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Abstract Rapid mixing and precise timing are key for accurate biomedical assay measurement, particularly when the result is determined as the rate of a reaction: for example rapid immunoassay in which the amount of captured target is kinetically determined; determination of the concentration of an enzyme or enzyme substrate; or as the final stage in any procedure that involves a capture reagent when an enzyme reaction is used as the indicator. Rapid mixing and precise timing are however difficult to achieve in point-of-care devices designed for small sample volumes and fast time to result. By using centrifugal microfluidics and transposing the reaction surface from a chamber to a single mm-scale bead we demonstrate an elegant and easily manufacturable solution. Reagents (which may be, for example, an enzyme, enzyme substrate, antibody or antigen) are immobilised on the surface of a single small bead (typically 1–2 mm in diameter) contained in a cylindrical reaction chamber subjected to periodically changing rotational accelerations which promote both mixing and uniform mass-transfer to the bead surface. The gradient of Euler force across the chamber resulting from rotational acceleration of the disc, dΩ disc /dt, drives circulation of fluid in the chamber. Oscillation of Euler force by oscillation of rotational acceleration with period, T, less than that of the hydrodynamic relaxation time of the fluid, folds the fluid streamlines. Movement of the bead in response to the fluid and the changing rotational acceleration provides a dynamically changing chamber shape, further folding and expanding the fluid. Bead rotation and translation driven by fluid flow and disc motion give uniformity of reaction over the surface. Critical parameters for mixing and reaction uniformity are the ratio of chamber radius to bead radius, r chamber /r bead , and the product Tr chamber (dΩ disc /dt), of oscillation period and Euler force gradient across the fluid. We illustrate application of the concept using the reaction of horse radish peroxidase (HRP) immobilised on the bead surface with its substrate tetramethylbenzidine (TMB) in solution. Acceleration from rest to break a hydrophobic valve provided precise timing for TMB contact with the bead. Solution uniformity from reaction on the surface of the bead in volumes 20–50 uL was obtained in times of 2.5 s or less. Accurate measurement of the amount of surface-bound HRP by model fitting to the measured kinetics of colour development at 10 s intervals is demonstrated.

One of the primary obstacles in current cancer treatments lies in the extensive heterogeneity of genetic and epigenetic changes that occur in each arising tumour. However, an additional challenge persists, as certain types of cancer display shared immune deficiencies in the antigen processing machinery (APM). This includes the downregulation of human leukocyte antigen (HLA) class I molecules, which serve as peptide antigen receptors for T lymphocyte recognition that plays a crucial role in killing emerging tumours. Consequently, this contributes to immune escape in metastatic disease. Notably, current cell-based immunotherapies primarily focusing on T lymphocytes and the implementation of immune checkpoint inhibitor modalities have largely ignored the crucial task of reversing immune escape. This oversight may explain the limited success of these approaches becoming more effective cancer immunotherapies. Hence, there is a critical need to prioritize the discovery of new therapeutic candidates that can effectively address immune escape and synergize with evolving immunotherapy strategies. In this context, we identified curcuphenol in a cell-based screen from a library of marine extracts as a chemical entity that reverses the immune-escape phenotype of metastatic cancers. To advance these findings toward clinical efficacy, the present study describes the synthesis of analogues of naturally occurring curcuphenol with enhanced chemical properties and biological efficacy. Here we test the hypothesis that these curcuphenol analogues can evoke the power of the immune system to reduce the growth of metastatic disease in tumour bearing animals. Our findings indicate that these compounds effectively restore the expression of APM genes in metastatic tumours and inhibit the growth of highly invasive tumours in preclinical models, thereby counteracting the common immune evasion phenomenon observed in metastatic cancers. We conclude that cancer immunotherapies capable of boosting APM expression, hold great potential in maximizing the effectiveness of immune blockade inhibitors and eradicating invasive tumours.

Curcuphenol, a common component of the culinary spices, naturally found in marine invertebrates and plants, has been identified as a novel candidate for reversing immune escape by restoring expression of the antigen presentation machinery (APM) in invasive cancers, thereby resurrecting the immune recognition of metastatic tumours. Two synthetic curcuphenol analogues, were prepared by informed design that demonstrated consistent induction of APM expression in metastatic prostate and lung carcinoma cells. Both analogues were subsequently found to possess a previously undescribed histone deacetylase (HDAC)-enhancing activity. Remarkably, the H3K27ac ChIPseq analysis of curcuphenol-treated cells reveals that the induced epigenomic marks closely resemble the changes in genome-wide pattern observed with interferon-γ, a cytokine instrumental for orchestrating innate and adaptive immunity. These observations link dietary components to modifying epigenetic programs that modulate gene expression guiding poised immunity.

Genetic and epigenetic events have been implicated in the downregulation of the cellular antigen processing and presentation machinery (APM), which in turn, has been associated with cancer evasion of the immune system. When these essential components are lacking, cancers develop the ability to subvert host immune surveillance allowing cancer cells to become invisible to the immune system and, in turn, promote cancer metastasis. Here we describe and validate the first high-throughput cell-based screening assay to identify chemical extracts and unique chemical entities that reverse the downregulation of APM components in cell lines derived from metastatic tumours. Through the screening of a library of 480 marine invertebrate extracts followed by bioassay-guided fractionation, curcuphenol, a common sesquiterpene phenol derived from turmeric, was identified as the active compound of one of the extracts. We demonstrate that curcuphenol induces the expression of the APM components, TAP-1 and MHC-I molecules, in cell lines derived from both metastatic prostate and lung carcinomas. Turmeric and curcumins that contain curcuphenol have long been utilized not only as a spice in the preparation of food, but also in traditional medicines for treating cancers. The remarkable discovery that a common component of spices can increase the expression of APM components in metastatic tumour cells and, therefore reverse immune-escape mechanisms, provides a rationale for the development of foods and advanced nutraceuticals as therapeutic candidates for harnessing the power of the immune system to recognize and destroy metastatic cancers.

Abstract The increase of COVID-19 breakthrough infection risk with time since vaccination has a clear relationship to the decrease of antibody concentration with time. The empirically-observed dependence on blood IgG anti-receptor binding domain antibody concentration of SARS-CoV-2 vaccine efficacy against infection has a rational explanation in the statistics of binding of antibody to spike proteins on the virus surface, leading to blocking of binding to the receptor: namely that the probability of infection is the probability that a critical number of the spike proteins protruding from the virus are unblocked. The model is consistent with the observed antibody concentrations required to induce immunity and with the observed dependence of vaccine efficacy on antibody concentration and thus is a useful tool in the development of models to relate, for an individual person, risk of infection given measured antibody concentration. It can be used to relate population breakthrough infection risk to the distribution across the population of antibody concentration, and its variation with time.

En este estudio para maní, se diferencian para Ecuador 51 razas de maní cultivados, de las cuales 18 pertenecen a Arachis hypogaea subsp. hypogaea var. hypogaea, una Arachis hypogaea subsp. hypogaea var. hirsuta, 13 a Arachis hypogaea subsp. fastigiata var. fastigiata, 10 a Arachis hypogaea subsp. fastigiata var. peruviana, 9 a Arachis hypogaea subsp. fastigiata var. aequatoriana; la var. aequatoriana, es casi exclusiva de la República del Ecuador, lo que indica otro genocentro importante

Benzo[a]pyrene (BaP) is formed by incomplete combustion of organic materials (petroleum, coal, tobacco, etc.). BaP is designated by the International Agency for Research on Cancer as a group 1 known human carcinogen; a classification supported by numerous studies in preclinical models and epidemiology studies of exposed populations. Risk assessment relies on toxicokinetic and cancer studies in rodents at doses 5–6 orders of magnitude greater than average human uptake. Using a dose–response design at environmentally relevant concentrations, this study follows uptake, metabolism, and elimination of [14C]-BaP in human plasma by employing UPLC - accelerator mass spectrometry (UPLC-AMS). Volunteers were administered 25, 50, 100, and 250 ng (2.7–27 nCi) of [14C]-BaP (with interceding minimum 3-week washout periods) with quantification of parent [14C]-BaP and metabolites in plasma measured over 48 h. [14C]-BaP median Tmax was 30 min with Cmax and area under the curve (AUC) approximating dose-dependency. Marked inter-individual variability in plasma pharmacokinetics following a 250 ng dose was seen with 7 volunteers as measured by the Cmax (8.99 ± 7.08 ng × mL−1) and AUC0-48hr (68.6 ± 64.0 fg × hr−1 × mL−1). Approximately 3–6% of the [14C] recovered (AUC0-48 hr) was parent compound, demonstrating extensive metabolism following oral dosing. Metabolite profiles showed that, even at the earliest time-point (30 min), a substantial percentage of [14C] in plasma was polar BaP metabolites. The best fit modeling approach identified non-compartmental apparent volume of distribution of BaP as significantly increasing as a function of dose (p = 0.004). Bay region tetrols and dihydrodiols predominated, suggesting not only was there extensive first pass metabolism but also potentially bioactivation. AMS enables the study of environmental carcinogens in humans with de minimus risk, allowing for important testing and validation of physiologically based pharmacokinetic models derived from animal data, risk assessment, and the interpretation of data from high-risk occupationally exposed populations.

Biofouling on surfaces, caused by the assimilation of proteins, peptides, lipids and microorganisms, leads to contamination, deterioration and failure of biomedical devices and causes implants rejection. To address these issues, various antifouling strategies have been extensively studied, including polyethylene glycol-based polymer brushes. Conducting polymers-based biointerfaces have emerged as advanced surfaces for interfacing biological tissues and organs with electronics. Antifouling of such biointerfaces is a challenge. In this study, we fabricated electrospun fibre mats from sulphonated polystyrene-block-poly(ethylene-ran-butylene)-block-polystyrene (sSEBS), infused with conducting polymer poly(3,4-ethylenedioxythiophene) (PEDOT) (sSEBS-PEDOT), to produce a conductive (2.06 ± 0.1 S/cm), highly porous, fibre mat that can be used as a biointerface in bioelectronic applications. To afford antifouling, here the poly(oligo (ethylene glycol) methyl ether methacrylate) (POEGMA) brushes were grafted onto the sSEBS-PEDOT conducting fibre mats via surface-initiated atom transfer radical polymerization technique (SI-ATRP). For that, a copolymer of EDOT and an EDOT derivative with SI-ATRP initiating sites, 3,4-ethylenedioxythiophene) methyl 2-bromopropanoate (EDOTBr), was firstly electropolymerized on the sSEBS-PEDOT fibre mat to provide sSEBS-PEDOT/P(EDOT-co-EDOTBr). The POEGMA brushes were grafted from the sSEBS-PEDOT/P(EDOT-co-EDOTBr) and the polymerization kinetics confirmed the successful growth of the brushes. Fibre mats with 10-mers and 30-mers POEGMA brushes were studied for antifouling using a BCA protein assay. The mats with 30-mers grafted brushes exhibited excellent antifouling efficiency, ~82% of proteins repelled, compared to the pristine sSEBS-PEDOT fibre mat. The grafted fibre mats exhibited cell viability >80%, comparable to the standard cell culture plate controls. Such conducting, porous biointerfaces with POEGMA grafted brushes are suitable for applications in various biomedical devices, including biosensors, liquid biopsy, wound healing substrates and drug delivery systems.

Brassica vegetables contain a multitude of bioactive compounds that prevent and suppress cancer and promote health. Evidence suggests that the gut microbiome may be essential in the production of these compounds; however, the relationship between specific microbes and the abundance of metabolites produced during cruciferous vegetable digestion are still unclear. We utilized an ex vivo human fecal incubation model with in vitro digested broccoli sprouts (Broc), Brussels sprouts (Brus), a combination of the two vegetables (Combo), or a negative control (NC) to investigate microbial metabolites of cruciferous vegetables. We conducted untargeted metabolomics on the fecal cultures by LC-MS/MS and completed 16S rRNA gene sequencing. We identified 72 microbial genera in our samples, 29 of which were significantly differentially abundant between treatment groups. A total of 4499 metabolomic features were found to be significantly different between treatment groups (q ≤ 0.05, fold change > 2). Chemical enrichment analysis revealed 45 classes of compounds to be significantly enriched by brassicas, including long-chain fatty acids, coumaric acids, and peptides. Multi-block PLS-DA and a filtering method were used to identify microbe–metabolite interactions. We identified 373 metabolites from brassica, which had strong relationships with microbes, such as members of the family Clostridiaceae and genus Intestinibacter, that may be microbially derived.

Hydrolysis of glucobrassicin by plant or bacterial myrosinase produces multiple indoles predominantly indole-3-carbinol (I3C). I3C and its major in vivo product, 3,3'-diindolylmethane (DIM), are effective cancer chemopreventive agents in pre-clinical models and show promise in clinical trials. The pharmacokinetics/pharmacodynamics of DIM have been studied in both rodents and humans and urinary DIM is a proposed biomarker of dietary intake of cruciferous vegetables. Recent clinical studies at Oregon State University show surprisingly robust metabolism of DIM in vivo with mono- and di-hydroxylation followed by conjugation with sulfate or glucuronic acid. DIM has multiple mechanisms of action, the most well-characterized is modulation of aryl hydrocarbon receptor (AHR) signaling. In rainbow trout dose-dependent cancer chemoprevention by dietary I3C is achieved when given prior to or concurrent with aflatoxin B1, polycyclic aromatic hydrocarbons, nitrosamines or direct acting carcinogens such as N-methyl-N'-nitro-nitrosoguanidine. Feeding pregnant mice I3C inhibits transplacental carcinogenesis. In humans much of the focus has been on chemoprevention of breast and prostate cancer. Alteration of cytochrome P450-dependent estrogen metabolism is hypothesized to be an important driver of DIM-dependent breast cancer prevention. The few studies done to date comparing glucobrassicin-rich crucifers such as Brussels sprouts with I3C/DIM supplements have shown the greater impact of the latter is due to dose. Daily ingestion of kg quantities of Brussels sprouts is required to produce in vivo levels of DIM achievable by supplementation. In clinical trials these supplement doses have elicited few if any adverse effects. Sulforaphane from glucoraphanin can act synergistically with glucobrassicin-derived DIM and this may lead to opportunities for combinatorial approaches (supplement and food-based) in the clinic.

We are investigating plants from the prairie ecological zone of Canada to identify natural products that inhibit mitosis in cancer cells. Investigation of plant parts from the Canadian plant species Hymenoxys richardsonii (Asteraceae) revealed that leaf extracts (PP-360A) had anti-mitotic activity on human cancer cell lines. Cells treated with leaf extracts acquired a rounded morphology, similar to that of cells in mitosis. We demonstrated that the rounded cells contained mitotic spindles and phospho-histone H3 using the techniques of immunofluorescence microscopy. By biology-guided fractionation of H. richardsonii leaves, we isolated a sesquiterpene lactone named hymenoratin, which had not been previously assigned a biological activity. Cells treated with hymenoratin have phospho-histone H3 positive chromosomes, a mitotic spindle, and enter a prolonged mitotic arrest in which the spindles become distorted. By Western blot analysis, hymenoratin treated cells acquire high levels of cyclin B and dephosphorylated Cdk1. There is a growing body of evidence that select members of the sesquiterpene lactone chemical family have anti-mitotic activity.

Laboratory cultures of two ‘biosynthetically talented’ bacterial strains harvested from tropical and temperate Pacific Ocean sediment habitats were examined for the production of new natural products. Cultures of the tropical Salinispora arenicola strain RJA3005, harvested from a PNG marine sediment, produced salinorcinol (3) and salinacetamide (4), which had previously been reported as products of engineered and mutated strains of Amycolatopsis mediterranei, but had not been found before as natural products. An S. arenicola strain RJA4486, harvested from marine sediment collected in the temperate ocean waters off British Columbia, produced the new aminoquinone polyketide salinisporamine (5). Natural products 3, 4, and 5 are putative shunt products of the widely distributed rifamycin biosynthetic pathway.

Twenty distinct endophytic fungi were isolated from the surface-sterilized plant parts of Nymphaea nouchali and were identified using morphological and molecular techniques. At 300 µg/disc concentration, eight of the 20 fungal extracts exhibited antimicrobial activities against Staphylococcus aureus (ATCC 25923) and Bacillus cereus (ATCC 11778) while two within the eight showed activity against Pseudomonas aeruginosa (ATCC 9027) and Escherichia coli (ATCC 35218). Furthermore, investigation of the crude extract of Chaetomium globosum resulted in the isolation of two known cytochalasans, chaetoglobosin A and C, and their structures were elucidated and confirmed by mass and nuclear magnetic resonance (NMR) (1H, 13C, COSY, HSQC, HMBC and tROESY) spectral data. Chaetoglobosin A showed antibacterial activities against Bacillus subtilis (MIC 16 µg mL−1), Staphylococcus aureus (MIC 32 µg mL−1) and methicillin-resistant Staphylococcus aureus (MRSA, MIC 32 µg mL−1). This is the first study to report the isolation, identification and antimicrobial properties of endophytic fungi of N. nouchali in Sri Lanka.

The phytochemical sulforaphane can induce cell cycle arrest and apoptosis in metastatic prostate cancer cells, though the mechanism of action is not fully known. We conducted a global proteome analysis in LNCaP metastatic prostate cancer cells to characterize how global protein signature responds to sulforaphane. We conducted parallel analyses to evaluate semi-quantitative 1-dimensional versus 2-dimensional liquid chromatography tandem mass spectrometry (LC–MS/MS) and their utility in characterizing whole cell lysate. We show that 2-dimensional LC–MS/MS can be a useful tool for characterizing global protein profiles and identify TRIAP1 as a novel regulator of cell proliferation in LNCaP metastatic prostate cancer cells. Keywords: Prostate cancer, Sulforaphane, Mass spectrometry

The increasing prevalence of drug-resistant influenza viruses emphasizes the need for new antiviral countermeasures. The M2 protein of influenza A is a proton-gated, proton-selective ion channel, which is essential for influenza replication and an established antiviral target. However, all currently circulating influenza A virus strains are now resistant to licensed M2-targeting adamantane drugs, primarily due to the widespread prevalence of an M2 variant encoding a serine to asparagine 31 mutation (S31N). To identify new chemical leads that may target M2(S31N), we performed a virtual screen of molecules from two natural product libraries and identified chebulagic acid as a candidate M2(S31N) inhibitor and influenza antiviral. Chebulagic acid selectively restores growth of M2(S31N)-expressing yeast. Molecular modeling also suggests that chebulagic acid hydrolysis fragments preferentially interact with the highly-conserved histidine residue within the pore of M2(S31N) but not adamantane-sensitive M2(S31). In contrast, chebulagic acid inhibits in vitro influenza A replication regardless of M2 sequence, suggesting that it also acts on other influenza targets. Taken together, results implicate chebulagic acid and/or its hydrolysis fragments as new chemical leads for M2(S31N) and influenza-directed antiviral development.

In a study designed to determine the antibacterial potential of endophytic fungi inhabiting plants of Cyperaceae family, 72 morphologically distinct endophytic fungi were isolated from six plants and their antibacterial activities against two Gram positive and three Gram negative pathogenic bacterial species were examined. Two antibacterial metabolites from two endophytes were also isolated and their chemical structures and minimum inhibitory concentrations were determined. Sixty six fungal extracts (92%) were active against at least one bacterium tested. Butyrolactone I isolated from the Aspergillus terreus and 9-epi viridol isolated from Trichoderma virens exhibited MIC values in the range 128-256 µg/mL against Gram positive Bacillus subtilits, Staphylococcus aureus, methicillin resistant S. aureus (MRSA) and Escherichia coli. Both compounds were inactive against other micro-organisms tested. This study confirmed that Cyperaceae plants harbor numerous endophytes that produce antibacterial metabolites active against both Gram positive and, to a lesser extent, Gram negative bacteria. Video Clip of Methodology: Column Chromatogrpahy: 15 min 34 sec Full Screen Alternate

Multiple drug resistance (MDR) in Mycobacterium tuberculosis (mTB), the causative agent for tuberculosis (TB), has led to increased use of second-line drugs, including ethionamide (ETA). ETA is a prodrug bioactivated by mycobacterial and mammalian flavin-containing monooxygenases (FMOs). FMO2 is the major isoform in the lungs of most mammals, including primates. In humans a polymorphism exists in the expression of FMO2. FMO2.2 (truncated, inactive) protein is produced by the common allele, while the ancestral allele, encoding active FMO2.1, has been documented only in individuals of African and Hispanic origin, at an incidence of up to 50% and 7%, respectively. We hypothesized that FMO2 variability in TB-infected individuals would yield differences in concentrations and ratios of ETA prodrug and metabolites. In this study we assessed the impact of the FMO2 genetic polymorphism on the pharmacokinetics of ETA after administration of a single oral dose of ETA (125 mg/kg) to wild type and triple Fmo1/2/4-null mice, measuring levels of prodrug vs. metabolites in plasma collected from 0 to 3.5 h post-gavage. All mice metabolized ETA to ETA S-oxide (ETASO) and 2-ethyl-4-amidopyridine (ETAA). Wild type mice had higher plasma concentrations of metabolites than of parent compound (p = 0.001). In contrast, Fmo1/2/4-null mice had higher plasma concentrations of parent compound than of metabolites (p = 0.0001). Thus, the human FMO2 genotype could impact the therapeutic efficacy and/or toxicity of ETA.