Adhesion advances: from nanomaterials to biomimetic adhesion and applications.

The field of adhesion has revealed a significant impact on numerous applications such as wound healing, drug delivery, electrically conductive adhesive, dental adhesive, and wood industry. Nanotechnology has continued to be the primary means to achieve adhesion. Among them, biological systems based on the unique structure of the nano-levels have developed excellent adhesion capabilities after billions of years of evolution and natural selection. Therefore, the research on bionic adhesion inspired by biological systems has gradually emerged. This review firstly focuses on the mechanism of adhesion, and secondly reports the effects of different nanomaterials on adhesion properties. Then based on the structure of mussels, geckos, tree frogs, octopuses, and other organisms, the research progress of biomimetic nanotechnology to achieve adhesion is summarized. Finally, the applications, challenges, and future directions of nanotechnology in new adhesive materials are provided.

Abiotic Mimic of Endogenous Tissue Inhibitors of Metalloproteinases: Engineering Synthetic Polymer Nanoparticles for Use as a Broad-Spectrum Metalloproteinase Inhibitor.

We describe a process for engineering a synthetic polymer nanoparticle (NP) that functions as an effective, broad-spectrum metalloproteinase inhibitor. Inhibition is achieved by incorporating three functional elements in the NP: a group that interacts with the catalytic zinc ion, functionality that enhances affinity to the substrate-binding pocket, and fine-tuning of the chemical composition of the polymer to strengthen NP affinity for the enzyme surface. The approach is validated by synthesis of a NP that sequesters and inhibits the proteolytic activity of snake venom metalloproteinases from five clinically relevant species of snakes. The mechanism of action of the NP mimics that of endogenous tissue inhibitors of metalloproteinases. The strategy provides a general design principle for synthesizing abiotic polymer inhibitors of enzymes.

Photo-polymerizable ferrous sulfate liposomes as vehicles for iron fortification of food.

The development of photo-polymerizable ferrous sulfate liposomes has been perused for iron fortification of food. Iron fortification is accompanied by several limitations that reduce its quality as it provokes sensory problems due to the oxidation of Fe2+ into Fe3+. To overcome such undesirable effects and improve the bioavailability of iron, photo-polymerizable 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DC8,9PC) phospholipids have been employed as iron delivery system. DC8,9PC possesses diacetylene groups that undergo intramolecular cross-linking following UV treatment, resulting in enhanced stability, high encapsulation efficiency (~91%) without inducing sensory changes during milk fortification, along with less oxidation and con-tent leakage after long term storage in ethanol. Furthermore, polymeric Fe-DC8,9PC liposomes present high in vivo iron bioavailability in hemoglobin (Hb)-repletion study in rats, with no indications of toxicity examined by hematoxylin-eosin test. This methodology offers great promise for a simple, reliable, cost-effective, and robust platform for treating iron deficiency anemia, seeking to bring its application toward market.

Inhalable dry powder prepared from folic acid-conjugated docetaxel liposomes alters pharmacodynamic and pharmacokinetic properties relevant to lung cancer chemotherapy.

Pulmonary delivery of anti-cancer drugs in the form of nanoparticulate dry powders is considered a promising modality for treating lung cancer. However, it is not known whether the pharmacodynamics and pharmacokinetics of nano-preparations are altered after co-spray drying. In this study, we compared the physicochemical property, anti-cancer activity, tumor targeting and pharmacokinetic behavior of docetaxel-loaded folic acid-conjugated liposomes (LPs-DTX-FA) with those of dry powder prepared by co-spray-drying LPs-DTX-FA. The particle size and PDI after re-dispersion of the powder were increased. The re-dispersed liposomes showed increased cellular uptake via micropinocytosis and exhibited higher cytotoxicity than LPs-DTX-FA. Tumor targeting of re-dispersed liposomes was less effective compared with LPs-DTX-FA but the metabolism of re-dispersed liposomes was decreased. Tracheal administration resulted in a 45-fold higher concentration of docetaxel in the lung of Sprague Dawley rats at 30 min as compared with intravenous administration. Our results indicated that co-spray drying did change the properties, while tracheal administration of the dry powder provided higher drug exposure at the tumor site without increasing the exposure of other organs. Thus, inhaled dry powders might be clinically effective for treatment of lung cancer.

Fixed-jaw technique to improve IMRT plan quality for the treatment of cervical and upper thoracic esophageal cancer.

The purpose of this study was to investigate the potential advantages of the fixed-jaw technique (FJT) over the conventional split-field technique (SFT) for cervical and upper thoracic esophageal cancer (EC) patients treated with intensity-modulated radiotherapy. The SFT and FJT plans were generated for 15 patients with cervical and upper thoracic EC. Dosimetric parameters and delivery efficiency were compared. An area ratio (AR) of the jaw opening to multileaf collimator (MLC) aperture weighted by the number of monitor units (MUs) was defined to evaluate the impact of the transmission through the MLC on the dose gradient outside the PTV50.4, and the correlation between the gradient index (GI) and AR was analyzed. The FJT plans achieved a better GI and AR (P < 0.001). There was a positive correlation between the GI and AR in the FJT (r = 0.883, P < 0.001) and SFT plans (r = 0.836, P < 0.001), respectively. Moreover, the mean dose (Dmean ), V5Gy -V40Gy for the lungs and the Dmean , V5Gy -V50Gy for the body-PTV50.4 in the FJT plans were lower than those in the SFT plans (P < 0.05). The FJT plans demonstrated a reduction trend in the doses to the spinal cord PRV and heart, but only the difference in the heart Dmean reached statistical significance (P < 0.05). The FJT plans reduced the number of MUs and subfields by 5.5% and 17.9% and slightly shortened the delivery time by 0.23 min (P < 0.05). The gamma-index passing rates were above 95% for both plans. The FJT combined with target splitting can provide superior organs at risk sparing and similar target coverage without compromising delivery efficiency and should be a preferred intensity-modulated radiotherapy planning method for cervical and upper thoracic EC patients.

Metals in paints on chopsticks: Solubilization in simulated saliva, gastric, and food solutions and implication for human health.

Paints are often used on chopsticks, however, a paucity of studies has assessed metals in the paints and the associated health risk. In this study, total Pb, Cd, Cr, Co, and Ni concentrations in paints were assessed for 72 wood and 29 stainless steel chopsticks with different colors while metal solubilization from paints in simulated saliva, 0.07 M HCl, and 1% citric acid solutions was measured for 9 samples having total Pb > 90 mg kg-1, representing exposure scenarios of mouthing, incidental paint ingestion, and metal migration in food. Results showed that Pb (0.12-500,000 mg kg-1), Cd (0.002-120,000 mg kg-1), Cr (2.2-8400 mg kg-1), Co (0.004-2600 mg kg-1), and Ni (0.10-150,000 mg kg-1) concentrations varied considerably among paint samples. Most samples showed low metal concentrations, however, high metal concentrations were observed in red and green paints on stainless steel chopsticks, while paints on wood chopsticks showed lower metal concentrations. Estimation of daily metal intakes incorporating metal solubilization data in saliva, 0.07 M HCl, and 1% citric acid solutions suggested that the health risk via saliva contact was negligible, while unacceptable health risk was observed for Pb and Cd via their solubilization in 0.07 M HCl and 1% citric acid solutions from paints with high Pb and Cd concentrations. To avoid Pb and Cd exposure, use of metal-based paints on chopsticks should be banned, particularly in countries where food is regularly consumed with chopsticks.

In vitro cytotoxicity, pharmacokinetics and tissue distribution in rats of MXN-004, a novel conjugate of polyethylene glycol and SN38.

1. MXN-004 is a water-soluble PEGylated 7-ethyl-10-hydroxy-camptothecin (SN38). The aim of this study was to evaluate the in vitro cytotoxicity of MXN-004 and investigate pharmacokinetics and tissue distribution of MXN-004 and its active metabolite SN38 in rats. 2. In vitro cytotoxicity of MXN-004 was tested in A549, HepG2 and Caco-2 cancer cell lines by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and compared with irinotecan. The pharmacokinetics and tissue distribution of MXN-004, irinotecan and their identical active metabolite SN38 were investigated after intravenous administration of MXN-004 and irinotecan at a same dose level of 16 µmol/kg in rats. 3. In vitro cytotoxicity study showed that MXN-004 was more potent in comparison with irinotecan. In rats, MXN-004 exhibited a longer half-life (sixfold) and much greater Vss as compared with irinotecan. The AUC0-∞, T1/2 and Cmax of SN38 after intravenous administration of MXN-004 were higher than those of irinotecan (3.5-, 1.92- and 10.6-fold, respectively). In addition, the concentrations of SN38 released from MXN-004 were significantly higher in all tissues than those from irinotecan, especially in the lung. 4. These results suggested that MXN-004 might be a more potential water-soluble antitumor agent with prolonged half-life of SN38 compared to irinotecan.

Mussel-inspired chitosan and its applications in the biomedical field.

Chitosan (CS) has physicochemical properties including solubility, crystallinity, swellability, viscosity, and cohesion, along with biological properties like biocompatibility, biodegradation, antioxidant, antibacterial, and antitumor effects. However, these characteristics of CS are greatly affected by its degree of deacetylation, molecular weight, pH and other factors, which limits the application of CS in biomedicine. The modification of CS with catechol-containing substances inspired by mussels can not only improve these properties of CS, but also endow it with self-healing property, providing an environmentally friendly and sustainable way to promote the application of CS in biomedicine. In this paper, the properties of CS and its limitation in the biomedical filed are introduced in detail. Then, the modification methods and properties of substances with catechol groups inspired by mussels on CS are reviewed. Finally, the applications of modified CS in the biomedical field of wound healing, drug delivery, anticancer therapy, biosensor and 3D printing are further discussed. This review can provide valuable information for the design and exploitation of mussel-inspired CS in the biomedical field.

Selective extraction and analysis of phenolic acids in herbal plants using Fe3O4@MXene@PEI aerogel.

A magnetic MXene aerogel (Fe3O4@MXene@PEI) was prepared by crosslinking amino modified MXene with polyethyleneimine using epichlorohydrin as a cross-linker. Adsorption properties of Fe3O4@MXene@PEI aerogel for phenolic acids were evaluated by adsorption kinetics and isotherms experiments, showing that the high adsorption affinity was governed by multilayer chemisorption process. An efficient MSPE/HPLC method was developed for the determination of phenolic acids with excellent selectivity, good linearity (0.025-5.0 µg mL-1), low LODs (0.007-0.017 µg mL-1), and satisfactory recoveries (80.0-120.0 %). Moreover, the antioxidant activity of the Fe3O4@MXene@PEI purified compounds was superior to that of the conventional method as demonstrated by the results of scavenging experiments on 2,2 -diphenyl-1-picrylhydrazyl radical scavenging assay. Finally, 65 organic acids were identified in the Fe3O4@MXene@PEI treated honeysuckle extracts by UHPLC-Q-Exactive Orbitrap MS/MS analysis. The proposed sorbent exhibits remarkable promise for the selective separation and purification of organic acids from herbal products.

Asymmetric chitosan-derivative/carboxymethylcellulose layer-by-layer film combining antimicrobial and vascular regeneration for the repair of infected wounds.

Bacterially infected wounds are a serious threat to patients' lives and health, and multifunctional dressings with antimicrobial properties and healing promotion are urgently needed. Thus, we used the cationic and anionic properties of chitosan (CS)-nerol (N) derivative (CSN) and carboxymethylcellulose (CMC) to prepare asymmetric layer-by-layer self-assembled (LBL) composite films (CSN-CMC LBL films) with antibacterial and healing properties using a spin-coating method. SEM images showed that the CSN-CMC LBL films had completely different degrees of roughness at the bottom (hydrophilic layer) and at the top (hydrophobic layer), with the roughness at the top increasing as the number of layers increased. The CSN and CMC were used to prepare asymmetric LBL films via the electrostatic attraction of -COO- and NH3+. In addition, adhesion and water contact angle tests showed that the CSN-CMC LBL films had enhanced tissue adhesion and good hydrophobicity. These materials had excellent antimicrobial activity and good biocompatibility. Importantly, the animal infection model results showed that CSN-CMC-8 LBL films effectively eliminated the infection in vivo, inhibited inflammation, promoted vascular regeneration, accelerated the epithelialization process, and achieved high quality healing. Overall, the CSN-CMC LBL films in this study showed considerable potential for application in infected wound healing.

Osteoblast-specific down-regulation of NLRP3 inflammasome by aptamer-functionalized liposome nanoparticles improves bone quality in postmenopausal osteoporosis rats.

Rationale: NLRP3 inflammasome is critical in the development and progression of many metabolic diseases driven by chronic inflammation, but its effect on the pathology of postmenopausal osteoporosis (PMOP) remains poorly understood. Methods: We here firstly examined the levels of NLRP3 inflammasome in PMOP patients by ELISA. Then we investigated the possible mechanisms underlying the effect of NLRP3 inflammasome on PMOP by RNA sequencing of osteoblasts treated with NLRP3 siRNA and qPCR. Lastly, we accessed the effect of decreased NLRP3 levels on ovariectomized (OVX) rats. To specifically deliver NLRP3 siRNA to osteoblasts, we constructed NLRP3 siRNA wrapping osteoblast-specific aptamer (CH6)-functionalized lipid nanoparticles (termed as CH6-LNPs-siNLRP3). Results: We found that the levels of NLRP3 inflammasome were significantly increased in patients with PMOP, and were negatively correlated with estradiol levels. NLRP3 knock-down influenced signal pathways including immune system process, interferon signal pathway. Notably, of the top ten up-regulated genes in NLRP3-reduced osteoblasts, nine genes (except Mx2) were enriched in immune system process, and five genes were related to interferon signal pathway. The in vitro results showed that CH6-LNPs-siNLRP3 was relatively uniform with a dimeter of 96.64 ± 16.83 nm and zeta potential of 38.37 ± 1.86 mV. CH6-LNPs-siNLRP3 did not show obvious cytotoxicity and selectively delivered siRNA to bone tissue. Moreover, CH6-LNPs-siNLRP3 stimulated osteoblast differentiation by activating ALP and enhancing osteoblast matrix mineralization. When administrated to OVX rats, CH6-LNPs-siNLRP3 promoted bone formation and bone mass, improved bone microarchitecture and mechanical properties by decreasing the levels of NLRP3, IL-1ß and IL-18 and increasing the levels of OCN and Runx2. Conclusion: NLRP3 inflammasome may be a new biomarker for PMOP diagnosis and plays a key role in the pathology of PMOP. CH6-LNPs-siNLRP3 has potential application for the treatment of PMOP.

Identifying symptom clusters among pregnant women during early and late pregnancy.

OBJECTIVES: To investigate the status of multiple psychosomatic symptoms among women and identify symptom clusters at different gestational stages. METHODS: This was a cross-sectional study and a consecutive sample of 557 pregnant women were recruited. They were asked to complete the Memorial Symptom Assessment Scale, and to supply sociodemographic and clinical information. Spearman correlation analysis, partial correlation networks, and hierarchical cluster analysis were carried out to identify the optimal number of symptom clusters. RESULTS: The top five scores for symptoms in early pregnancy were nausea, lack of appetite, feeling drowsy, lack of energy, and vomiting. Weight gain, difficulty sleeping, sweating, lack of energy, and dry mouth scored high in late pregnancy. Four clusters were identified in early pregnancy, and three clusters were determined in late pregnancy. CONCLUSIONS: Women experience multiple psychosomatic symptoms during pregnancy, and the manifestation of symptoms varies at different stages. The present study has provided new insights into symptom clusters in pregnant women.

Classification and research progress of implant surface antimicrobial techniques.

Due to the good biocompatibility and ideal mechanical property, titanium implants have been widely used in dental clinic and orthopedic surgery. However, bacteria induced infection can cause per-implant inflammation and decrease the success rate of implant surgery. Therefore, developing antimicrobial techniques is essential to successful application of titanium implants. Many surface antimicrobial techniques, including antimicrobial coating and surface modifications, have been explored and they always exert antimicrobial effect by reducing bacterial adhesion, inhibiting their metabolism, or destructing cell structure. In this paper, different surface antimicrobial techniques and their recent research progress are reviewed to provide a brief insight on this area.

Stimulus responsive microcapsules and their aromatic applications.

Fragrances and essential oils are promising for a wide range of applications due to their pleasant odors and diverse effects. However, direct addition to consumer products has the disadvantages of short retention time and easy deterioration of odor. At the same time, releasing a large amount of odor in a short time may be an unpleasant experience, which severely limits the practical application of aromatic substances. Microencapsulation perfectly solves these problems. Stimuli-responsive microcapsules, which combine environmental stimulation with microencapsulation, can not only effectively prevent the rapid decomposition and evaporation of aroma components, but also realize the "on-off" intelligent release of aroma substances to environmental changes, which have great promise in the field of fragrances. In this review, the application of stimuli-responsive microcapsules in fragrances is highlighted. Firstly, various encapsulation materials used to prepare stimuli-responsive aromatic microcapsules are described, mainly including some natural polymers, synthetic polymers, and inorganic materials. Subsequently, there is a detailed description of the common release mechanisms of stimuli-responsive aromatic microcapsules are described in detail. Finally, the application and future research directions are given for stimuli-responsive aromatic microcapsules in new textiles, food, paper, and leather.

Limited diagnostic value of liver stiffness for clinically significant portal hypertension in HBV-related cirrhosis.

PURPOSE: Hepatic venous pressure gradient (HVPG) is the gold standard for portal pressure in cirrhosis, but most previous studies focused on the diagnostic value of clinically significant portal hypertension (CSPH) based on the correlation between liver stiffness (LS) and HVPG in hepatitis C virus (HCV) patients and alcoholic liver. Therefore, it is necessary to clarify the diagnostic value of LS for CSPH and the correlation with HVPG in hepatitis B virus (HBV) patients. METHODS: A total of 137 patients from the Fifth Medical Center of PLA General Hospital were divided into HBV group and non-HBV group according to etiology. Correlation analysis and ROC were used to analyze the correlation between LS and HVPG and the diagnostic value of CSPH. RESULTS: There was a good correlation between LS and HVPG in the total cohort and non-HBV cohort (r = 0.398, P < 0.001; r = 0.575, P < 0.001, respectively). However, the correlation between LS and HVPG was acceptable in the HBV cohort (r = 0.316, P = 0.002). When adjustment for age, MELD score, and INR, the result was still the same. Similar results were observed in the prediction for CSPH. LS showed good diagnostic value for CSPH in the total cohort and non-HBV cohort (AUC = 0.732, AUC = 0.829, respectively). However, it performed poorly in the HBV cohort (AUC = 0.689). CONCLUSION: The etiology of HBV might affect the diagnostic performance of LS for predicting CSPH.

In situ formed thermogelable hydrogel photonic crystals assembled by thermosensitive IPNs.

In this paper, soft thermosensitive photonic crystals are immobilized via a reversible temperature-triggered in situ sol-gel transition above their phase transition temperature (Tp), which may be a significant advance in the field. Specifically, a library of thermosensitive poly(N-isopropylacrylamide)/poly(acrylic acid) (PNIPAm/PAA) interpenetrating nanogels (IPNs) is synthesized, which can achieve a reversible temperature-induced sol-gel transition at a low concentration (1.1 wt%). More interestingly, as the temperature is increased above Tp, the photonic crystals assembled by these IPNs do not disappear but are "immobilized" in the in situ formed hydrogel matrix. Moreover, these colorful IPN dispersions exhibit outstanding syringe-injectability, immediately turning from an aqueous solution into an insoluble hydrogel as they are injected into PBS at 37 °C. Plus, a protein-release study showed that these injectable hydrogels show extended release times and slower release rates in comparison with dilute nanogel dispersions. In brief, these in situ formed hydrogels with brilliant structural colors have potential in optical applications, e.g., color displays, crystal immobilization, and biological applications, e.g., 3D cell culture and drug delivery.

Out-of-the-Box Nanocapsules Packed with On-Demand Hydrophobic Anticancer Drugs for Lung Targeting, Esterase Triggering, and Synergy Therapy.

Most anticancer drugs, particularly paclitaxel (PTX), are suffering the challenges of cancer chemotherapy due to their poor water-solubility, high toxicity under effective therapeutic dosages, and multi-drug resistance. Currently, nanoscale drug delivery systems (DDSs) represent an efficient platform to overcome the above challenges. However, those DDSs generally need a careful design of conjugation, complexation, or co-self-assembly. Herein, a facile out-of-the-box nanocapsule is developed not only to be easily packed with on-demand hydrophobic anticancer drugs (up to 76% of loading efficiency for PTX), but also to be loaded with other concomitant drugs for synergy therapy (Itraconazole (ITA) here as P-glycoprotein inhibitor for drug resistance and antiangiogenic agent for combination therapy with PTX). Three kinds of biocompatible poly(ethylene glycol) dimethacrylates (PEGDM) derivatives usually as cross-linking agents are selected and successfully constructed adequate nanocapsules with single monomer as shell materials. More importantly, as-prepared nanocapsules have abilities of esterase triggering and lung targeting. Both in vitro and in vivo studies showed that the drug-loaded nanocapsules can effectively inhibit tumor growth and vascular proliferation in PTX-resistant tumor models without apparent systemic toxicity. The above results demonstrate that the nanocapsule system provides an effective and universal strategy for lung targeting, esterase triggering, and synergy therapy.

Electromagnetized-Nanoparticle-Modulated Neural Plasticity and Recovery of Degenerative Dopaminergic Neurons in the Mid-Brain.

The degeneration of dopaminergic neurons is a major contributor to the pathogenesis of mid-brain disorders. Clinically, cell therapeutic solutions, by increasing the neurotransmitter dopamine levels in the patients, are hindered by low efficiency and/or side effects. Here, a strategy using electromagnetized nanoparticles to modulate neural plasticity and recover degenerative dopamine neurons in vivo is reported. Remarkably, electromagnetic fields generated by the nanoparticles under ultrasound stimulation modulate intracellular calcium signaling to influence synaptic plasticity and control neural behavior. Dopaminergic neuronal functions are reversed by upregulating the expression tyrosine hydroxylase, thus resulting in ameliorating the neural behavioral disorders in zebrafish. This wireless tool can serve as a viable and safe strategy for the regenerative therapy of the neurodegenerative disorders.

Metal/Metalloid Levels in Electronic Cigarette Liquids, Aerosols, and Human Biosamples: A Systematic Review.

BACKGROUND: Electronic cigarettes (e-cigarettes) have become popular, in part because they are perceived as a safer alternative to tobacco cigarettes. An increasing number of studies, however, have found toxic metals/metalloids in e-cigarette emissions. OBJECTIVE: We summarized the evidence on metal/metalloid levels in e-cigarette liquid (e-liquid), aerosols, and biosamples of e-cigarette users across e-cigarette device systems to evaluate metal/metalloid exposure levels for e-cigarette users and the potential implications on health outcomes. METHODS: We searched PubMed/TOXLINE, Embase®, and Web of Science for studies on metals/metalloids in e-liquid, e-cigarette aerosols, and biosamples of e-cigarette users. For metal/metalloid levels in e-liquid and aerosol samples, we collected the mean and standard deviation (SD) if these values were reported, derived mean and SD by using automated software to infer them if data were reported in a figure, or calculated the overall mean (mean ± SD) if data were reported only for separate groups. Metal/metalloid levels in e-liquids and aerosols were converted and reported in micrograms per kilogram and nanograms per puff, respectively, for easy comparison. RESULTS: We identified 24 studies on metals/metalloids in e-liquid, e-cigarette aerosols, and human biosamples of e-cigarette users. Metal/metalloid levels, including aluminum, antimony, arsenic, cadmium, cobalt, chromium, copper, iron, lead, manganese, nickel, selenium, tin, and zinc, were present in e-cigarette samples in the studies reviewed. Twelve studies reported metal/metalloid levels in e-liquids (bottles, cartridges, open wick, and tank), 12 studies reported metal/metalloid levels in e-cigarette aerosols (from cig-a-like and tank devices), and 4 studies reported metal/metalloid levels in human biosamples (urine, saliva, serum, and blood) of e-cigarette users. Metal/metalloid levels showed substantial heterogeneity depending on sample type, source of e-liquid, and device type. Metal/metalloid levels in e-liquid from cartridges or tank/open wicks were higher than those from bottles, possibly due to coil contact. Most metal/metalloid levels found in biosamples of e-cigarette users were similar or higher than levels found in biosamples of conventional cigarette users, and even higher than those found in biosamples of cigar users. CONCLUSION: E-cigarettes are a potential source of exposure to metals/metalloids. Differences in collection methods and puffing regimes likely contribute to the variability in metal/metalloid levels across studies, making comparison across studies difficult. Standardized protocols for the quantification of metal/metalloid levels from e-cigarette samples are needed. https://doi.org/10.1289/EHP5686.