Limited Commercial Availability Research Articles

Development of user-friendly protocols for single-cell arachidonic acid production in developing countries

Arachidonic acid (ARA) is a sorely needed nutrient and an effective and safe schistosomicide and tumoricide. Consumption levels are low in developing countries because of unawareness of its critical impact on health and limited commercial availability. The research work aims to encourage production in developing countries via providing user-friendly definitive protocols for polyunsaturated fatty acids production by the fungus Mortierella alpina and ARA isolation. Fatty acids content was determined by gas chromatography-flame ionization detector. The optimal cultivation conditions included culture medium to flask volume 1:5; shake-flask culture at 25 °C, then at 20 °C, and stationary aging at 10 °C, each over 7 days, while addition of trace minerals and vitamins was dispensable. The ARA yield attained >1.8 g per liter culture. Repeat experiments led to the selection of an optimal lipid extraction method from dried biomass, and efficacious urea complexation-based protocol for obtention of high ARA yield and purity.

Secondary piggyback intraocular lens for posterior microphthalmos

We describe a rare case of posterior microphthalmos (PM) submitted to cataract surgery and highlight the problem of high residual hyperopia in these eyes due to the limited commercial availability of a high-powered intraocular lens (IOL). A 50-year-old highly hyperopic man who pretended refractive surgery presented in our hospital. Pre-operative Snellen best-corrected visual acuity (BCVA) was 20/125 in the right eye with a spherical correction of +17.00 D and 20/40 in the left eye with a spherical correction of +18.75. The anterior segment and fundus exam were typical for PM. Optical axial length was 15.50 mm in the right eye and 15.53 mm in the left eye and anterior segment dimensions were within normal limits. IOL power calculation for emmetropia ranged between +51.5 and +62.5 D using modern IOL formulas. Cataract surgery was performed in a bilateral and sequential manner with the implantation of monofocal foldable acrylic +45.0 D IOL with the improvement of spherical correction to +11.00 D bilaterally. Secondary implantation of supplementary sulcus IOL with the maximum +10.0 D power was then performed bilaterally and sequentially with a final BCVA of 20/125 in the right eye with spherical correction of +5.75 D and 20/40 in the left eye with spherical correction of +5.25 D. Piggyback IOL implantation is beneficial and, in most times, necessary in PM since one IOL will not be sufficient to achieve emmetropia. Different piggyback strategies in regard to timing (primary vs. secondary) and chosen IOLs (in-the-bag, sulcus, and iris-claw) can be used.

Methods for controlled preparation and dosing of microplastic fragments in bioassays

Microplastic fragments (microfragments) are among the most abundant microplastic shapes found in marine ecosystems throughout the world. Due to their limited commercial availability, microfragments are rarely used in laboratory experiments. Here a novel method of microfragment production has been developed and validated. Polyethylene and polypropylene plastic stock (2 and 3 mm thick respectively) was ground using a cryomill, washed, and rinsed through a stack of sieves. Microfragments were prepared at three distinct size classes (53–150, 150–300, 300–1000 μm) and were confirmed to be accurate and consistent in size. Employing a novel ice cap dosing technique, microfragments were accurately dosed into experimental vials while excluding headspace, facilitating particle suspension without the aid of chemical surfactants. A proof of principle ingestion experiment confirmed the bioavailability of 53–150 μm polyethylene microfragments to brine shrimp Artemia sp. Together, these methods provide a controlled way to produce and dose microplastic fragments for experimental and analytical research.

Evaluation of alternative gas chromatographic and mass spectrometric behaviour of trimethylsilyl-derivatives of non-hydrolysed sulfated anabolic steroids.

Sulfated metabolites have shown to have potential as long-term markers (LTMs) of anabolic-androgenic steroid (AAS) abuse. The compatibility of gas chromatography-mass spectrometry (GC-MS) with trimethylsilyl (TMS)-derivatives of non-hydrolysed sulfated steroids has been demonstrated, where, after derivatisation, generally, two closely eluting isomers are formed that both have the same molecular ion [M-H2 SO4 ]•+ . Sulfated reference standards are in limited commercial availability, and therefore, the current knowledge of the GC-MS behaviour of these compounds is mainly based on sulfating and analysing the available standard reference material. This procedure can unfortunately not cover all of the current known LTMs as these are often not available as pure substance. Therefore, in theory, some metabolites could be missed as they exhibit alternative behaviour. To investigate the matter, in-house sulfated reference materials that bear resemblance to known sulfated LTMs were analysed on GC-MS in their TMS-derivatised non-hydrolysed state. The (alternative) gas chromatographic and mass spectrometric behaviour was mapped, evaluated and linked to the corresponding steroid structures. Afterwards, using fraction collection, known sulfated LTMs were isolated from excretion urine to confirm the observed findings. The categories that were selected were mono-hydroxy-diones, 17-methyl-3,17-diols and 17-keto-3,16-diols as these are commonly encountered AAS conformations. The ability to predict the GC-MS behaviour of non-hydrolysed sulfated AAS metabolites is the corner stone of finding new metabolites. This knowledge is also essential, for example, for understanding AAS detection analyses, for the mass spectrometric characterization of metabolites of new designer steroids or when one needs to characterize an unknown steroid structure.

Dynamics of Nutrients in Jujube (Ziziphus jujuba Mill.) at Different Maturity Stages, Cultivars, and Locations in the Southwest United States

Jujube (Ziziphus jujuba Mill.) is also called Chinese date. There are ∼100 jujube cultivars with limited commercial availability, and the majority of them have scant details in the United States. In this study, nutrient dynamics during fruit maturation of different jujube cultivars grown at Las Cruces, Los Lunas, and Alcalde, NM, were examined in 2018 and 2019. Cultivars varied by location and year, and included ‘Li’, ‘Lang’, ‘Sugarcane’, ‘September Late’, and ‘Sherwood’. Parameters tested were total phenolic content (TPC), proanthocyanidins (PAs), vitamin C, cyclic adenosine monophosphate (cAMP), and antioxidant capacity: 2,2-diphenylpicrylhydrazyl radical scavenging capacity and ferric reducing antioxidant potential (FRAP). Moisture, TPC, PAs, FRAP, and vitamin C content decreased with fruit maturity; however, the latter stage of fruit maturity showed an increase in cAMP. Compared with fruit at full-red maturity, creamy fruit had TPC, PA, FRAP, and vitamin C concentrations that were 1.0 to 1.8, 4.4 to 12.4, 1.9 to 2.6, and 0.1 to 1.3 times higher, respectively, depending on location (P < 0.05). From creamy to full-red maturity, cAMP increased by 0.9 to 4.5 times. At full-red maturity, estimated TPC in jujube fruit ranged from 10.6 to 16.8 mg gallic acid equivalent per gram dry weight (DW), whereas estimated PAs ranged from 1.8 to 5.3 mg PA B2/g DW. Jujube fruit at full-red maturity had a vitamin C content that ranged from 649.0 to 1153.3 mg/100 g DW. At full-red maturity, the concentration of cAMP ranged from 148.1 to 277.6 μg/g DW in Las Cruces samples.

Branching Ionizable Lipids Can Enhance the Stability, Fusogenicity, and Functional Delivery of mRNA

Ionizable lipids with branched tails have been used in lipid nanoparticles (LNPs)‐based messenger RNA (mRNA) therapeutics like COVID‐19 vaccines. However, due to the limited commercial availability of branched ingredients, a systematic analysis of how the branched tails affect LNP quality has been lacking to date. Herein, α‐branched tail lipids are focused, as they can be synthesized from simple commercially available chemicals, and the length of each chain can be independently controlled. Furthermore, symmetry and total carbon number can be used to describe α‐branched tails, facilitating the design of a systematic lipid library to elucidate “structure–property–function” relationships. Consequently, a lipid library is developed containing 32 different types of α‐branched tails. This library is used to demonstrate that branched chains increase LNP microviscosity and headgroup ionization ability in an acidic environment, which in turn enhances the stability and in vivo efficacy of mRNA‐LNPs. Of the branched lipids, CL4F 8‐6 LNPs carrying Cas9 mRNA and sgRNA could achieve 54% genome editing and 77% protein reduction with a single dose of 2.5 mg kg−1. This mechanism‐based data on branched lipids is expected to provide insights into rational lipid design and effective gene therapy in the future.

A Combined Bayesian and Similarity-Based Approach for Predicting E. coli Biofilm Inhibition by Phenolic Natural Compounds.

Screening for biofilm inhibition by purified natural compounds is difficult due to compounds' chemical diversity and limited commercial availability, combined with time- and cost-intensiveness of the laboratory process. In silico prediction of chemical and biological properties of molecules is a widely used technique when experimental data availability is of concern. At the same time, the performance of predictive models directly depends on the amount and quality of experimental data. Driven by the interest in developing a model for prediction of the antibiofilm effect of phenolic natural compounds such as flavonoids, we performed experimental assessment of antibiofilm activity of 320 compounds from this subset of chemicals. The assay was performed once on two Escherichia coli strains on agar in 24-well microtiter plates. The inhibition was assessed visually by detecting morphological changes in macrocolonies. Using the data obtained, we subsequently trained a Bayesian logistic regression model for prediction of biofilm inhibition, which was combined with a similarity-based method in order to increase the overall sensitivity (at the cost of accuracy). The quality of the predictions was subsequently validated by experimental assessment in three independent experiments with two resistant E. coli strains of 23 compounds absent in the initial data set. The validation demonstrated that the model may successfully predict the targeted effect as compared to the baseline accuracy. Using a randomly selected database of commercially available natural phenolics, we obtained approximately 6.0% of active compounds, whereas using our prediction-based substance selection, the percentage of phenolics found to be active increased to 34.8%.

Microwave-assisted C–H oxidation of methylpyridylheteroarenes via a Kornblum-Type reaction

Expansion of the operational capacity of soft-Lewis basic complexant scaffolds towards improved physical properties for the chemoselective sequestration of minor actinides from the electronically similar lanthanides necessitates rapid access to synthons for efficient complexant construction for downstream employment in separations assays. Pursuant to the aforementioned, we were interested in exploring the potential utility of advanced, unsymmetric heteroarene constructs for separations which required access to pyridyl carbaldehydes. Limited commercial availability of synthetic precursors inspired our effort to define a chemoselective, microwave assisted, Kornblum-type reaction via C–H functionalization. This efficient reaction sequence uses I2 as a mild oxidant without acidic or basic additives, in concert with DMSO as the solvent and putative oxygen source to afford a diverse array of heteroaryl products devoid of competitive remote methyl group oxidation, or degradation of the heteroaryl N atom. Method development, substrate scope, and a preliminary mechanistic hypothesis supported by Density Functional Theory are presented herein.

Non-Antibody-Based Binders for the Enrichment of Proteins for Analysis by Mass Spectrometry

There is often a need to isolate proteins from body fluids, such as plasma or serum, prior to further analysis with (targeted) mass spectrometry. Although immunoglobulin or antibody-based binders have been successful in this regard, they possess certain disadvantages, which stimulated the development and validation of alternative, non-antibody-based binders. These binders are based on different protein scaffolds and are often selected and optimized using phage or other display technologies. This review focuses on several non-antibody-based binders in the context of enriching proteins for subsequent liquid chromatography-mass spectrometry (LC-MS) analysis and compares them to antibodies. In addition, we give a brief introduction to approaches for the immobilization of binders. The combination of non-antibody-based binders and targeted mass spectrometry is promising in areas, like regulated bioanalysis of therapeutic proteins or the quantification of biomarkers. However, the rather limited commercial availability of these binders presents a bottleneck that needs to be addressed.

3D‐Printed Bioreactor with Integrated Impedance Spectroscopy for Cell Barrier Monitoring

AbstractCell culture experiments often suffer from limited commercial availability of laboratory‐scale bioreactors, which allow experiments to be conducted under flow conditions and additional online monitoring techniques. A novel 3D‐printed bioreactor with a homogeneously distributed flow field enabling epithelial cell culture experiments and online barrier monitoring by integrated electrodes through electrical impedance spectroscopy (EIS) is presented. Transparent and conductive indium tin oxide glass as current‐injecting electrodes allows direct visualization of the cells, while measuring EIS simultaneously. The bioreactor's design considers the importance of a homogeneous electric field by placing the voltage pick‐up electrodes in the electrical field. The device's functionality is demonstrated by the cultivation of the epithelial cell line Caco‐2 under continuous flow and monitoring of the cell layer by online EIS. The collected EIS data were fitted by an equivalent electric circuit, resulting in the cell layer's resistance and capacitance. This data is used to monitor the cell layer's reaction to ethylene glycol‐bis‐(2‐aminoethyl ether)‐N,N,N′,N′‐tetraacetic acid and forskolin. These two model substances show the power of impedance spectroscopy as a non‐invasive way to characterize cell barriers. In addition, the bioreactor design is available as a print‐ready file in the Appendix, enabling its use for other scientific institutions.

Supramolecular ultra-short carboxybenzyl-protected dehydropeptide-based hydrogels for drug delivery

Self-assembled peptide-based hydrogels are promising materials for biomedical research owing to biocompatibility and similarity to the extracellular matrix, amenable synthesis and functionalization and structural tailoring of the rheological properties. Wider developments of self-assembled peptide-based hydrogels in biomedical research and clinical translation are hampered by limited commercial availability allied to prohibitive costs.In this work a focused library of Cbz-protected dehydrodipeptides Cbz-L-Xaa-Z-ΔPhe-OH (Xaa= Met, Phe, Tyr, Ala, Gly) was synthesised and evaluated as minimalist hydrogels. The Cbz-L-Met-Z-ΔPhe-OH and Cbz-L-Phe-Z-ΔPhe-OH hydrogelators were comprehensively evaluated regarding molecular aggregation and self-assembly, gelation, biocompatibility and as drug carriers for delivery of the natural compound curcumin and the clinically important antitumor drug doxorubicin. Drug release profiles and FRET studies of drug transport into small unilamellar vesicles (as biomembrane models) demonstrated that the Cbz-protected dehydropeptide hydrogels are effective nanocarriers for drug delivery. The expedite and scalable synthesis (in 3 steps), using commercially available reagents and amenable reaction conditions, makes Cbz-protected dehydrodipeptide hydrogels, widely available at affordable cost to the research community.

Navigating the COVID-19 pandemic: Initial impacts and responses of the Organ Procurement and Transplantation Network in the United States.

COVID‐19 has been sweeping the globe, hitting the United States particularly hard with a state of emergency declared on March 13, 2020. Transplant hospitals have taken various precautions to protect patients from potential exposure. OPTN donor, candidate, and transplant data were analyzed from January 5, 2020 to September 5, 2020. The number of new waiting list registrations decreased, with the Northeast seeing over a 50% decrease from the week of 3/8 versus the week of 4/5. The national transplant system saw near cessation of living donor transplantation (−90%) from the week of 3/8 to the week of 4/5. Similarly, deceased donor kidney transplant volume dropped from 367 to 202 (−45%), and other organs saw similar decreases: lung (−70%), heart (−43%), and liver (−37%). Deceased donors recovered dropped from 260 to 163 (−45%) from 3/8 compared to 4/5, including a 67% decrease for lungs recovered. The magnitude of this decrease varied by geographic area, with the largest percent change (−67%) in the Northeast. Despite the pandemic, discard rates across organ has remained stable. Although the COVID‐19 pandemic continues to evolve, OPTN data show recent evidence of stabilization, an indication that an early recovery of the number of living and deceased donors and transplants has ensued.

A practical and scalable synthesis of KRN7000 using glycosyl iodide as the glycosyl donor

KRN7000 is particularly useful because it is a powerful and specific CD1d agonist and has prompted intense interest in the context of immunology in the past 25 years. Its limited commercial availability and high price has led to the publication of many different syntheses. However, almost all of them focused on the methodology development rather than a scalable synthesis. Herein, we have described a practical and scalable procedure for the synthesis of KRN7000 basing on the glycosyl iodide method. This procedure involves total of eight steps to obtain the highly pure product KNR7000 on gram scale from the commercially available starting materials (d-galactose and the phytosphingosine) with only three column chromatographic purifications.

Rearing and Maintenance of Galleria mellonella and Its Application to Study Fungal Virulence

Galleria mellonella larvae have been widely used as alternative non-mammalian models for the study of fungal virulence and pathogenesis. The larvae can be acquired in small volumes from worm farms, pet stores, or other independent suppliers commonly found in the United States and parts of Europe. However, in countries with no or limited commercial availability, the process of shipping these larvae can cause them stress, resulting in decreased or altered immunity. Furthermore, the conditions used to rear these larvae including diet, humidity, temperature, and maintenance procedures vary among the suppliers. Variation in these factors can affect the response of G. mellonella larvae to infection, thereby decreasing the reproducibility of fungal virulence experiments. There is a critical need for standardized procedures and incubation conditions for rearing G. mellonella to produce quality, unstressed larvae with the least genetic variability. In order to standardize these procedures, cost-effective protocols for the propagation and maintenance of G. mellonella larvae using an artificial diet, which has been successfully used in our own laboratory, requiring minimal equipment and expertise, are herein described. Examples for the application of this model in fungal pathogenicity and gene knockout studies as feasible alternatives for traditionally used animal models are also provided.

Biosynthesis of alkaloids in Amaryllidaceae plants: a review

Amaryllidaceae alkaloids are a group of specialized metabolites found predominantly in the Amaryllidaceae plant family. Approximately 600 naturally occurring Amaryllidaceae alkaloids have been identified, many of which possess a variety of potent biological and pharmacological properties. However, only the Amaryllidaceae alkaloid galanthamine, an acetylcholinesterase inhibitor used to treat symptoms of neurodegenerative disorders such as Alzheimer’s disease, is currently on the market. Despite promising pharmacological potential, the clinical development of Amaryllidaceae alkaloids is hindered by limited commercial availability. In contrast to the large body of knowledge on the pharmacological and phytochemical aspects of Amaryllidaceae alkaloids, their molecular and physiological features are less explored. A better understanding of Amaryllidaceae alkaloid biosynthesis and metabolic regulation is crucial to take advantage of new metabolic engineering technologies for improving the efficiency and sustainability of plant or microbial Amaryllidaceae alkaloid production. Although there is still much to learn, over the past several years comparative transcriptomic and traditional biochemical approaches have led to significant advances in the identification of the molecular players involved in producing Amaryllidaceae alkaloids including enzymes from the shikimate and phenylpropanoid pathways. The identity of few Amaryllidaceae alkaloid biosynthetic enzymes and several genes encoding them has been reported. In this review, an overview of the current knowledge on the biosynthesis of Amaryllidaceae alkaloids is presented.

Direct Carotid Cavernous Fistulas.

A direct carotid cavernous fistula (CCF) is an abnormal shunt between the internal carotid artery (ICA) and the cavernous sinus (CS). Traumatic CCF is the most common type, accounting for up to 75% of all CCFs. For the management of direct CCF, endovascular therapy has become the standard. For successful endovascular therapy, evaluation of the size and location of orifice of the CCF, venous drainage, and tolerance for ICA occlusion on cerebral angiography is necessary. Multi-planner reformatted images of 3D rotation angiography are useful to visualize the fistula and compartments of the CS precisely. Due to the limited commercial availability of detachable balloons, detachable coils have become a widely employed endovascular tool for the treatment of direct CCFs. The advantageous aspects of coil application are their easy retrievability and better control. In the case of large/multiple fistulas, adjunctive techniques, including balloon- and stent-assisted techniques, are often needed to occlude the CCF while preserving the ICA. To avoid cranial nerve palsy related to over-packing of the CS with detachable coils or a detachable balloon, selective embolization of the fistula portion is required. Use of liquid embolic materials and covered stents was recently reported as another adjunctive technique. In cases in which it is impossible to occlude the CCF while preserving the ICA, parent artery occlusion (PAO) is considered. The selection of additional/alternative techniques and devices depends on the anatomy and hemodynamics of each CCF, and the skill and experience of individual operators.

High-intensity and low-divergence THz laser with 1D autofocusing symmetric Airy beams

Single-mode THz quantum-cascade lasers (QCLs) have been realized using a wide variety of techniques to obtain a combination of large power output, good beam quality with single-lobed beams, and low far-field divergence. Beam shaping using external components has not been previously exploited due to limited commercial availability of THz optical components and also the accompanying large loss from most THz optical materials. Here, we demonstrate that excellent beam characteristics could be obtained for a THz QCL by integration of a surface-emitting distributed-feedback (DFB) QCL with a simple lens within the vacuum chamber of a cryocooler. Plano-convex lenses are made from inexpensive plastic balls and integrated in proximity with a 3.4THz DFB QCL. With appropriately chosen lens parameters, dual-lobed Airy beams are generated that autofocus into a high-intensity single-lobed beam with large focusing efficiency. A simple and general method to generate one-dimensional autofocusing Airy beams is thus demonstrated that is applicable at any wavelength. THz laser beams with high peak intensity (57mW/mm2 in a spot-size of 1mm2) or low-divergence (1.4∘×1.8∘ for a beam with 118mW peak power) are realized at 62K in a compact electrically operated Stirling cooler. A high brightness of 5.4×106Wsr-1m-2 is estimated for the focused beams by measuring the beam propagation ratios (M2 parameters).

Synthesis and Characterization of Acridinium Dyes for Photoredox Catalysis.

Photoredox catalysis is a rapidly evolving platform for synthetic methods development. The prominent use of acridinium salts as a sustainable option for photoredox catalysts has driven the development of more robust and synthetically useful versions based on this scaffold. However, more complicated syntheses, increased cost, and limited commercial availability have hindered the adoption of these catalysts by the greater synthetic community. By utilizing the direct conversion of a xanthylium salt into the corresponding acridinium as the key transformation, we present an efficient and scalable preparation of the most synthetically useful acridinium reported to date. This divergent strategy also enabled the preparation of a suite of novel acridinium dyes, allowing for a systematic investigation of substitution effects on their photophysical properties.

ISiCLE: A Quantum Chemistry Pipeline for Establishing in Silico Collision Cross Section Libraries.

High-throughput, comprehensive, and confident identifications of metabolites and other chemicals in biological and environmental samples will revolutionize our understanding of the role these chemically diverse molecules play in biological systems. Despite recent technological advances, metabolomics studies still result in the detection of a disproportionate number of features that cannot be confidently assigned to a chemical structure. This inadequacy is driven by the single most significant limitation in metabolomics, the reliance on reference libraries constructed by analysis of authentic reference materials with limited commercial availability. To this end, we have developed the in silico chemical library engine (ISiCLE), a high-performance computing-friendly cheminformatics workflow for generating libraries of chemical properties. In the instantiation described here, we predict probable three-dimensional molecular conformers (i.e., conformational isomers) using chemical identifiers as input, from which collision cross sections (CCS) are derived. The approach employs first-principles simulation, distinguished by the use of molecular dynamics, quantum chemistry, and ion mobility calculations, to generate structures and chemical property libraries, all without training data. Importantly, optimization of ISiCLE included a refactoring of the popular MOBCAL code for trajectory-based mobility calculations, improving its computational efficiency by over 2 orders of magnitude. Calculated CCS values were validated against 1983 experimentally measured CCS values and compared to previously reported CCS calculation approaches. Average calculated CCS error for the validation set is 3.2% using standard parameters, outperforming other density functional theory (DFT)-based methods and machine learning methods (e.g., MetCCS). An online database is introduced for sharing both calculated and experimental CCS values ( metabolomics.pnnl.gov ), initially including a CCS library with over 1 million entries. Finally, three successful applications of molecule characterization using calculated CCS are described, including providing evidence for the presence of an environmental degradation product, the separation of molecular isomers, and an initial characterization of complex blinded mixtures of exposure chemicals. This work represents a method to address the limitations of small molecule identification and offers an alternative to generating chemical identification libraries experimentally by analyzing authentic reference materials. All code is available at github.com/pnnl .

Isoelectric Focusing in Agarose Gel for Detection of Oligoclonal Bands in Cerebrospinal and Other Biological Fluids.

Isoelectric focusing (IEF) coupled with immunodetection (immunofixation or immunoblotting) has become the leading technique for the detection and study of oligoclonal bands (OCBs) in cerebrospinal fluid (CSF) and also is increasingly used in other body fluids such as the tear and serum. Limited commercial availability of precast agarose IEF gels for research and a need for customization prompted reporting a detailed general protocol for the preparation and casting of agarose IEF gel along with sample, control, and isoelectric point marker preparation and carrying out the focusing itself for CSF OCBs. However, the method is readily adaptable to the use of other body fluid specimens and, possibly, research specimens such as culture fluids as well.