Identification of fragments targeting SMYD3 using highly sensitive kinetic and multiplexed biosensor-based screening.

A 1056-membered fragment library has been screened against SMYD3 using a novel multiplexed experimental design implemented in a grating coupled interferometry (GCI)-based biosensor. SMYD3 is a prospective target for anticancer drugs and the focus has initially been on discovery of inhibitors of its lysine methyl transferase activity. However, it has multiple protein interaction partners and several potential roles in carcinogenesis. It therefore remains unclear what mode of action ligands targeting the protein should have. Our goal was therefore to identify new ligands and discriminate hits that interact with the active site and those that interact with other sites. In addition, we were interested in selecting hits based on kinetic features rather than affinity. Screening was done in parallel against SMYD3 alone or SMYD3 with the active site blocked by a tight binding inhibitor. Hit selection was primarily based on dissociation rates. In total, 20 fragments were selected as hits, of which half apparently targeted the active site and half targeted other sites. Twelve of the hits were selected for structural analysis using X-ray crystallography in order to identify binding sites and modes of binding. Four of the hits were successfully identified in crystal structures with SMYD3; the others did not show any electron densities for ligands in the crystals. Although it might be possible to optimize the crystallography approach for a better success rate, it was clear that the sensitivity and time resolution of the biosensor assay was exceptional and enabled kinetic rate constants to be estimated for fragments. Fragments are typically considered to interact too rapidly for such quantification to be possible. This approach consequently represents a paradigm shift. In addition, the multiplexed approach allows ligands targeting different sites to be rationally selected already in the fragment library screening stage.

Accelerating BRPF1b hit identification with BioPhysical and Active Learning Screening (BioPALS).

We report the development of BioPhysical and Active Learning Screening (BioPALS); a rapid and versatile hit identification protocol combining AI-powered virtual screening with a GCI-driven biophysical confirmation workflow. Its application to the BRPF1b bromodomain afforded a range of novel micromolar binders with favorable ADMET properties. In addition to the excellent in silico/in vitro confirmation rate demonstrated with BRPF1b, binding kinetics were determined, and binding topologies predicted for all hits. BioPALS is a lean, data-rich, and standardized approach to hit identification applicable to a wide range of biological targets.

Multiplexed experimental strategies for fragment library screening against challenging drug targets using SPR biosensors.

Surface plasmon resonance (SPR) biosensor methods are ideally suited for fragment-based lead discovery.  However, generally applicable experimental procedures and detailed protocols are lacking, especially for structurally or physico-chemically challenging targets or when tool compounds are not available. Success depends on accounting for the features of both the target and the chemical library, purposely designing screening experiments for identification and validation of hits with desired specificity and mode-of-action, and availability of orthogonal methods capable of confirming fragment hits. The range of targets and libraries amenable to an SPR biosensor-based approach for identifying hits is considerably expanded by adopting multiplexed strategies, using multiple complementary surfaces or experimental conditions. Here we illustrate principles and multiplexed approaches for using flow-based SPR biosensor systems for screening fragment libraries of different sizes (90 and 1056 compounds) against a selection of challenging targets. It shows strategies for the identification of fragments interacting with 1) large and structurally dynamic targets, represented by acetyl choline binding protein (AChBP), a Cys-loop receptor ligand gated ion channel homologue, 2) targets in multi protein complexes, represented by lysine demethylase 1 and a corepressor (LSD1/CoREST), 3) structurally variable or unstable targets, represented by farnesyl pyrophosphate synthase (FPPS), 4) targets containing intrinsically disordered regions, represented by protein tyrosine phosphatase 1B  (PTP1B), and 5) aggregation-prone proteins, represented by an engineered form of human tau  (tau K18M). Practical considerations and procedures accounting for the characteristics of the proteins and libraries, and that increase robustness, sensitivity, throughput and versatility are highlighted. The study shows that the challenges for addressing these types of targets is not identification of potentially useful fragments per se, but establishing methods for their validation and evolution into leads.

Discovery of an Allosteric Ligand Binding Site in SMYD3 Lysine Methyltransferase.

SMYD3 is a multifunctional epigenetic enzyme with lysine methyltransferase activity and various interaction partners. It is implicated in the pathophysiology of cancers but with an unclear mechanism. To discover tool compounds for clarifying its biochemistry and potential as a therapeutic target, a set of drug-like compounds was screened in a biosensor-based competition assay. Diperodon was identified as an allosteric ligand; its R and S enantiomers were isolated, and their affinities to SMYD3 were determined (KD =42 and 84 µM, respectively). Co-crystallization revealed that both enantiomers bind to a previously unidentified allosteric site in the C-terminal protein binding domain, consistent with its weak inhibitory effect. No competition between diperodon and HSP90 (a known SMYD3 interaction partner) was observed although SMYD3-HSP90 binding was confirmed (KD =13 µM). Diperodon clearly represents a novel starting point for the design of tool compounds interacting with a druggable allosteric site, suitable for the exploration of noncatalytic SMYD3 functions and therapeutics with new mechanisms of action.

Discovery of fragments inducing conformational effects in dynamic proteins using a second-harmonic generation biosensor.

Biophysical screening of compound libraries for the identification of ligands that interact with a protein is efficient, but does typically not reveal if (or how) ligands may interfere with its functional properties. For this a biochemical/functional assay is required. But for proteins whose function is dependent on a conformational change, such assays are typically complex or have low throughput. Here we have explored a high-throughput second-harmonic generation (SHG) biosensor to detect fragments that induce conformational changes upon binding to a protein in real time and identify dynamic regions. Multiwell plate format SHG assays were developed for wild-type and six engineered single-cysteine mutants of acetyl choline binding protein (AChBP), a homologue to ligand gated ion channels (LGICs). They were conjugated with second harmonic-active labels via amine or maleimide coupling. To validate the assay, it was confirmed that the conformational changes induced in AChBP by nicotinic acetyl choline receptor (nAChR) agonists and antagonists were qualitatively different. A 1056 fragment library was subsequently screened against all variants and conformational modulators of AChBP were successfully identified, with hit rates from 9-22%, depending on the AChBP variant. A subset of four hits was selected for orthogonal validation and structural analysis. A time-resolved grating-coupled interferometry-based biosensor assay confirmed the interaction to be a reversible 1-step 1 : 1 interaction, and provided estimates of affinities and interaction kinetic rate constants (K D = 0.28-63 µM, k a = 0.1-6 µM-1 s-1, k d = 1 s-1). X-ray crystallography of two of the fragments confirmed their binding at a previously described conformationally dynamic site, corresponding to the regulatory site of LGICs. These results reveal that SHG has the sensitivity to identify fragments that induce conformational changes in a protein. A selection of fragment hits with a response profile different to known LGIC regulators was characterized and confirmed to bind to dynamic regions of the protein.

Proteomic and Metabolomic Characterization of Human Neurovascular Unit Cells in Response to Methamphetamine.

The functional state of the neurovascular unit (NVU), composed of the blood-brain barrier and the perivasculature that forms a dynamic interface between the blood and the central nervous system (CNS), plays a central role in the control of brain homeostasis and is strongly affected by CNS drugs. Human primary brain microvascular endothelium, astrocyte, pericyte, and neural cell cultures are often used to study NVU barrier functions as well as drug transport and efficacy; however, the proteomic and metabolomic responses of these different cell types are not well characterized. Culturing each cell type separately, using deep coverage proteomic analysis and characterization of the secreted metabolome, as well as measurements of mitochondrial activity, the responses of these cells under baseline conditions and when exposed to the NVU-impairing stimulant methamphetamine (Meth) are analyzed. These studies define the previously unknown metabolic and proteomic profiles of human brain pericytes and lead to improved characterization of the phenotype of each of the NVU cell types as well as cell-specific metabolic and proteomic responses to Meth.

Impact of geo-imputation on epidemiologic associations in a study of outdoor air pollution and respiratory hospitalization.

Imputation of missing spatial attributes in health records may facilitate linkages to geo-referenced environmental exposures, but few studies have assessed geo-imputation impacts on epidemiologic inference. We imputed patient Census tracts in a case-crossover analysis of fine particulate matter (PM2.5) and respiratory hospitalizations in New York State (2000-2005). We observed non-significantly higher PM2.5 exposures, high accuracy of binary exposure assignment (89 to 99%), and marginally different hazard ratios (HRs) (-0.2 to 0.7%). HR differences were greater in urban versus rural areas. Given its efficiency and nominal influence on accuracy of exposure classification and measures of association, geo-imputation is a candidate method to address missing spatial attributes for health studies.

Robotic fluidic coupling and interrogation of multiple vascularized organ chips.

Organ chips can recapitulate organ-level (patho)physiology, yet pharmacokinetic and pharmacodynamic analyses require multi-organ systems linked by vascular perfusion. Here, we describe an 'interrogator' that employs liquid-handling robotics, custom software and an integrated mobile microscope for the automated culture, perfusion, medium addition, fluidic linking, sample collection and in situ microscopy imaging of up to ten organ chips inside a standard tissue-culture incubator. The robotic interrogator maintained the viability and organ-specific functions of eight vascularized, two-channel organ chips (intestine, liver, kidney, heart, lung, skin, blood-brain barrier and brain) for 3 weeks in culture when intermittently fluidically coupled via a common blood substitute through their reservoirs of medium and endothelium-lined vascular channels. We used the robotic interrogator and a physiological multicompartmental reduced-order model of the experimental system to quantitatively predict the distribution of an inulin tracer perfused through the multi-organ human-body-on-chips. The automated culture system enables the imaging of cells in the organ chips and the repeated sampling of both the vascular and interstitial compartments without compromising fluidic coupling.

A longitudinal study of polychlorinated biphenyls and neuropsychological function among older adults from New York State.

BACKGROUND: Cross-sectional studies have linked greater polychlorinated biphenyl (PCB) exposure to adverse neuropsychological effects in older adults, including learning, memory, and depressive symptoms. However, no studies among older adults have evaluated the association over time. OBJECTIVES: To assess the effect of serum PCB levels on neuropsychological function over a 14-year period in a cohort of older men and women from a PCB-contaminated area of New York State. METHODS: In 2000-2002, we assessed serum PCB levels and neuropsychological function (including the California Verbal Learning Test Trial 1 (CVLTT1) for verbal memory and learning, and the Beck Depression Index (BDI) for depressive symptoms) in 253 men and women, ages 55-74 years. A total of 116 (46%) persons repeated the PCB and neuropsychological assessment 14 years later. To assess the association over time, we used generalized estimating equations with clustering variables time, total PCB (∑PCB), and ∑PCB × time, and adjusted for baseline age, sex, smoking, and total serum-lipids. For statistically significant ∑PCB × time interactions, we evaluated the association between PCBs and either verbal memory and learning or depressive symptoms while holding ∑PCB constant at the 10th and 90th percentiles to clarify the direction of the interaction. RESULTS: Over the study period, serum ∑PCB levels (wet-weight) declined by 22%, and were associated with different patterns of change over time for memory (∑PCB × Time ß = 0.08 p = 0.009) and depressive symptoms (∑PCB × Time ß = -0.16 p = 0.013). Specifically, verbal memory and learning decreased (ß = -0.08 p = 0.008) and depressive symptoms increased (ß = 0.17 p = 0.008) among persons with low exposure (∑PCB levels at the 10th percentile), while persons with high exposure (90th percentile) showed non-significant improvements. DISCUSSION: In this cohort, declining ∑PCB levels were likely due at least in part to low rates of local fish consumption in recent decades, given the ban since 1976. The decreased verbal memory and learning and increased depressive symptoms over time among persons with low serum ∑PCB levels is consistent with studies of normative aging. However, the small improvements in those outcomes among those with high serum ∑PCB levels was unexpected. Healthy survivor selection bias or uncontrolled confounding may explain this result. It may also indicate that the neurotoxic impacts of PCBs in older adults are not permanent, but future studies are needed to confirm this possibility.

Medical equipment donation in low-resource settings: a review of the literature and guidelines for surgery and anaesthesia in low-income and middle-income countries.

BACKGROUND: Medical equipment donation to low-resource settings is a frequently used strategy to address existing disparities, but there is a paucity of reported experience and evaluation. Challenges such as infrastructure gaps, lack of technological and maintenance capabilities, and non-prioritisation of essential supplies have previously been highlighted. This pragmatic review summarises existing guidelines and literature relevant to surgical and anaesthesia equipment, with recommendations for future initiatives and research. METHODS: Retrospective literature review including both academic and grey literature from 1980 to 2018. We conducted a narrative synthesis to identify key factors that were condensed thematically. RESULTS: Thirty-three biomedical equipment donation guidelines were identified from governments, WHO, World Bank, academic colleges and non-governmental organisations, and 36 relevant studies in peer-reviewed literature. These highlighted the need to consider all stages of the donation process, including planning, sourcing, transporting, training, maintaining and evaluating equipment donation. Donors were advised to consult national guidelines to ensure equipment was appropriate, desirable and non-costly to both parties. User training and access to biomechanical engineers were suggested as necessary for long-term sustainability. Finally, equitable partnerships between donors and recipients were integral to reducing inappropriate donations and to improve follow-up and evaluation. CONCLUSION: There is a paucity of evidence on the causes of success or failure in medical equipment donation, despite its domination of equipment sourcing across many low-resource settings. Equitable partnerships, consultation of policies and guidelines, and careful planning may improve equipment usability and life span. A concerted effort is required to increase awareness of guidelines among health professionals worldwide.

Hypoxia-enhanced Blood-Brain Barrier Chip recapitulates human barrier function and shuttling of drugs and antibodies.

The high selectivity of the human blood-brain barrier (BBB) restricts delivery of many pharmaceuticals and therapeutic antibodies to the central nervous system. Here, we describe an in vitro microfluidic organ-on-a-chip BBB model lined by induced pluripotent stem cell-derived human brain microvascular endothelium interfaced with primary human brain astrocytes and pericytes that recapitulates the high level of barrier function of the in vivo human BBB for at least one week in culture. The endothelium expresses high levels of tight junction proteins and functional efflux pumps, and it displays selective transcytosis of peptides and antibodies previously observed in vivo. Increased barrier functionality was accomplished using a developmentally-inspired induction protocol that includes a period of differentiation under hypoxic conditions. This enhanced BBB Chip may therefore represent a new in vitro tool for development and validation of delivery systems that transport drugs and therapeutic antibodies across the human BBB.

A linked organ-on-chip model of the human neurovascular unit reveals the metabolic coupling of endothelial and neuronal cells.

The neurovascular unit (NVU) regulates metabolic homeostasis as well as drug pharmacokinetics and pharmacodynamics in the central nervous system. Metabolic fluxes and conversions over the NVU rely on interactions between brain microvascular endothelium, perivascular pericytes, astrocytes and neurons, making it difficult to identify the contributions of each cell type. Here we model the human NVU using microfluidic organ chips, allowing analysis of the roles of individual cell types in NVU functions. Three coupled chips model influx across the blood-brain barrier (BBB), the brain parenchymal compartment and efflux across the BBB. We used this linked system to mimic the effect of intravascular administration of the psychoactive drug methamphetamine and to identify previously unknown metabolic coupling between the BBB and neurons. Thus, the NVU system offers an in vitro approach for probing transport, efficacy, mechanism of action and toxicity of neuroactive drugs.

Maternal ambient heat exposure during early pregnancy in summer and spring and congenital heart defects - A large US population-based, case-control study.

BACKGROUND/OBJECTIVE: Few studies have assessed the effect of ambient heat during the fetal development period on congenital heart defects (CHDs), especially in transitional seasons. We examined and compared the associations between extreme heat and CHD phenotypes in summer and spring, assessed their geographical differences, and compared different heat indicators. METHODS: We identified 5848 CHD cases and 5742 controls (without major structural defects) from the National Birth Defects Prevention Study, a US multicenter, population-based case-control study. Extreme heat events (EHEs) were defined by using the 95th (EHE95) or 90th (EHE90) percentile of daily maximum temperature and its frequency and duration during postconceptional weeks 3-8. We used a two-stage Bayesian hierarchical model to examine both regional and study-wide associations. Exposure odds ratios (ORs) were calculated using multivariate logistic regression analyses, while controlling for potential confounding factors. RESULTS: Overall, we observed no significant relationships between maternal EHE exposure and CHDs in most regions during summer. However, we found that 3-11 days of EHE90 during summer and spring was significantly associated with ventricular septal defects (VSDs) study-wide (ORs ranged: 2.17-3.24). EHE95 in spring was significantly associated with conotruncal defects and VSDs in the South (ORs: 1.23-1.78). Most EHE indicators in spring were significantly associated with increased septal defects (both VSDs and atrial septal defects (ASDs)) in the Northeast. CONCLUSION: While generally null results were found, long duration of unseasonable heat was associated with the increased risks for VSDs and ASDs, mainly in South and Northeast of the US. Further research to confirm our findings is needed.

Engagement and role of surgical trainees in global surgery: Consensus statement and recommendations from the Association of Surgeons in Training.

BACKGROUND: There is a wide chasm in access to essential and emergency surgery between high and low/middle income countries (LMICs). Surgeons worldwide are integral to solutions needed to address this imbalance. Involving surgical trainees, who represent the future of surgery, is vital to this endeavour. The Association of Surgeons in Training (ASiT) is an independent charity that support surgical trainees of all ten surgical specialties in the UK and Ireland. ASiT convened a consensus meeting at the ASiT conference in Liverpool 2016 to discuss trainee engagement with global surgery, including potential barriers and solutions. METHODS: A face-to-face consensus meeting reviewed the engagement of, and roles for, surgical trainees in global surgery at the ASiT Conference (Liverpool, England), March 2016. Participants self-identified based on experience and interest in the field, and included trainees (residents and students) and consultants (attending grade). Following expert review, seven pre-determined core areas were presented for review and debate. Extensive discussion was facilitated by a consultant and a senior surgical trainee, with expertise in global surgery. The draft derived from these initial discussions was circulated to all those who had participated, and an iterative process of revision was undertaken until a final consensus and recommendations were reached. RESULTS: There is increasing interest from trainee surgeons to work in LMICs. There are however, ethical considerations, and it is important that trainees working in LMICs undertake work appropriate to their training stage and competencies. Visiting surgeons must consider the requirements of the hosting centres rather than just their own objectives. If appropriately organised, both short and long-term visits, can enable development of transferable clinical, organisational, research and education skills. A central repository of information on global surgery would be useful to trainees, to complement existing resources. Challenges to trainees considering a global surgery placement include approval for placements while on a training program, financial cost and dangers inherent in working in a resource poor setting. Currently global surgery experience is generally as an out of program experience and does not count for certificate of completion of training (CCT). Methods to recognise surgical trainee global surgery experience as an integrated part of training should be explored, similar to that seen in other specialties. CONCLUSION: There is a role for surgical trainees to become involved in Global Surgery, especially in partnership with local surgeons and with appropriate ethical consideration. Trainees develop translational skills in resource poor settings. Development of appropriate pathways for recognition of global surgery experience for CCT should be considered.

Occupational exposure to perfluoroalkyl substances and serum levels of perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) in an aging population from upstate New York: a retrospective cohort study.

PURPOSE: Perfluoroalkyl substances (PFASs) are environmentally persistent amphiphilic compounds. Exposure to two PFASs, perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) is linked to specific occupations and industries. This study examines the contribution of past occupational PFAS exposure to serum PFOS and PFOA levels among 154 older adults in New York State. METHODS: Serum PFOS and PFOA levels were compared to data from the National Health and Nutrition Examination Survey (NHANES). Potential occupational exposure to any PFAS was determined from work histories, reviewed by an industrial hygienist, and assessed in relation to current serum PFOS and PFOA levels using exposure probability, duration and cumulative exposure. RESULTS: We observed 25% higher serum PFOS and 80% higher PFOA levels in study participants compared to NHANES. No participants reported PFAS chemical manufacturing work, but n = 68 reported work in occupations and industries known to use PFASs. We found that participants with high cumulative workplace exposure had 34% higher serum PFOS levels compared to participants without occupational exposure, adjusted for age, sex and income. Serum PFOS levels were 26% higher for participants with longer occupational exposure durations. The probability of occupational PFAS exposure metric was not associated with serum PFOS. Serum PFOA was not associated with any measure of occupational exposure. CONCLUSION: Occupational exposure may contribute to total PFOS body burden in this study population, even among workers not directly involved in manufacturing PFASs. PFAS exposure assessments should evaluate the workplace as a potential source, even when workplace exposures are assumed to be low or moderate.

Organs-on-Chips with combined multi-electrode array and transepithelial electrical resistance measurement capabilities.

Here we demonstrate that microfluidic cell culture devices, known as Organs-on-a-Chips can be fabricated with multifunctional, real-time, sensing capabilities by integrating both multi-electrode arrays (MEAs) and electrodes for transepithelial electrical resistance (TEER) measurements into the chips during their fabrication. To prove proof-of-concept, simultaneous measurements of cellular electrical activity and tissue barrier function were carried out in a dual channel, endothelialized, heart-on-a-chip device containing human cardiomyocytes and a channel-separating porous membrane covered with a primary human endothelial cell monolayer. These studies confirmed that the TEER-MEA chip can be used to simultaneously detect dynamic alterations of vascular permeability and cardiac function in the same chip when challenged with the inflammatory stimulus tumor necrosis factor alpha (TNF-α) or the cardiac targeting drug isoproterenol. Thus, this Organ Chip with integrated sensing capability may prove useful for real-time assessment of biological functions, as well as response to therapeutics.

Perfluoroalkyl substances, thyroid hormones, and neuropsychological status in older adults.

Minimal data exist regarding the neurotoxicity of perfluoroalkyl substances (PFASs) in aging populations and the possible mediating effects of thyroid hormones (THs). Hence, the aims of this study were to: (i) assess associations between PFASs and neuropsychological function, and (ii) determine if such associations are mediated by changes in circulating THs in an aging population. We measured perfluorooctanoic acid (PFOA), perfluorooctane sulfonate (PFOS), total thyroxine (T4) and free thyroxine (fT4) in serum and performed neuropsychological tests in 126 men and women aged 55-74 years and living in upper Hudson River communities. Multivariable linear regressions were conducted to assess associations between PFASs and neuropsychological test scores. Mediation analyses were performed in a subset of 87 participants for whom information was available on both PFASs and THs. We calculated TH-mediated, non-TH mediated, and total effects of PFASs on neuropsychological test scores. Higher PFOA was associated with better performance in tasks of the California Verbal Learning Test and the Wisconsin Card Sorting Test. Higher PFOS was associated with improved performance in a Wechsler Memory Scale subtest and Block Design Subtest (BDT) total scores. There was no evidence of mediation by THs for PFOA-neuropsychological function associations. However, T4 and fT4 partially mediated the protective effect of PFOS on BDT total scores. Our findings do not suggest that PFASs are associated with poor neuropsychological function. There was some evidence of mediation for the association between PFASs and neuropsychological functions by THs, although some other modes of action also appear likely.

Low-level arsenic exposure via drinking water consumption and female fecundity - A preliminary investigation.

High level arsenic exposure is associated with reproductive toxicity in experimental and observational studies; however, few data exist to assess risks at low levels. Even less data are available to evaluate the impact of low level arsenic exposure on human fecundity. Our aim in this pilot study was a preliminary evaluation of associations between low level drinking water arsenic contamination and female fecundity. This retrospective study was conducted among women previously recruited to a hospital-based case-control study of spontaneous pregnancy loss in Timis County, Romania. Women (n=94) with planned pregnancies of 5-20 weeks gestation completed a comprehensive physician-administered study questionnaire and reported the number of menstrual cycles attempting to conceive as the time to pregnancy (TTP). Drinking water samples were collected from residential drinking water sources and we determined arsenic levels using hydride generation-atomic absorption spectrometry (HG-AAS). Multivariable Cox-proportional hazards regression with Efron approximation was employed to evaluate TTP as a function of drinking water arsenic concentrations among planned pregnancies, adjusted for covariates. There was no main effect for drinking water arsenic exposure, yet the conditional probability for pregnancy was modestly lower among arsenic exposed women with longer TTPs, relative to women with shorter TTPs, and relative to unexposed women. For example, 1µg/L average drinking water arsenic conferred 5%, 8%, and 10% lower likelihoods for pregnancy in the 6th, 9th, and 12th cycles, respectively (P=0.01). While preliminary, our results suggest that low level arsenic contamination in residential drinking water sources may further impair fecundity among women with longer waiting times; however, this hypothesis requires confirmation by a future, more definitive study.