Screening Methodology Research Articles

Expedient synthesis and luminescence sensing of the inositol pyrophosphate cellular messenger 5-PP-InsP5.

Inositol pyrophosphates are important biomolecules associated with apoptosis, cell growth and kinase regulation, yet their exact biological roles are still emerging and probes do not exist for their selective detection. We report the first molecular probe for the selective and sensitive detection of the most abundant cellular inositol pyrophosphate 5-PP-InsP5, as well as an efficient new synthesis. The probe is based on a macrocyclic Eu(iii) complex bearing two quinoline arms providing a free coordination site at the Eu(iii) metal centre. Bidentate binding of the pyrophosphate group of 5-PP-InsP5 to the Eu(iii) ion is proposed, supported by DFT calculations, giving rise to a selective enhancement in Eu(iii) emission intensity and lifetime. We demonstrate the use of time-resolved luminescence as a bioassay tool for monitoring enzymatic processes in which 5-PP-InsP5 is consumed. Our probe offers a potential screening methodology to identify drug-like compounds that modulate the activity of enzymes of inositol pyrophosphate metabolism.

High-throughput screening of glycosynthases using azido sugars for oligosaccharides synthesis.

Glycosynthases are mutant glycosyl hydrolases that can synthesize glycosidic bonds between acceptor glycone/aglycone groups and activated donor sugars with suitable leaving groups (e.g., azido, fluoro). However, it has been challenging to rapidly detect glycosynthase reaction products involving azido sugars as donor sugars. This has limited our ability to apply rational engineering and directed evolution methods to rapidly screen for improved glycosynthases that are capable of synthesizing bespoke glycans. Here, we outline our recently developed screening methodologies for rapidly detecting glycosynthase activity using a model fucosynthase enzyme engineered to be active on fucosyl azide as donor sugar. We created a diverse library of fucosynthase mutants using semi-random and random error prone mutagenesis and then identified improved fucosynthase mutants with desired activity using two distinct screening methods developed by our group to detect glycosynthase activity (i.e., by detecting azide formed upon completion of fucosynthase reaction); (a) pCyn-GFP regulon method, and (b) Click chemistry method. Finally, we provide some proof-of-concept results illustrating the utility of both these screening methods to rapidly detect products of glycosynthase reactions involving azido sugars as donor groups.

Cholesterol-dependent activity of dapsone against non-replicating persistent mycobacteria.

One-third of the world's population is estimated to be latently infected with Mycobacterium tuberculosis. This reservoir of bacteria is largely resistant to antimicrobial treatment that often only targets actively replicating mycobacteria, with current treatment for latent infection revolving around inhibiting the resuscitation event rather than preventing or treating latent infection. As a result, antimicrobials that target latent infection often have little to no activity in vivo. Here we report a method of in vitro analysis of physiologically relevant non-replicating persistence (NRP) utilizing cholesterol as the sole carbon source, alongside hypoxia as a driver of Mycobacterium bovis BCG into the NRP state. Using the minimal cholesterol media NRP assay, we observed an increased state of in vitro resistance to front-line anti-tubercular compounds. However, following a phenotypic screen of an approved-drug library, we identified dapsone as a bactericidal active molecule against cholesterol-dependent NRP M. bovis BCG. Through an overexpression trial of probable antimicrobial target enzymes, we further identified FolP2, a non-functional dihydropteroate synthase homologue, as the likely target of dapsone under cholesterol-NRP due to a significant increase in bacterial resistance when overexpressed. These results highlight the possible reason for little in vivo activity seen for current front-line anti-NRP drugs, and we introduce a new methodology for future drug screening as well as a potential role for dapsone inclusion within the current treatment regime.

Structure-Based Virtual Screening of Natural Products and Optimization for the Design and Synthesis of Novel CeCht1 Inhibitors as Nematicide Candidates.

Nematode chitinases are critical components of the nematode life cycle, and CeCht1 is a potential target for developing novel nematicides. Herein, lunidonine, a natural quinoline alkaloid, was first discovered to have inhibitory activity against CeCht1, which was acquired from a library of over 16,000 natural products using a structure-based virtual screening methodology. A pocket-based lead optimization strategy was employed based on the predicted binding mode of lunidonine. Subsequently, a series of benzo[d][1,3]dioxole-5-carboxylate derivatives were designed and synthesized, and their inhibitory activities against CeCht1 as well as in vitro nematicidal activities against Caenorhabditis elegans were assessed. The analysis of structure-activity relationship and inhibitory mechanisms provided insights into their interactions with the CeCht1 active site, which could facilitate future research in improving the potency of the inhibitory activity. Especially, compound a12 interacted well with CeCht1 and exhibited excellent in vitro nematicidal activity against C. elegans with a LC50 value of 41.54 mg/L, suggesting that it could be a promising candidate for a novel chemical nematicide targeting CeCht1. The known binding modes and structural features of these inhibitors will contribute to the design of stronger CeCht1-based nematicides to control nematodes in agriculture.

An in-silico pharmacophore-based molecular docking study to evaluate the inhibitory potentials of novel fungal triterpenoid Astrakurkurone analogues against a hypothetical mutated main protease of SARS-CoV-2 virus

BackgroundThe main protease is an important structural protein of SARS-CoV-2, essential for its survivability inside a human host. Considering current vaccines' limitations and the absence of approved therapeutic targets, Mpro may be regarded as the potential candidate drug target. Novel fungal phytocompound Astrakurkurone may be studied as the potential Mpro inhibitor, considering its medicinal properties reported elsewhere. MethodsIn silico molecular docking was performed with Astrakurkurone and its twenty pharmacophore-based analogues against the native Mpro protein. A hypothetical Mpro was also constructed with seven mutations and targeted by Astrakurkurone and its analogues. Furthermore, multiple parameters such as statistical analysis (Principal Component Analysis), pharmacophore alignment, and drug likeness evaluation were performed to understand the mechanism of protein-ligand molecular interaction. Finally, molecular dynamic simulation was done for the top-ranking ligands to validate the result. ResultWe identified twenty Astrakurkurone analogues through pharmacophore screening methodology. Among these twenty compounds, two analogues namely, ZINC89341287 and ZINC12128321 showed the highest inhibitory potentials against native and our hypothetical mutant Mpro, respectively (−7.7 and −7.3 kcal mol−1) when compared with the control drug Telaprevir (−5.9 and −6.0 kcal mol−1). Finally, we observed that functional groups of ligands namely two aromatic and one acceptor groups were responsible for the residual interaction with the target proteins. The molecular dynamic simulation further revealed that these compounds could make a stable complex with their respective protein targets in the near-native physiological condition. ConclusionTo conclude, Astrakurkurone analogues ZINC89341287 and ZINC12128321 can be potential therapeutic agents against the highly infectious SARS-CoV-2 virus.

Screening and identification of potential MERS-CoV papain-like protease (PLpro) inhibitors; Steady-state kinetic and Molecular dynamic studies

MERS-CoV belongs to the coronavirus group. Recent years have seen a rash of coronavirus epidemics. In June 2012, MERS-CoV was discovered in the Kingdom of Saudi Arabia, with 2,591 MERSA cases confirmed by lab tests by the end of August 2022 and 894 deaths at a case-fatality ratio (CFR) of 34.5% documented worldwide. Saudi Arabia reported the majority of these cases, with 2,184 cases and 813 deaths (CFR: 37.2%), necessitating a thorough understanding of the molecular machinery of MERS-CoV. To develop antiviral medicines, illustrative investigation of the protein in coronavirus subunits are required to increase our understanding of the subject. In this study, recombinant expression and purification of MERS-CoV (PLpro), a primary goal for the development of 22 new inhibitors, were completed using a high throughput screening methodology that employed fragment-based libraries in conjunction with structure-based virtual screening. Compounds 2, 7, and 20, showed significant biological activity. Moreover, a docking analysis revealed that the three compounds had favorable binding mood and binding free energy. Molecular dynamic simulation demonstrated the stability of compound 2 (2-((Benzimidazol-2-yl) thio)-1-arylethan-1-ones) the strongest inhibitory activity against the PLpro enzyme. In addition, disubstitutions at the meta and para locations are the only substitutions that may boost the inhibitory action against PLpro. Compound 2 was chosen as a MERS-CoV PLpro inhibitor after passing absorption, distribution, metabolism, and excretion studies; however, further investigations are required.

Next-generation sequencing in Newborn Screening: A review on clinical and economic prospects

Next-generation sequencing (NGS) technology has revamped the area of genetics with improve the sequence of a substantial number of genes with high accuracy and short turn-around time. It may allow being used of NGS as a first-tier diagnostic test for inborn errors of metabolic or other genetic disorders. Early diagnosis of genetic disorders may help to improve the clinical condition of the child. The advantages of NGS included panel-specific gene sequencing, which targets disease-specific genes to confirm the genetic conditions. This review discussed the advantage and potential challenges of the NGS in newborn screening, other methodologies for newborn screening v/s NGS, application of NGS in various disorders, in comparison to the other, clinical importance, and economic aspects. Gene-specific panels and whole exome sequencing have shortened the clinical diagnosis of complex medical conditions at an early age. Furthermore, gene sequencing facilitates to recognize the novel mutations. There are innumerable gaps in between knowledge, as well as the views of varied populations, abilities of public health, and health economics. DNA sequencing through NGS is nowadays frequently used in some clinical diagnoses, and its execution in newborn screening can provide us with better outcomes. Although inferences across the countries additional rigorous cost-effectiveness studies towards NGS have to be piloted and it is a favour to use NGS for newborn screening. In conclusion, NGS is a rapid, robust, and accurate diagnostic tool that can be used for newborn screening which helps the clinician to make a correct diagnosis and help in prior prevention and surveillance of disorder conditions.

Machine Learning Styles for Diabetic Retinopathy Detection: A Review and Bibliometric Analysis

Diabetic retinopathy (DR) is a medical condition caused by diabetes. The development of retinopathy significantly depends on how long a person has had diabetes. Initially, there may be no symptoms or just a slight vision problem due to impairment of the retinal blood vessels. Later, it may lead to blindness. Recognizing the early clinical signs of DR is very important for intervening in and effectively treating DR. Thus, regular eye check-ups are necessary to direct the person to a doctor for a comprehensive ocular examination and treatment as soon as possible to avoid permanent vision loss. Nevertheless, due to limited resources, it is not feasible for screening. As a result, emerging technologies, such as artificial intelligence, for the automatic detection and classification of DR are alternative screening methodologies and thereby make the system cost-effective. People have been working on artificial-intelligence-based technologies to detect and analyze DR in recent years. This study aimed to investigate different machine learning styles that are chosen for diagnosing retinopathy. Thus, a bibliometric analysis was systematically done to discover different machine learning styles for detecting diabetic retinopathy. The data were exported from popular databases, namely, Web of Science (WoS) and Scopus. These data were analyzed using Biblioshiny and VOSviewer in terms of publications, top countries, sources, subject area, top authors, trend topics, co-occurrences, thematic evolution, factorial map, citation analysis, etc., which form the base for researchers to identify the research gaps in diabetic retinopathy detection and classification.

Analysis of unknown fentanyl analogs using high resolution mass spectrometry with mass defect filtering

Drug overdoses in the United States have increased over the past several years due to the increase in the highly potent fentanyl and fentanyl analogs on the illicit drug market. Fentanyl analogs are created by altering the functional groups on the core fentanyl molecule. These molecules are continuously being tweaked which creates a challenge for forensic laboratories trying to identify these substances with traditional methods. Current screening methods of immunoassay or mass spectrometry target known standards for analysis and may not identify new analogs. Herein, we demonstrate a screening methodology that takes advantage of the mass accuracy afforded by a commercial high resolution mass spectrometer. A three-step, post-acquisition data analysis process identifies fentanyl analogs through mass defect filtering of unfragmented data, common fragments, and mass defect filtering of the fragmented data. Using this method, analysts can identify potential fentanyl analog peaks in the spectra and can then perform further structural elucidation and identification.

Lysosomal Acid Lipase Deficiency: Genetics, Screening, and Preclinical Study.

Lysosomal acid lipase (LAL) is a lysosomal enzyme essential for the degradation of cholesteryl esters through the endocytic pathway. Deficiency of the LAL enzyme encoded by the LIPA gene leads to LAL deficiency (LAL-D) (OMIM 278000), one of the lysosomal storage disorders involving 50-60 genes. Among the two disease subtypes, the severe disease subtype of LAL-D is known as Wolman disease, with typical manifestations involving hepatomegaly, splenomegaly, vomiting, diarrhea, and hematopoietic abnormalities, such as anemia. In contrast, the mild disease subtype of this disorder is known as cholesteryl ester storage disease, with hypercholesterolemia, hypertriglyceridemia, and high-density lipoprotein disappearance. The prevalence of LAL-D is rare, but several treatment options, including enzyme replacement therapy, are available. Accordingly, a number of screening methodologies have been developed for this disorder. This review summarizes the current discussion on LAL-D, covering genetics, screening, and the tertiary structure of human LAL enzyme and preclinical study for the future development of a novel therapy.

Improvement to Gravity Drainage Recovery by Repressurization as a Criterion to Screen and Rank Naturally Fractured Reservoirs for Gas Injection

Summary Many of the naturally fractured carbonate reservoirs of the Middle East exhibit low natural-depletion recoveries. The reason is that most of their oil reserves are stored in the low-permeability host rocks and are left behind by the fast advancing gas/oil contact (GOC) and water/oil contact (WOC) in fractures. Producing the remaining oil in the large gas-invaded zone of these reservoirs has been a crucial reservoir management issue. We show in this study using experimental observations, analytical calculations, and numerical investigations that repressurizing naturally fractured reservoirs (NFRs) by crestal immiscible gas injection has the potential to produce a large portion of this remaining oil by improving gravity drainage (GD) through two main mechanisms. One is that at higher pressures, the gas-oil interfacial tension (IFT) and hence the capillary forces that control recovery by GD are lessened, allowing additional recovery. This mechanism is aided by the other one, which is swelling of the oil at higher pressures. In this way, repressurization is thought to be not only a means for pressure maintenance but also a methodology for enhanced-oil recovery (EOR). This is confirmed by both laboratory studies and field performance of large-scale gas injection projects. Despite the desire for implementation of projects of repressurization, gas availability and cost of these projects are important concerns, requiring a cost-benefit analysis. Screening and ranking methodologies have been previously presented for some EOR techniques but not for repressurization by gas in NFRs. Evaluating the performance of gas injection in NFRs is often done using methodologies such as numerical simulations, which are in-depth, costly, and tedious. The methodology developed here is simple, requiring spreadsheet calculations. To develop the methodology, we first obtain simple relations to calculate additional GD recovery by considering the interplay of capillary and gravity forces in a matrix block subjected to pressurization by equilibrium gas injection and then use experimental data from literature to show that these relations can predict primary and secondary GD recoveries to a good approximation. We also show by mechanistic studies using a history-matched numerical model that IFT reduction and oil swelling are the main mechanisms contributing to additional oil recovery. Then, we propose a methodology to screen and rank candidate NFRs for gas injection that uses commonly available reservoir data and is based upon two criteria, these being additional oil recovered from a matrix block by pressurization and required volume of gas to produce an additional barrel of oil. We then implement this methodology to more than 20 Iranian NFRs and identify six reservoirs with potential for additional recovery of more than 20%. By quantifying and including the uncertainties associated with the reservoir data, we illustrate that for the reservoirs under study, capillary pressure parameters along with matrix-block height are the main parameters affecting GD recovery and should be characterized more accurately.

Looking at Diabetes-Related Distress through a New Lens: The Socio-Ecological Health Model

Diabetes-related distress (DRD) is defined as an emotional state experienced by people with diabetes (PWD) who are worried about their disease management, the emotional burden from the condition, and/or potential difficulties accessing care or support. The psychosocial aspect of diabetes management is a factor that directly influences patients’ well-being as well as the chronic management of the condition yet is not a primary clinical problem being addressed within the healthcare setting. This review advocates for a re-evaluation and subsequent adjustment of the current DRD screening methodology by implementing the five primary components (Intrapersonal, Interpersonal, Organizational, Community, and Public Policy) of the Socio-Ecological Model of Health (SEMH), bridging the gaps from a public-health perspective. We searched two electronic databases for studies published in the United States from 1995 to 2020 reporting the effects of social determinants of health (SDOH) on DRD. Articles that contained at least one of the five elements of the SEMH and focused on adults aged 18 years or older were included. SDOH, which include circumstances where individuals grow, work, and age, are highly influenced by external factors, such as the distribution of wealth, power, and resources. Current DRD screening tools lack the capacity to account for all major components of SDOH in a comprehensive manner. By applying the SEMH as a theory-based framework, a novel DRD screening tool addressing sex, ethnicity, and socioeconomic background should be implemented to better improve diabetes management outcomes. By exploring the relationships between each level of the SEMH and DRD, healthcare professionals will be better equipped to recognize potential stress-inducing factors for individuals managing diabetes. Further efforts should be invested with the goal of developing a novel screening tool founded on the all-encompassing SEMH in order to perpetuate a more comprehensive diabetes treatment plan to address barriers within the SDOH framework.

High-throughput screening of 2D van der Waals crystals with plastic deformability

Inorganic semiconductors exhibit multifarious physical properties, but they are prevailingly brittle, impeding their application in flexible and hetero-shaped electronics. The exceptional plasticity discovered in InSe crystal indicates the existence of abundant plastically deformable two-dimensional van der Waals (2D vdW) materials, but the conventional trial-and-error method is too time-consuming and costly. Here we report on the discovery of tens of potential 2D chalcogenide crystals with plastic deformability using a nearly automated and efficient high-throughput screening methodology. Seven candidates e.g., famous MoS2, GaSe, and SnSe2 2D materials are carefully verified to show largely anisotropic plastic deformations, which are contributed by both interlayer and cross-layer slips involving continuous breaking and reconstruction of chemical interactions. The plasticity becomes a new facet of 2D materials for deformable or flexible electronics.

SeqFLoW: A systematic approach to identify and select food waste valorisation opportunities

Food loss and waste (FLW) represents a global challenge with complex and multifaceted environmental, economic and social implications. There are many options to manage FLW, some of which extract value that would otherwise be lost and mitigate environmental impacts. Decision making frameworks aid the selection of FLW management options by comparing the associated environmental, social and economic impacts. However, these frameworks generally utilise in-depth assessments and multi-criteria decision making (MCDM) methods that are time consuming and require specialist training or software. Consequently, it is generally not feasible to apply in-depth analyses to multiple FLW management options, which limits the scope of the assessment, as there are many potential FLW management options for any given FLW, and new processes and products are being developed regularly. The Sequential decision making for Food Loss and Waste (SeqFLoW) framework provides a screening methodology, to assess and rank a broad range of FLW management options, based on critical success factors. The application of the SeqFLoW framework was demonstrated for a citrus waste case study, which systematically downselected five FLW management options from over 80 alternatives. The SeqFLoW framework offers industry and academic professionals a methodology to justify the downselection of FLW management alternatives for further analysis.

Investigating Biomarkers for Primary Immunodeficiency Disorders in Patients with Autoimmune Cytopenia

Introduction: Autoimmune cytopenias (AICs) occur in the setting of immune dysregulation and autoimmunity. AICs are known to precede the classical presentation and subsequent diagnosis of primary immunodeficiency disorders (PIDs) in some cases, with a PID spectrum involving B, T, and Treg dysregulation. Conversely, PID patients are also at a markedly increased risk of AICs and patients co-diagnosed with AICs and PIDs face higher mortality rates likely due to delayed diagnosis or improper/delayed therapy. Further, co-diagnosed patients are often refractory to standard treatment. Early screening and diagnosis of PIDs in the setting of AICs could lead to more targeted therapies and potentially better patient outcomes. We intend to investigate biomarkers that differentiate immune dysregulation among those with or without PID in the setting of AIC. Methods: A retrospective analysis comparing clinical history, including symptoms, presence of PID, genetic testing and diagnosis and treatment outcomes was performed on AIC patients in our hospital from 2016 to 2019. We also performed extensive immune phenotyping by flow cytometric analysis. Patients with expansion of CD19hi CD21lo B cells were further evaluated for Tbet+ CD11chi features for potential autoreactivity. Results: Our original cohort of 103 AIC patients included patients who were diagnosed with AIC only (n=52) or co-diagnosed with AIC-PID (n=51). From this cohort, a total of 43 were evaluated for further immune phenotyping, including 35 AIC-PID patients and 8 AIC only patients. Of the AIC-PID patients, 23 (65.7%) had a genetically coded PID, including pathogenic variants in CTLA4, NFKB1, PI3K or partial DiGeorge, while 12 (34.3%) were diagnosed with CVID/CID. Flow cytometry analysis revealed significantly increased T-follicular helper cell (Tfh) population in the AIC-PID group compared to AIC only (p=0.03) and healthy donor (HD) (p=0.004). We saw a similar expansion of CD19hi CD21lo B cells within the AIC-PID group. Conversely, patients with AIC-PID had decreased percentage of T-regulatory cells (Tregs) when compared to AIC only and HD groups. These differences were amplified within the genetically coded AIC-PID patients in Treg and Tfh populations and maintained in CD19hi CD21lo B cells. Further investigation of clinical history within the symptomatic, genetically coded AIC-PID group yielded 3 (33.3%) patients responsive to treatment and 6 (66.7%) patients unresponsive or partially responsive to therapy. Interestingly, nonresponsive patients showed an increased percentage of CD19hi CD21lo B cells. Conclusions: Our analysis demonstrates the potential utility of biomarkers, notably CD19hi CD21lo B cells and their immunological function for detecting underlying PIDs within the setting of AIC patients. Though this B cell population needs further assessment regarding Tbet and CD11c expression, with studies ongoing, these biomarkers may lead to a screening methodology to increase clinical visibility and detection as well as hasten diagnosis of underlying PID and potentially predict response to therapy, therefore increasing effective treatment outcomes for patients co-diagnosed with AIC PID.

Therapeutic Strategies Targeting Pancreatic Islet β-Cell Proliferation, Regeneration, and Replacement.

Diabetes results from insufficient insulin production by pancreatic islet β-cells or a loss of β-cells themselves. Restoration of regulated insulin production is a predominant goal of translational diabetes research. Here, we provide a brief overview of recent advances in the fields of β-cell proliferation, regeneration, and replacement. The discovery of therapeutic targets and associated small molecules has been enabled by improved understanding of β-cell development and cell cycle regulation, as well as advanced high-throughput screening methodologies. Important findings in β-cell transdifferentiation, neogenesis, and stem cell differentiation have nucleated multiple promising therapeutic strategies. In particular, clinical trials are underway using in vitro-generated β-like cells from human pluripotent stem cells. Significant challenges remain for each of these strategies, but continued support for efforts in these research areas will be critical for the generation of distinct diabetes therapies.

Factors Predictive of Psychiatric Consultation-Liaison Practice in the Gynecology Clinic of a Nigerian Teaching Hospital

There is a high rate of psychological distress among women attending gynecology clinics. Psychiatric morbidity among this group of patients has been studied but no clear-cut delineations in the association between psychiatric morbidity, established gynecological diagnosis and ongoing gynecological symptoms especially as guidelines for consultation-liaison practice in the gynecological unit. The objectives of this study therefore were to determine the prevalence of psychiatric disorders in this group of patients; to determine the association of psychiatric morbidity with ongoing gynecological complaints, gynecological diagnosis and psychosocial factors; and to delineate factors predictive of psychiatric consultation-liaison need in the gynecological clinic. Using structural questionnaires and employing a 2-stage screening methodology for patients attending the gynecological clinic of Ladoke Akintola University of Technology Teaching Hospital for about 12 weeks consecutively, a total of seventy eight patients were recruited invariably. The mean age of the study population was 35.9 years. Overall prevalence of psychiatric disorder was 55.3%. Depressive illness occurred in about 42.8% of the study population. No significant association between psychiatric morbidity, ongoing gynecological symptoms and established gynecological diagnosis. The commonest diagnosis was infertility. However, specific ongoing gynaecological complaints with psychiatric morbidity had 49.7% of those with inability to conceive (primary), 57.1% of those who had pains during intercourse, 71.4% of those with cessation of menses and 60% of those with irregular menses as opposed to lower scores among those with established gynecological diagnosis. Specific on-going gynecological complaints may impose graver consequences on the psychological wellbeing of a woman than established gynecological diagnosis. The consideration and management of ongoing gynecological complaints and social support may be issues for psychiatric consultation-liaison crucial in the management of such patients to reduce psychiatric morbidity amongst them.

Determination of grated hard cheese adulteration by digital image analysis and multivariate analysis

Hard-cheeses are foods with high commercial value, thus fraudulent practices are frequent and difficult to detect, which reduce hard-cheese quality and consequently, decreases nutritional value. This work presents a novel and feasible tool to identify the adulteration of grated-cheese with higher levels of additives than those permitted (cellulose and silicon dioxide), and foreign substances used to increase weight and volume (wheat-flour, wheat-semolina and sawdust). The methodology is based on the use of colour histograms (obtained from digital images) and multivariate classification analysis. Satisfactory results were obtained with support vector machines with average values of accuracy and sensitivity above 82%, as adulterated samples were discriminated from original (unadulterated) samples. The proposed screening methodology represents a fast, simple, reliable and affordable way to perform quality control of grated-cheeses to avoid food fraud and nutritional detriments of worldwide consumption foods such as hard-cheeses.

Advanced throughput analytical strategies for the comprehensive HRMS screening of organic micropollutants in eggs of different bird species

Raptors are ideal indicators for biomonitoring studies using wildlife in order to assess the environmental pollution in the terrestrial ecosystem, since they are placed in the highest trophic position in the food webs and their life expectancy is relatively long. In this study, 26 eggs of 4 bird species (Peregrine falcon, Eurasian curlew, Little owl and Eagle owl) collected in Germany, were investigated for the presence of persistent organic pollutants (POPs) and thousands of contaminants of emerging concern (CECs). Generic sample preparation protocols were followed for the extraction of the analytes and the purification of the extracts, and the samples were analyzed both by liquid (LC) and gas chromatography (GC) coupled to high resolution mass spectrometry (HRMS), for capturing a wide range of organic micropollutants with different physicochemical properties. State-of-the-art screening methodologies were applied in the acquired HRMS data, including wide-scope target analysis of 2448 known pollutants and suspect screening of over 65,000 environmentally relevant compounds. Overall, 58 pollutants from different chemical classes, such as plant protection products, per- and polyfluoroalkyl substances and medicinal products, as well as their transformation products, were determined through target analysis. Most of the detected compounds were lipophilic (logP>2), although the presence of (semi)polar contaminants should not be overlooked, underlying the need for holistic analytical approaches in environmental monitoring studies. p,p’-DDE, PCB 153 and PCB138, PFOS and methylparaben were the most frequently detected compounds. 50 additional substances were identified and semi-quantified through suspect screening workflows, including mainly compounds of industrial use with high production volume.

Highly Efficient CRISPR/Cas9-Mediated Genome Editing in Human Pluripotent Stem Cells.

Human pluripotent stem cells hold tremendous potential for both basic biology and cell-based therapies for a wide variety of diseases. The ability to manipulate the genome of these cells using the CRISPR/Cas9 technology has expanded this potential by providing a valuable tool to engineer or correct disease-associated mutations. Because of the high efficiency with which CRISPR/Cas9 creates targeted double-strand breaks, a major challenge has been the introduction of precise genetic modifications on one allele without indel formation on the non-targeted allele. To overcome this obstacle, we describe use of two oligonucleotide repair templates: one expressing the sequence change and the other maintaining the normal sequence. In addition, we have streamlined both the transfection and screening methodologies to make the protocols efficient, with small numbers of cells and a limited amount of labor-intensive clone passaging. This article provides a technically simple approach for generating valuable tools to model human disease in stem cells. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Application and optimization of CRISPR-based genome editing in human pluripotent stem cells Basic Protocol 2: Genetic modification of human pluripotent stem cells using a double-oligonucleotide CRISPR/Cas9 recombination system.