagent-saving, efficient, and flexible way for protein network research and related applications.

Rhodium-catalyzed synthesis of 1-silabenzonorbornenes via 1,4-rhodium migration

An efficient synthesis of 1-silabenzonorbornenes was developed from a simple substrate combination of alkynyl(2-alkylallyl)silanes and arylboron reagents under rhodium catalysis through the use of 1,4-rhodium migration. Three carbon–carbon bonds were newly formed during the catalytic process and the reaction pathway was probed by a deuterium labeling experiment as well.

An external quality assessment for the molecular testing of the SARS-CoV-2 virus genome in Zhejiang Province, China

The COVID-19 pandemic has necessitated the rapid expansion of laboratories that conduct SARS-CoV-2 tests. A provincial external quality assessment (EQA) scheme on SARS-CoV-2 tests was organized by Zhejiang Provincial CDC to assess the accuracy of the tests in individual CDC municipal and county laboratories in Zhejiang Province, China. Three positive samples in high, medium, and low concentrations, respectively, were prepared using the serial dilutions from the culture with the viral titer concentration of 1×106.3 TCID50/mL, and one negative sample were included. A total of 93 laboratories participated, contributing results from 36 distinct combinations of nucleic acid extraction methods and PCR reagents. There was 100% concordance among all laboratories for all EQA samples, and no false-positive or false-negative results were observed. The EQA survey provides confidence in the identification of infected individuals or asymptomatic populations and assurance for clinical and public health decision-making based on test results.

Synthesis of Phosphorodiamidate Morpholino Oligonucleotides Using Trityl and Fmoc Chemistry in an Automated Oligo Synthesizer.

Phosphorodiamidate morpholino oligonucleotides (PMOs) constitute 3 out of the 11 FDA-approved oligonucleotide-based drugs in the last 6 years. PMOs can effectively silence disease-causing genes and modify splicing. However, PMO synthesis has remained challenging for a variety of reasons: inefficient deprotection and coupling methods and instability of monomers. Here, we report the development of a suitable combination of resin supports, deblocking and coupling reagents for synthesizing PMOs using either trityl or Fmoc-protected chlorophosphoramidate monomers. The synthesized PMOs using both the methods on a solid support have been validated for gene silencing in a zebrafish model. The protocol was successfully transferred into an automated DNA synthesizer to make several sequences of PMOs, demonstrating for the first time the adaptation of regular PMOs in a commercial DNA synthesizer. Moreover, PMOs with longer than 20-mer sequences, including FDA-approved Eteplirsen (30-mer), were achieved in >20% overall yield that is superior to previous reports. Hybridization study shows that PMOs exhibit a higher binding affinity toward complementary DNA relative to the DNA/DNA duplex (>6 °C). Additionally, the introduction of Fmoc chemistry into PMOs opens up the possibility for PMO synthesis in commercial peptide synthesizers for future development.