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Pectin from the citrus peel waste has novel applications in food and biomedical industries. The present work focused on addressing iron deficiency, which is a global health concern, by developing a functional ingredient using pectin extracted from Assam lemon (Citrus limon Burm. F) and supplementing iron via the pectin‑iron complex (PIC). Extracted pectin was incubated with iron chloride hexahydrate (0.90-1.80 mM) for 180 h to optimize the complexation conditions, with the optimal concentration being 1.36 mM. The iron bioavailability and its absorption in the PIC was assessed using in-vitro simulation digestion and Caco-2 cell monolayers. The bioaccessible form of iron in the developed PIC during the intestinal phase was 5.34 ± 0.16%, which was negligible in pectin. The absorption of bioaccessible iron in the PIC was found to be 2.93 ± 0.03%. The results demonstrated that PIC could reduce iron deficiency and increase fibre intake, leading to several health benefits., Competing Interests: Declaration of competing interest The authors assert that they have no conflicting interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The targeted organisms include mosquito vectors, bacterial pathogens and non-targeted organisms. Preliminary mosquito larvicidal activity was conducted using cell-free supernatants (CFSs) from 11 gut bacteria. Among them, the bacterium SS11 exhibited promising results and was identified as Kurthia gibsonii based on its 16S rRNA sequence (1350 bp). The diethyl ether extract (DEE) of K. gibsonii demonstrated significant larvicidal effects, with LC50 values of 5.59 µL/mL and 8.59 µL/mL for 3rd instar larvae of Aedes aegypti and 2nd instar larvae of Anopheles stephensi , respectively. Analysis of the DEE using FT-IR, and GC-MS revealed the presence of 16 functional groups, and 7 bioactive compounds, respectively. A molecular docking study identified GC-MS compounds against odorant receptors from A. aegypti and odorant-binding proteins from A. stephensi was performed to assess the interaction and binding affinity. Overall, these findings suggest that the bioactive compounds 2, 4, 6-tribromoaniline from the DEE of K. gibsonii hold potential as an environmentally compatible alternative for biocontrol purposes, and compounds 9-tricosene and didecyl phthalate can be used for mosquito traps.

COVID-19 appeared as a highly contagious disease after its outbreak in December 2019 by the virus, named SARS-CoV-2. The threat, which originated in Wuhan, China, swiftly became an international emergency. Among different genomic products, spike protein of virus plays a crucial role in the initiation of the infection by binding to the human lung cells, therefore, SARS-CoV-2's spike protein is a promising therapeutic target. Using a combination of a structure-based virtual screening and biochemical assay, this study seeks possible therapeutic candidates that specifically target the viral spike protein. A database of ~ 850 naturally derived compounds was screened against SARS-CoV-2 spike protein to find natural inhibitors. Using virtual screening and inhibitory experiments, we identified acetyl 11-keto-boswellic acid (AKBA) as a promising molecule for spike protein, which encouraged us to scan the rest of AKBA derivatives in our in-house database via 2D-similarity searching. Later 19 compounds with > 85% similarity with AKBA were selected and docked with receptor binding domain (RBD) of spike protein. Those hits declared significant interactions at the RBD interface, best possess and excellent drug-likeness and pharmacokinetics properties with high gastrointestinal absorption (GIA) without toxicity and allergenicity. Our in-silico observations were eventually validated by in vitro bioassay, interestingly, 10 compounds (A3, A4, C3, C6A, C6B, C6C, C6E, C6H, C6I, and C6J) displayed significant inhibitory ability with good percent inhibition (range: > 72-90). The compounds C3 (90.00%), C6E (91.00%), C6C (87.20%), and C6D (86.23%) demonstrated excellent anti-SARS CoV-2 spike protein activities. The docking interaction of high percent inhibition of inhibitor compounds C3 and C6E was confirmed by MD Simulation. In the molecular dynamics simulation, we observed the stable dynamics of spike protein inhibitor complexes and the influence of inhibitor binding on the protein's conformational arrangements. The binding free energy ΔG TOTAL of C3 (-38.0 ± 0.08 kcal/mol) and C6E (-41.98 ± 0.08 kcal/mol) respectively indicate a strong binding affinity to Spike protein active pocket. These findings demonstrate that these molecules particularly inhibit the function of spike protein and, therefore have the potential to be evaluated as drug candidates against SARS-CoV-2., (© 2024. The Author(s).)

Uridine 5'-diphospho-glucuronosyltransferases (UGTs) have been considered as a family of enzymes responsible for the glucuronidation process, a crucial phase II detoxification reaction. Among the various UGT isoforms, UGTs A10 and B7 have garnered significant attention due to their broad substrate specificity and involvement in the metabolism of numerous compounds. Recent studies have suggested that certain vitamins may exert inhibitory effects on UGT activity, thereby influencing the metabolism of drugs, environmental toxins, and endogenous substances, ultimately impacting their biological activities. In the present study, the inhibition potential of vitamins (A, B1, B2, B3, B5, B6, B7, B9, D3, E, and C) on UGT1A10 and UGT2B7 was determined using in silico and in vitro approaches. A 3-dimensional model of UGT1A10 and UGT2B7 enzymes was built using Swiss Model, ITASSER, and ROSETTA and verified using Ramachandran plot and SAVES tools. Molecular docking studies revealed that vitamins interact with UGT1A10 and UGT2B7 enzymes by binding within the active site pocket and interacting with residues. Among all vitamins, the highest binding affinity predicted by molecular docking was - 8.61 kcal/mol with vitamin B1. The in vitro studies results demonstrated the inhibition of the glucuronidation activity of UGTs by vitamins A, B1, B2, B6, B9, C, D, and E, with IC 50 values of 3.28 ± 1.07 µg/mL, 24.21 ± 1.11 µg/mL, 3.69 ± 1.02 µg/mL, 23.60 ± 1.08 µg/mL, 6.77 ± 1.08 µg/mL, 83.95 ± 1.09 µg/ml, 3.27 ± 1.13 µg/mL and 3.89 ± 1.12 µg/mL, respectively. These studies provided the valuable insights into the mechanisms underlying drug-vitamins interactions and have the potential to guide personalized medicine approaches, optimizing therapeutic outcomes, and ensuring patient safety. Indeed, further research in the area of UGT (UDP-glucuronosyltransferase) inhibition by vitamins is essential to fully understand the clinical relevance and implications of these interactions. UGTs play a crucial role in the metabolism and elimination of various drugs, toxins, and endogenous compounds in the body. Therefore, any factors that can modulate UGT activity, including vitamins, can have implications for drug metabolism, drug-drug interactions, and overall health., Supplementary Information: The online version contains supplementary material available at 10.1007/s40203-023-00182-0., Competing Interests: Conflict of interestThe authors have no conflicts of interest to declare that are relevant to the content of this article., (© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2024. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

The horizon of immunotherapy using CAR-T cells is continuously extending to treat solid tumors beyond the success in the treatment of liquid tumors. Precise in-vitro evaluations of CAR-T cells for their phenotypes, quantity and quality of activation in various tumor microenvironments including different antigen densities, and the resulting effector functions are critical for the successful development of CAR-T therapies and safe translation to clinics. Unfortunately, the development of methods and tools to accommodate these needs have been lagging behind. Here, we developed a novel biomaterial platform, acellular artificial target particles (aaTPs) against CAR-T cells, using magnetic microbeads that are already widely employed in the manufacturing of T cell products. By devising a simple and standardized procedure, we precisely controlled the antigen surface densities presented on the aaTPs for a wide range. By co-incubation of aaTPs with CAR-T cells followed by flow cytometry and cytokine assays, we quantitatively determined the antigen-specific and dose-dependent activation of anti-HER2 CAR-T cells. We also demonstrated that the aaTP can serve as a clean target cell in in-vitro assays to prove the proposed mechanism of action of a next-generation CAR-T product. Overall, the simple, inexpensive, modular and precisely controllable synthetic nature of aaTPs enables the development of clean and standardized in-vitro assays for CAR-T cells, which provides critical advantages over the conventional assays using target cell lines. The design of aaTPs can be extended to include other tumor antigens and relevant surface molecules of physiological target cells. Thus, the aaTP platform has great potential as a standardized tool for the development and evaluation of both conventional and new CAR-T products in the context of approval from regulatory agencies and clinical translation., Competing Interests: Authors RH-S, LC, and FM were employed by the company Refuge Biotechnologies. The authors declare that this study received funding from Refuge Biotechnologies. The funder had the following involvement in the study: the study design, collection, analysis, interpretation of data, and the writing of this article., (Copyright © 2022 Harari-Steinfeld, Abhinav Ayyadevara, Cuevas, Marincola and Roh.)

The adaptive foam reticulation technique combines the foam reticulation and freeze casting methodologies of fabricating bone reparative scaffolds to offer a potential alternative to autografts. For the first time this paper studies the effect of processing on the mechanical properties and in-vitro cell growth of controllably generating a hierarchical structure of macro- (94 ± 6 to 514 ± 36 μm) and microporosity (2-30 μm) by the inclusion of camphene as a porogen during processing. Scaffolds were produced with porogen additions of 0-25 wt%. Porosity values of the structures of 85-96% were determined using the Archimedes technique and verified using X-ray Computed Tomography. The strength of the hydroxyapatite scaffolds, 5.70 ± 1.0 to 159 ± 61 kPa, correlated to theoretically determined values, 3.71 ± 0.8 to 134 ± 12 kPa, calculated by the novel incorporation of a shape factor into a standard equation. Fibroblast (3T3) and pre-osteoblast (MC3T3) cell growth was found to be significantly (P < 0.005) improved using 25 wt% porogen. This was supported by increased levels of alkaline phosphatase and was thought to result from greater dissolution as quantified by increased calcium levels in incubating media. The combination of these properties renders adaptive foam reticulation-fabricated scaffolds suitable for non-structural bone regenerative applications in non-load bearing bone defects., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

To date, the production and development of portable analytical devices for environmental and healthcare applications are rapidly growing. Herein, a portable electrochemical sensor for monitoring of noradrenaline (NA) secreted from living cells using mesoporous carbon-based materials was fabricated. The modification of the interdigitated electrode array (IDA) by nitrogen-doped mesoporous carbon spheres (N-doped MCS) and nitrogen-doped carbon hollow trunk-like structure (N-doped CHT) was used to fabricate the NA sensor. The N-doped CHT surface shows multiple holes distributed with micrometer-sized open holes (1-2 μm) and nanometer-sized thin walls (∼98 nm). The N-doped CHT surface heterogeneity of wrinkled and twisted hollow trunk structures improve the diffusion pathway and the NA molecules loading. The N-doped CHT/IDA showed a highly selective assay for monitoring of NA with high sensitivity (1770 μA/μM × cm 2 ), a low detection limit (0.005 μM), and a wide linear range (0.01-0.3 μM). The N-doped CHT/IDA monitored the NA secreted from PC12 cells under various concentrations of simulation agents (KCl). The designed N-doped CHT/IDA provides a portable NA-sensor assay with facile signaling, good stability, high biocompatibility, in-vitro assay compatibility, and good reproducibility. Therefore, the designed sensor can be used as a portable sensor for NA detection in live cells and can be matched with portable smartphones after further developments., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Diagnostic testing plays a critical role in addressing the coronavirus disease 2019 (COVID-19) pandemic, caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Rapid and accurate diagnostic tests are imperative for identifying and managing infected individuals, contact tracing, epidemiologic characterization, and public health decision making. Laboratory testing may be performed based on symptomatic presentation or for screening of asymptomatic people. Confirmation of SARS-CoV-2 infection is typically by nucleic acid amplification tests (NAAT), which requires specialized equipment and training and may be particularly challenging in resource-limited settings. NAAT may give false-negative results due to timing of sample collection relative to infection, improper sampling of respiratory specimens, inadequate preservation of samples, and technical limitations; false-positives may occur due to technical errors, particularly contamination during the manual real-time polymerase chain reaction (RT-PCR) process. Thus, clinical presentation, contact history and contemporary phyloepidemiology must be considered when interpreting results. Several sample-to-answer platforms, including high-throughput systems and Point of Care (PoC) assays, have been developed to increase testing capacity and decrease technical errors. Alternatives to RT-PCR assay, such as other RNA detection methods and antigen tests may be appropriate for certain situations, such as resource-limited settings. While sequencing is important to monitor on-going evolution of the SARS-CoV-2 genome, antibody assays are useful for epidemiologic purposes. The ever-expanding assortment of tests, with varying clinical utility, performance requirements, and limitations, merits comparative evaluation. We herein provide a comprehensive review of currently available COVID-19 diagnostics, exploring their pros and cons as well as appropriate indications. Strategies to further optimize safety, speed, and ease of SARS-CoV-2 testing without compromising accuracy are suggested. Access to scalable diagnostic tools and continued technologic advances, including machine learning and smartphone integration, will facilitate control of the current pandemic as well as preparedness for the next one., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mardian, Kosasih, Karyana, Neal and Lau.)

Alzheimer's disease (AD), is among the most growing neurodegenerative diseases, which is mainly caused by the acetylcholine neurotransmitter loss in the hippocampus and cortex. Emerging of the dual Acetylcholinesterase (AChE)/Butyrylcholinesterase (BuChE) inhibitors has increased for treating Alzheimer disease. In this study, we would like to report the design and synthesis of a new sequence of 1-benzyl-4-((4-oxoquinazolin-3(4 H )-yl)methyl) pyridin-1-ium derivatives (BOPs) assessed as BuChE and AChE inhibitors. Ellman's approach was used for the evaluation of AChE and BuChE inhibitory activities. Moreover, docking research was conducted to predict the action mechanism. Among all synthesized compounds, 1-(3-bromobenzyl)-3-((4-oxoquinazolin-3(4 H )-yl)methyl) pyridin-1-ium bromide (BOP-1) was found to be the most active compound with dual activity for inhibition of AChE (IC 50 = 5.90 ± 0.07μM), and BuChE (IC 50 = 6.76 ± 0.04μM) and 1-(4-chlorobenzyl)-3-((6,7-dimethoxy-4-oxoquinazolin-3(4 H )-yl)methyl) pyridin-1-ium chloride (BOP-8) showed the highest AChE inhibitory activity (IC 50 s = 1.11 ± 0.09 μM). The synthesized compounds BOP-1 and BOP-8 could be proposed as valuable lead compounds for further drug discovery development against AD., Competing Interests: The authors declare no conflict of interest., (© 2021 The Author(s).)

Objectives: Direct toxic effects of transthyretin amyloid in patient plasma upon cardiomyocytes are discussed. However, no data regarding the relevance of this putative effect for clinical outcome are available. In this monocentric prospective study, we analyzed cellular hypertrophy after phenylephrine stimulation in vitro in the presence of patient plasma and correlated the cellular growth response with phenotype and prognosis., Methods and Results: Progress in automated microscopy and image analysis allows high-throughput analysis of cell morphology. Using the InCell microscopy system, changes in cardiomyocyte's size after treatment with patient plasma from 89 patients suffering from transthyretin amyloidosis and 16 controls were quantified. For this purpose, we propose a novel metric that we named Hypertrophic Index, defined as difference in cell size after phenylephrine stimulation normalized to the unstimulated cell size. Its prognostic value was assessed for multiple endpoints (HTX: death/heart transplantation; DMP: cardiac decompensation; MACE: combined) using Cox proportional hazard models. Cells treated with plasma from healthy controls and hereditary transthyretin amyloidosis with polyneuropathy showed an increase in Hypertrophic Index after phenylephrine stimulation, whereas stimulation after treatment with hereditary cardiac amyloidosis or wild-type transthyretin patient plasma showed a significantly attenuated response. Hypertrophic Index was associated in univariate analyses with HTX (hazard ratio (HR) high vs low: 0.12 [0.02-0.58], p = 0.004), DMP: (HR 0.26 [0.11-0.62], p = 0.003) and MACE (HR 0.24 [0.11-0.55], p < 0.001). Its prognostic value was independent of established risk factors, cardiac TroponinT or N-terminal prohormone brain natriuretic peptide (NTproBNP)., Conclusions: Attenuated cardiomyocyte growth response after stimulation with patient plasma in vitro is an independent risk factor for adverse cardiac events in ATTR patients.

Background: Acetylcholine deficiencies in hippocampus and cortex, aggregation of β-amyloid, and β-secretase over activity have been introduced as main reasons in pathogenesis of Alzheimer's disease., Methods: Colorimetric Ellman's method was used for determination of IC 50 value in AChE and BChE inhibitory activity. The kinetic studies, neuroprotective and β-secretase inhibitory activities, evaluation of inhibitory potency on β-amyloid (Aβ) aggregations induced by AChE, and docking study were performed for prediction of the mechanism of action., Result and Discussion: A new series of cinnamic acids-tryptamine hybrid was designed, synthesized, and evaluated as dual cholinesterase inhibitors. These compounds demonstrated in-vitro inhibitory activities against acetyl cholinesterase (AChE) and butyryl cholinesterase (BChE). Among of these synthesized compounds, (E)-N-(2-(1H-indol-3-yl)ethyl)-3-(3,4-dimethoxyphenyl)acrylamide (5q) demonstrated the most potent AChE inhibitory activity (IC 50  = 11.51 μM) and (E)-N-(2-(1H-indol-3-yl)ethyl)-3-(2-chlorophenyl)acrylamide (5b) were the best anti-BChE (IC 50  = 1.95 μM) compounds. In addition, the molecular modeling and kinetic studies depicted 5q and 5b were mixed type inhibitor and bound with both the peripheral anionic site (PAS) and catalytic sites (CAS) of AChE and BChE. Moreover, compound 5q showed mild neuroprotective in PC12 cell line and weak β-secretase inhibitory activities. This compound also inhibited aggregation of β-amyloid (Aβ) in self-induced peptide aggregation test at concentration of 10 μM., Conclusion: It is worth noting that both the kinetic study and the molecular modeling of 5q and 5b depicted that these compounds simultaneously interacted with both the catalytic active site and the peripheral anionic site of AChE and BChE. These findings match with those resulted data from the enzyme inhibition assay. Graphical abstract A new series of cinnamic-derived acids-tryptamine hybrid derivatives were designed, synthesized and evaluated as butyrylcholinesterase (BChE) and acetylcholinesterase (AChE) inhibitors and neuroprotective agents. Compound 5b and 5q, as the more potent compounds, interacted with both the peripheral site and the choline binding site having mixed type inhibition. Results suggested that derivatives have a therapeutic potential for the treatment of AD.

Reliable and sensitive detection of antigen specific cells is essential in several fields of research, whether it concerns monitoring responses to infectious agents or exploring the auto-antigen repertoire in autoimmune diseases. Identification of these cells is however difficult, especially when the cells often are rare and methods not sensitive, specific or practical enough. We propose a novel method of processing antigens before stimulation of cells which consists of covalently binding protein antigen to superparamagnetic micro-beads and using denaturing washes to remove contaminants. Peripheral blood mononuclear cells (PBMCs) from healthy donors were stimulated using both cytomegalovirus and tetanus-diphtheria antigen-beads as well as non-antigenic protein-beads as negative control in an IFNγ FluoroSpot assay in order to detect Th1 and CD8 + responses. The responses toward the antigen beads were both antigen specific and sensitive, with a detection threshold of 1 IFNγ producing T-cell per 18,000 PBMCs. •Covalently binding antigen to paramagnetic beads allows for harsh denaturing washes without loss of antigen.•Microbeads are phagocytosed by antigen presenting cells, resulting in efficient uptake, processing and presentation of the antigens.•The method allows the usage of relatively impure starting antigen material and whole PBMC samples without high background levels in follow up cellular assays., Competing Interests: We wish to draw the attention of the editor to the following facts which may be considered as potential conflicts of interest. There are pending patents regarding parts of the methods presented in this manuscript, more precisely the usage of antigen-beads for detection of antigen-specific T-cells. Mattias Bronge and Hans Grönlund are the inventors of said patents which are held by the private company TCER AB. Hans Grönlund is a co-founder of TCER AB and Claudia Carvalho-Queiroz, Ola Nilsson and Andreas Kaiser hold positions at TCER AB. The study has been partly funded by grants from CBD solutions, Neuroförbundet, Swedish Research Council (2017-00777), Swedish Brain Foundation, Margareta af Ugglas Foundation and Stratneuro. None of the funding sources have had a role in the study design, collection, analysis or interpretation of the data.

An automatic flow-through dynamic extraction method is proposed for the first time for in vitro exploration, with high temporal resolution, of the transit of the chyme from the gastric to the duodenal compartment using the Versantvoort's fed-state physiologically relevant extraction test. The flow manifold was coupled on-line to an inductively coupled plasma optical emission spectrometer (ICP OES) for real-time elucidation of the bioaccessible elemental fraction of micronutrients (viz., Cu, Fe and Mn) in food commodities across the gastrointestinal tract. The simulated intestinal and bile biofluid (added to the gastric phase) was successively pumped at 1.0 mL min -1 through a large-bore column (maintained at 37.0 ± 2.0 °C) initially loaded with a weighed amount of linseed (250 mg) using a PVDF filter membrane (5.0 μm pore size) for retaining of the solid sample and in-line filtration of the extracts. The lack of bias (trueness) of the on-line gastrointestinal extraction method coupled to ICP OES was confirmed using mass balance validation following microwave assisted digestion of the residual (non-bioaccessible) elemental fraction. Mass balance validation yielded absolute recoveries spanning from 79 to 121% for the overall analytes and samples. On-line dynamic extraction was critically appraised against batch counterparts for both gastric and gastrointestinal compartments. Due to the lack of consensus in the literature regarding the agitation method for batch oral bioaccessibility testing, several extraction approaches (viz., magnetic stirring, end-over-end rotation and orbital shaking) were evaluated. Improved gastric extractability of Fe along with bioaccessible data comparable to the dynamic counterpart based on the continuous displacement of the extraction equilibrium was obtained with batchwise magnetic stirring, which is deemed most appropriate for ascertaining worst-case/maximum bioaccessibility scenarios., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Butyrylcholinesterase (BuChE) inhibitors have become interesting target for treatment of Alzheimer's disease (AD). A series of dual binding site BuChE inhibitors were designed and synthesized based on 2,3,4,9-tetrahydro-1H-carbazole attached benzyl pyridine moieties. In-vitro assay revealed that all of the designed compounds were selective and potent BuChE inhibitors. The most potent BuChE inhibitor was compound 6i (IC 50  = 0.088 ± 0.0009 μM) with the mixed-type inhibition. Docking study revealed that 6i is a dual binding site BuChE inhibitor. Also, Pharmacokinetic properties for 6i were accurate to Lipinski's rule. In addition, compound 6i demonstrated neuroprotective and β-secretase (BACE1) inhibition activities. This compound could also inhibit AChE-induced and self-induced Aβ peptide aggregation at concentration of 100 μM and 10 μM respectively. Generally, the results are presented as new potent selective BuChE inhibitors with a therapeutic potential for the treatment of AD., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Reconstitution of cellular organelles in vitro offers the possibility to perform quantitative and qualitative experiments in a controlled environment that cannot be done with the same accuracy in living cells. Following a previous report, the subsequent list of protocols describes how to reconstitute and quantify a tubular ER network in vitro based on purified microsomes from culture cells and cytosol from Xenopus laevis egg extracts. Biological material preparation and reconstitution assays require mostly basic laboratory instrumentation and chemicals, and can be executed without any specific training, making them appealing to a wide range of laboratories. Moreover, to promote conditions that are markedly more reflective of in vivo environments, this method describes for the first time in the literature, the purification of microsomes from HeLa cells in some detail. Basic Protocol 1 in this article describes the reconstitution process on different substrates including the associated fluorescence imaging process. Purification of ER microsomes and cytosol, both of which are needed for this approach, are described in detail in Support Protocols 1 and 2, respectively. Coating of surfaces with polyacrylamide gels is described in Support Protocol 3. Basic Protocol 2 outlines how to segment and skeletonize fluorescence images of ER networks, and how to quantify segment lengths between the network's branching points. The described quantitative evaluation provides a meaningful approach to analyze the topology and geometry of organelle structures. © 2017 by John Wiley & Sons, Inc., (Copyright © 2017 John Wiley & Sons, Inc.)

In-vitro physiologically relevant gastrointestinal extraction based on the validated Unified BARGE Method (UBM) is in this work hyphenated to inductively coupled plasma optical emission spectrometry in a batch-flow configuration for real-time monitoring of oral bioaccessibility assays with high temporal resolution. A fully automated flow analyzer is designed to foster in-line filtration of gastrointestinal extracts at predefined times (≤15 min) followed by on-line multi-elemental analysis of bioaccessible micro-nutrients, viz., Cu, Fe and Mn, in well-defined volumes of extracts (300 μL) of transgenic and non-transgenic soybean seeds taken as model samples. The hyphenated flow setup allows for recording of temporal extraction profiles to gain full knowledge of the kinetics of the gastrointestinal digestion processes, including element leaching and concomitant precipitation and complexation reactions hindering bioavailability. Simplification of the overall standard procedure is also feasible by identification of steady-state extraction conditions. Our findings indicate that reliable measurement of oral bioaccessible pools of Cu, Fe and Mn in soybean might be obtained in less than 180 min rather than 240 min as endorsed by UBM. Using a matrix-matched external calibration, limits of detection according to the 3s criteria were 0.5 μg/g for Mn, 0.6 μg/g for Cu and 2.3 μg/g for Fe. Trueness of the automatic bioaccessibility method was confirmed by mass balance validation with recoveries ranging from 87 to 116% regardless of the target element and sample. Cu was the micronutrient with the highest oral bioaccessibility ranging from 73% to 83% (7.5-7.9 μg/g) for non-transgenic and transgenic soybeans, respectively, followed by Mn and Fe within the ranges of 29-31% (10.8-11.4 μg/g) and 11-15% (8-14 μg/g), respectively, regardless of transgenesis. The proposed kinetic method is proven suitable for fast and expedient estimation of the nutritional value of soybeans and elucidation of the potential effect of transgenesis onto bioaccessible fractions of elements., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Development of an improved vaccine against tuberculosis (TB) is hindered by the lack of a surrogate of protection. Efficacy of new TB vaccines in humans can only be evaluated by expensive and time consuming efficacy trials within TB endemic areas. It is critical that vaccines with the greatest potential to protect are selected for these trials. Mycobacterial growth inhibition assays (MGIAs) have been developed with the hope that these in-vitro functional assays will correlate with protection, which could aid in the selection of the best vaccine candidates. The present study describes the use of the BACTEC system to perform MGIAs in mice. We demonstrate reproducible mycobacterial growth inhibition in splenocytes from BCG immunised mice compared with unimmunised mice (P < 0.023), which corresponded with in-vivo efficacy against Mycobacterium tuberculosis (M. tb) challenge. Microarray data showed extensive differential gene expression in splenocyte responses to ex-vivo BCG stimulation between unimmunised and BCG-immunised mice. TH1 responses, including IFN-γ, nitric oxide synthase (NOS2) and Interleukin -17 (IL-17) expression were enhanced in BCG immunised mice, indicating a possible mechanism for mycobacterial growth inhibition. Further investigation into whether the BACTEC MGIA can be used as a surrogate of protection in humans and preclinical animal models is now warranted., (Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.)

In-vitro methods were developed to test fungi for production of metabolites affecting nematode egg hatch and mobility of second-stage juveniles. Separate assays were developed for two nematodes: root-knot nematode (Meloidogyne incognita) and soybean cyst nematode (Heterodera glycines). For egg hatch to be successfully assayed, eggs must first be surface-disinfested to avoid the confounding effects of incidental microbial growth facilitated by the fungal culture medium. Sodium hypochlorite was more effective than chlorhexidine diacetate or formaldehyde solutions at surface-disinfesting soybean cyst nematode eggs from greenhouse cultures. Subsequent rinsing with sodium thiosulfate to remove residual chlorine from disinfested eggs did not improve either soybean cyst nematode hatch or juvenile mobility. Soybean cyst nematode hatch in all culture media was lower than in water. Sodium hypochlorite was also used to surface-disinfest root-knot nematode eggs. In contrast to soybean cyst nematode hatch, root-knot nematode hatch was higher in potato dextrose broth medium than in water. Broth of the fungus Fusarium equiseti inhibited root-knot nematode egg hatch and was investigated in more detail. Broth extract and its chemical fractions not only inhibited egg hatch but also immobilized second-stage juveniles that did hatch, confirming that the fungus secretes nematode-antagonistic metabolites.