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Spot fires pose a major risk and add to the already complex physics, which makes fire spread so hard to predict, especially in the wildland urban interface. Firebrands can not only cross fuel breaks and thwart other suppression efforts but also directly damage infrastructure and block evacuation routes. Transport models and computational fluid dynamics tools often make simplifications when predicting spot fire risk, but there is a relative lack of experimental data to validate such parameterizations. To this end, we present a field experiment performed at the University of California Berkeley Blodgett Research Forest in California where we recorded the flame and firebrands emanating from a nighttime hand-drawn pile fire using high-frequency imaging. We used image-processing to characterize the fire intensity and turbulence as well as particle tracking velocimetry to measure ejected firebrand kinematics as they are lofted by the plume. We further collected embers that settled around the fire at varying distances and measured their size, shape, density, and settling distributions. We also examine existing physics-based time-averaged models of firebrand lofting and note discrepancies between such models, often used due to their speed and simplicity, and our experimental observations. Finally, we discuss some implications our observations could have on future modeling efforts by considering the time-dependent fire dynamics, intermittency in the plume turbulence, and in the firebrand generation rate. To the best of our knowledge, these are the first in situ observations of firebrand generation and lofting from representative fuels, addressing a major source of data gap and uncertainty in the wildland fire literature.

Lipophilic aggregation using adamantanes is a widely exploited molecular property in medicinal and materials chemistry. Adamantanes are traditionally installed to molecular units via covalent bonds. However, the noncovalent installation of adamantanes has been relatively underexplored and presents the potential to bring properties associated with adamantanes to molecules without affecting their intrinsic properties (e.g., pharmacophores). Here, we systematically study a series of adamantanecarboxylic acids with varying substitution levels of methyl groups and their cocrystals with bipyridines. Specifically, single-crystal X-ray diffraction shows that while the directionality of single-component adamantanes is notably sensitive to changes in methyl substitution, hydrogen-bonded cocrystals with bipyridines show consistent and robust packing due to π-stacking predominance. Our observations are supported by Hirshfeld surface and energy framework analyses. The applicability of cocrystal formation of adamantanes bearing carboxylic acids was used to generate the first cocrystals of adapalene, an adamantane-bearing retinoid used for treating acne vulgaris. We envisage our study to inspire noncovalent (i.e., cocrystal) installation of adamantanes to generate lipophilic aggregation in multicomponent systems.

Superficial granulomatous pyoderma gangrenosum is a rare, superficial, vegetating form of pyoderma gangrenosum that tends to occur as a single lesion, most commonly on the trunk. Herein, we report a clinically confounding case of disseminated superficial granulomatous pyoderma gangrenosum in a patient with a 5-year history of painful and chronic ulcerations of the bilateral upper extremities and face in a sun exposed distribution. This was a diagnostically challenging case due to the treatment-refractory nature of our patient's skin lesions and the atypical clinical and histologic presentations encountered. We review our clinical decision process and acknowledge other entities that were considered during the clinical course of this case. Additionally, we discuss the lack of responsiveness to various treatment options with eventual successful clearance of this patient's active skin disease with initiation of adalimumab.

BackgroundPersons with HIV (PWH) have both more frequent depression and higher levels of plasma inflammatory biomarkers compared to persons without HIV (PWoH). Inflammation and depressive symptoms are linked, including in PWH; however, it is unclear whether these associations differ by HIV serostatus and biological sex.MethodsSix plasma inflammatory biomarkers were assessed using samples from PWH and PWoH who participated in six NIH-funded studies through the UCSD HIV Neurobehavioral Research Program (HNRP) from 2011 to 2019. Factor analysis was performed to identify intercorrelated groups of biomarkers. Factors and their components were then examined for relationships with Beck Depression Inventory-II (BDI-II) and modifying effects of sex or HIV serostatus using multivariable linear regression, adjusting for demographics, substance use diagnoses, and relevant co-morbidities.ResultsParticipants included 150 PWH (age = 48.3 ± 13.1 yr; 88% biologically male) and 138 PWoH (age = 46.3 ± 15.9; 56% male). Two inflammatory factors were identified: Factor 1 loaded on interleukin-6 (IL-6), C-reactive protein (CRP), and D-dimer; Factor 2 loaded on interleukin-8, chemokine C-C ligand 2 (CCL2), and chemokine C-X-C ligand 10 (CXCL10). Sex modified the effect of Factor 1 on BDI-II, with a more positive association for men than women (p = 0.04). No significant association between Factor 2 and BDI-II was found. Of the biomarkers in Factor 1, only IL-6 was significantly associated with BDI-II and was modified by sex (p = 0.003). In sex-stratified analysis, a positive association was found for men (β = 5.42; 95% confidence interval = [1.32, 9.52]) but not women (β = -3.88; 95% C.I. = [-11.02, 3.26]). No HIV-related interactions were detected.InterpretationWe identified a depression-associated inflammatory factor present in both PWH and PWoH, consistent with prior studies of PWH only. The association was driven by a correlation between IL-6 and depression exclusively in men, suggesting that the depression-inflammation link differs by sex. Future studies of depression etiology or treatment, including those on persons with HIV, should consider the impact of biological sex in both design and analysis.

Cognitive disorders are prevalent in people with HIV (PWH) despite antiretroviral therapy. Given the heterogeneity of cognitive disorders in PWH in the current era and evidence that these disorders have different etiologies and risk factors, scientific rationale is growing for using data-driven models to identify biologically defined subtypes (biotypes) of these disorders. Here, we discuss the state of science using machine learning to understand cognitive phenotypes in PWH and their associated comorbidities, biological mechanisms, and risk factors. We also discuss methods, example applications, challenges, and what will be required from the field to successfully incorporate machine learning in research on cognitive disorders in PWH. These topics were discussed at the National Institute of Mental Health meeting on "Biotypes of CNS Complications in People Living with HIV" held in October 2021. These ongoing research initiatives seek to explain the heterogeneity of cognitive phenotypes in PWH and their associated biological mechanisms to facilitate clinical management and tailored interventions.

Background: Loneliness during adolescence has adverse consequences for mental health, education and employment outcomes. Yet, we know little about common correlates of loneliness among adolescents, making intervention work difficult. Aims: In this study, we (1) explore individual-, school- and country-level correlates of loneliness to help identify potential intervention targets, and (2) examine the influence of loneliness on academic performance. Sample: A total of 518,210 students aged 15 years from 75 countries provided self-reported loneliness data. Results: Using multilevel modelling, we found individual-, school- and country-level correlates of self-reported school-based loneliness, and showed that loneliness negatively influenced academic performance. Conclusions: Based on the findings, interventions that focus on enhancing social and emotional skills, increasing trust between teachers and students and changing school climate to be more inclusive are likely to be the most effective for adolescents; they should also be culturally sensitive.

BackgroundCombination antiretroviral therapy (cART) has allowed for viral load (VL) suppression and increased life expectancy for persons with HIV (PWH). Altered brain integrity, measured by neuropsychological (NP) performance and neuroimaging, is still prevalent among virally suppressed PWH. Age-related conditions such as cardiovascular disease may also affect brain integrity. This study investigated the effects of cardiovascular risk, VL, and HIV serostatus on cerebral blood flow (CBF), brain volumetrics, and cognitive function in PWH and persons without HIV (PWoH).MethodsTen-year cardiovascular risk, using the Framingham Heart Study criteria, was calculated in PWH (n = 164) on cART with undetectable (≤20 copies/mL; n = 134) or detectable (>20 copies/mL; n = 30) VL and PWoH (n = 66). The effects of cardiovascular risk on brain integrity (CBF, volume, and cognition) were compared for PWH (undetectable and detectable VL) and PWoH.ResultsPWH had smaller brain volumes and worse NP scores than PWoH. PWH with detectable and undetectable VL had similar brain integrity measures. Higher cardiovascular risk was associated with smaller volumes and lower CBF in multiple brain regions for PWH and PWoH. Significant interactions between HIV serostatus and cardiovascular risk on brain volumes were observed in frontal, orbitofrontal, and motor regions. Cardiovascular risk was not associated with cognition for PWH or PWoH.ConclusionsNeuroimaging, but not cognitive measures, was associated with elevated cardiovascular risk. HIV serostatus was associated with diminished brain volumes and worse cognition while CBF remained unchanged, reflecting potential protective effects of cART. Neuroimaging measures of structure (volume) and function (CBF) may identify contributions of comorbidities, but future longitudinal studies are needed.

BackgroundPersons with human immunodeficiency virus (PWH) are characterized by altered brain structure and function. As they attain normal lifespans, it has become crucial to understand potential interactions between human immunodeficiency virus (HIV) and aging. However, it remains unclear how brain aging varies with viral load (VL).MethodsIn this study, we compare magnetic resonance imaging (MRI) biomarkers among PWH with undetectable VL (UVL; ≤50 genomic copies/mL; n = 230), PWH with detectable VL (DVL; >50 copies/mL; n = 93), and HIV-uninfected (HIV-) controls (n = 206). To quantify gray matter cerebral blood flow (CBF), we utilized arterial spin labeling. To measure structural aging, we used a publicly available deep learning algorithm to estimate brain age from T1-weighted MRI. Cognitive performance was measured using a neuropsychological battery covering 5 domains.ResultsAssociations between age and CBF varied with VL. Older PWH with DVL had reduced CBF vs PWH with UVL (P = .02). Structurally predicted brain aging was accelerated in PWH vs HIV- controls regardless of VL (P < .001). Overall, PWH had impaired learning, executive function, psychomotor speed, and language compared to HIV- controls. Structural brain aging was associated with reduced psychomotor speed (P < .001).ConclusionsBrain aging in HIV is multifaceted. CBF depends on age and current VL and is improved by medication adherence. By contrast, structural aging is an indicator of cognitive function and reflects serostatus rather than current VL.

In the current study, data collected from Wave 6 of the Millennium Cohort Study (n = 11,872), a nationally representative sample survey of youth aged 14 years in the UK, are used to examine the prevalence of loneliness among this age-group, investigate the feelings associated with the experience of loneliness among youth, explore the risk factors for loneliness among young people, and learn how they coped with loneliness. Given recent findings that youth are vulnerable to loneliness, the study assesses the prevalence of loneliness among adolescents across some important sociodemographic characteristics, such as nation of residence, gender, and ethnicity. We also identify the kinds of social experiences that accompany loneliness during adolescence, exploring friendship, relations with parents, social support, and bullying. Our key finding is that, in addition to the absence of desired social relationships, which has been typically identified as the ultimate source of loneliness, the presence of undesirable and even harmful social relationships is a major source of loneliness. This study uniquely brings together psychological and sociological perspectives to understand the experience of youth loneliness.

The focus on human development is foundational to the field of counseling, with its importance codified in guiding documents and frameworks, such as the American Counseling Association's Code of Ethics (2014). Many developmental theories have been established using single-gender or single-culture groups, yet they claim universal application to all humans. Although counseling students must learn these theories because of accreditation standards and licensure requirements, counselor educators need to prepare students for practice in a multicultural world. Counselors are now called to act as social justice advocates, and teaching strategies are needed to prepare students for this role. This study's focus is on the use of service learning with community counseling students in a human development course. Results from a content analysis demonstrate how service learning enhances learning and broadens students' perceptions of themselves, others, and social justice in counseling. Findings indicate a shift in participants' perception of social justice in counseling.

BackgroundTo date, there have not been any epidemiologic studies that have evaluated the association between swimming over a lifetime and knee health.ObjectiveThe study aimed to evaluate the relationship of a history of swimming with knee pain, radiographic knee OA (ROA), and symptomatic knee OA (SOA).DesignCross-sectional retrospective study.SettingFour academic centers in the United States.ParticipantsRespondents to the historical physical activity survey within the Osteoarthritis Initiative with knee radiographs and symptom assessments.MethodsIn this retrospective study nested within the Osteoarthritis Initiative, researchers performed logistic regression with the predictor being swimming over a lifetime and over particular age ranges.Main outcome measurementsPerson-based definitions of frequent knee pain, ROA, and SOA.ResultsA total of 2637 participants were included, with a mean age of 64.3 years (SD 8.9), body mass index of 28.4 kg/m2 (SD 4.9), and 44.2% male. Over a lifetime, the adjusted prevalence measures for frequent knee pain, ROA, and SOA for any versus no history of swimming were 36.4% (33.4% - 39.5%) v. 39.9% (37.4% - 42.5%), 54.3% (51.0% - 57.6%) v. 61.1% (58.4% - 63.7%), and 21.9% (19.4% - 24.7%) v. 27.0% (24.7% - 29.4%) respectively.ConclusionsThis is the first epidemiologic study to indicate that swimming is potentially beneficial toward knee health, particularly when performed earlier in life (before age 35). Future prospective studies are needed to confirm these findings and to better scrutinize the associations in older age groups.

One of the key components for environmental risk assessment of engineered nanomaterials (ENMs) is data on bioaccumulation potential. Accurately measuring bioaccumulation can be critical for regulatory decision making regarding material hazard and risk, and for understanding the mechanism of toxicity. This perspective provides expert guidance for performing ENM bioaccumulation measurements across a broad range of test organisms and species. To accomplish this aim, we critically evaluated ENM bioaccumulation within three categories of organisms: single-celled species, multicellular species excluding plants, and multicellular plants. For aqueous exposures of suspended single-celled and small multicellular species, it is critical to perform a robust procedure to separate suspended ENMs and small organisms to avoid overestimating bioaccumulation. For many multicellular organisms, it is essential to differentiate between the ENMs adsorbed to external surfaces or in the digestive tract and the amount absorbed across epithelial tissues. For multicellular plants, key considerations include how exposure route and the role of the rhizosphere may affect the quantitative measurement of uptake, and that the efficiency of washing procedures to remove loosely attached ENMs to the roots is not well understood. Within each organism category, case studies are provided to illustrate key methodological considerations for conducting robust bioaccumulation experiments for different species within each major group. The full scope of ENM bioaccumulation measurements and interpretations are discussed including conducting the organism exposure, separating organisms from the ENMs in the test media after exposure, analytical methods to quantify ENMs in the tissues or cells, and modeling the ENM bioaccumulation results. One key finding to improve bioaccumulation measurements was the critical need for further analytical method development to identify and quantify ENMs in complex matrices. Overall, the discussion, suggestions, and case studies described herein will help improve the robustness of ENM bioaccumulation studies.

Mental health is complex, comprising both mental distress and well-being. This study used latent class analysis to identify common combinations of mental distress and well-being ('mental health classes') among schoolchildren aged 8-9 years (N = 3340).Thirteen items, measuring a range of conduct problems, emotional symptoms, and subjective well-being, were included in the analysis. Four mental health classes were identified: (1) "complete mental health" (n = 1895, 57%), (2) "vulnerable" (n = 434, 13%), (3) "emotional symptoms but content" (n = 606, 18%), and (4) "conduct problems but content" (n = 404, 12%). The classes were reliably identified across different datasets, and for males and females. Differential relations with covariates indicated that mental health classes were distinct and externally valid. The results supported the dual-factor model of mental health, suggesting that mental distress and subjective well-being are separate continua. Three of the four possible combinations of high and low distress and subjective well-being posited by the dual-factor model were found using this inductive statistical method. Importantly, our analysis also revealed two 'symptomatic but content' groups, differentiated by symptom domain (internalising/externalising). The covariate analyses between mental health classes and sociodemographic factors, prior academic attainment, school connectedness, and peer support, indicated that there are nuanced relations between those variables and particular constellations of mental distress and well-being. As one of the few dual-factor studies to focus on middle childhood, the current study adds important new evidence that contributes to our understanding of the complexities of mental health among schoolchildren.

BackgroundAntimicrobial stewardship to combat the spread of antibiotic-resistant bacteria has become a national priority. This project focuses on reducing inappropriate use of antimicrobials for asymptomatic bacteriuria (ASB), a very common condition that leads to antimicrobial overuse in acute and long-term care. We previously conducted a successful intervention, entitled "Kicking Catheter Associated Urinary Tract Infection (CAUTI): the No Knee-Jerk Antibiotics Campaign," to decrease guideline-discordant ordering of urine cultures and antibiotics for ASB. The current objective is to facilitate implementation of a scalable version of the Kicking CAUTI campaign across four geographically diverse Veterans Health Administration facilities while assessing what aspects of an antimicrobial stewardship intervention are essential to success and sustainability.MethodsThis project uses an interrupted time series design with four control sites. The two main intervention tools are (1) an evidence-based algorithm that distills the guidelines into a streamlined clinical pathway and (2) case-based audit and feedback to train clinicians to use the algorithm. Our conceptual framework for the development and implementation of this intervention draws on May's General Theory of Implementation. The intervention is directed at providers in acute and long-term care, and the goal is to reduce inappropriate screening for and treatment of ASB in all patients and residents, not just those with urinary catheters. The start-up for each facility consists of centrally-led phone calls with local site champions and baseline surveys. Case-based audit and feedback will begin at a given site after the start-up period and continue for 12 months, followed by a sustainability assessment. In addition to the clinical outcomes, we will explore the relationship between the dose of the intervention and clinical outcomes.DiscussionThis project moves from a proof-of-concept effectiveness study to implementation involving significantly more sites, and uses the General Theory of Implementation to embed the intervention into normal processes of care with usual care providers. Aspects of implementation that will be explored include dissemination, internal and external facilitation, and organizational partnerships. "Less is More" is the natural next step from our prior successful Kicking CAUTI intervention, and has the potential to improve patient care while advancing the science of implementation.

Higher levels of moderate to vigorous physical activity improve all-cause mortality and cardiovascular events. However, the effect of running, a moderate to vigorous activity, in those with knee osteoarthritis (OA), a common arthritis that occurs with aging, a high-risk group for mortality and cardiovascular events, is unclear. Therefore, we aimed to evaluate the association of self-selected running on OA symptom and structure progression in people with knee OA. This nested cohort study within the Osteoarthritis Initiative (OAI) (2004-2014) included those at least 50 years old with OA in at least one knee. Runners were defined using a self-administered questionnaire at the 96-month visit. At baseline and 48-months, symptoms were assessed and radiographs were scored for Kellgren-Lawrence (KL) grade (2-4) and medial Joint Space Narrowing (JSN) score (0-3). We evaluated the association of self-selected running with outcomes: KL worsening, medial JSN worsening, new knee pain, and improved knee pain over 48 months, adjusting for baseline age, sex, body mass index (BMI), KL score, contralateral KL score, contralateral knee pain, and injury. If data were not available at the 48-month visit, then they were imputed from the 36-month visit. One thousand two hundred three participants had a mean age of 63.2 (7.9) years, BMI of 29.5 (4.6) kg/m2, 45.3% male, and 11.5% runners. Data from 8% of participants required imputation. Adjusted odds ratios for KL grade worsening and new frequent knee pain were 0.9 (0.6-1.3) and 0.9 (0.6-1.6) respectively. Adjusted odds ratio for frequent knee pain resolution was 1.7 (1.0-2.8). Among individuals 50 years old and older with knee OA, self-selected running is associated with improved knee pain and not with worsening knee pain or radiographically defined structural progression. Therefore, self-selected running, which is likely influenced by knee symptoms and may result in lower intensity and shorter duration sessions of exercise, need not be discouraged in people with knee OA.

An increase in production of commercial products containing graphene-family nanomaterials (GFNs) has led to concern over their release into the environment. The fate and potential ecotoxicological effects of GFNs in the environment are currently unclear, partially due to the limited analytical methods for GFN measurements. In this review, the unique properties of GFNs that are useful for their detection and quantification are discussed. The capacity of several classes of techniques to identify and/or quantify GFNs in different environmental matrices (water, soil, sediment, and organisms), after environmental transformations, and after release from a polymer matrix of a product is evaluated. Extraction and strategies to combine methods for more accurate discrimination of GFNs from environmental interferences as well as from other carbonaceous nanomaterials are recommended. Overall, a comprehensive review of the techniques available to detect and quantify GFNs are systematically presented to inform the state of the science, guide researchers in their selection of the best technique for the system under investigation, and enable further development of GFN metrology in environmental matrices. Two case studies are described to provide practical examples of choosing which techniques to utilize for detection or quantification of GFNs in specific scenarios. Because the available quantitative techniques are somewhat limited, more research is required to distinguish GFNs from other carbonaceous materials and improve the accuracy and detection limits of GFNs at more environmentally relevant concentrations.

ObjectiveRegular physical activity, including running, is recommended based on known cardiovascular and mortality benefits. However, controversy exists regarding whether running can be harmful to knees. The purpose of this study is to evaluate the relationship of running with knee pain, radiographic osteoarthritis (OA), and symptomatic OA.MethodsThis was a retrospective cross-sectional study of Osteoarthritis Initiative participants (2004-2014) with knee radiograph readings, symptom assessments, and completed lifetime physical activity surveys. Using logistic regression, we evaluated the association of history of leisure running with the outcomes of frequent knee pain, radiographic OA, and symptomatic OA. Symptomatic OA required at least 1 knee with both radiographic OA and pain.ResultsOf 2,637 participants, 55.8% were female, the mean ± SD age was 64.3 ± 8.9 years, and the mean ± SD body mass index was 28.5 ± 4.9 kg/m2 ; 29.5% of these participants ran at some time in their lives. Unadjusted odds ratios of pain, radiographic OA, and symptomatic OA for those prior runners and current runners compared to those who never ran were 0.83 and 0.71 (P for trend = 0.002), 0.83 and 0.78 (P for trend = 0.01), and 0.81 and 0.64 (P for trend = 0.0006), respectively. Adjusted models were similar, except radiographic OA results were attenuated.ConclusionThere is no increased risk of symptomatic knee OA among self-selected runners compared with nonrunners in a cohort recruited from the community. In those without OA, running does not appear to be detrimental to the knees.

Consumer goods contain multiwall carbon nanotubes (MWCNTs) that could be released during product life cycles into the environment, where their effects are uncertain. Here, we assessed MWCNT bioaccumulation in the protozoan Tetrahymena thermophila via trophic transfer from bacterial prey (Pseudomonas aeruginosa) versus direct uptake from growth media. The experiments were conducted using (14)C-labeled MWCNT ((14)C-MWCNT) doses at or below 1 mg/L, which proved subtoxic since there were no adverse effects on the growth of the test organisms. A novel contribution of this study was the demonstration of the ability to quantify MWCNT bioaccumulation at low (sub μg/kg) concentrations accomplished by employing accelerator mass spectrometry (AMS). After the treatments with MWCNTs at nominal concentrations of 0.01 mg/L and 1 mg/L, P. aeruginosa adsorbed considerable amounts of MWCNTs: (0.18 ± 0.04) μg/mg and (21.9 ± 4.2) μg/mg bacterial dry mass, respectively. At the administered MWCNT dose of 0.3 mg/L, T. thermophila accumulated up to (0.86 ± 0.3) μg/mg and (3.4 ± 1.1) μg/mg dry mass by trophic transfer and direct uptake, respectively. Although MWCNTs did not biomagnify in the microbial food chain, MWCNTs bioaccumulated in the protozoan populations regardless of the feeding regime, which could make MWCNTs bioavailable for organisms at higher trophic levels.

Engineered nanomaterials (ENMs) are increasingly entering the environment with uncertain consequences including potential ecological effects. Various research communities view differently whether ecotoxicological testing of ENMs should be conducted using environmentally relevant concentrations-where observing outcomes is difficult-versus higher ENM doses, where responses are observable. What exposure conditions are typically used in assessing ENM hazards to populations? What conditions are used to test ecosystem-scale hazards? What is known regarding actual ENMs in the environment, via measurements or modeling simulations? How should exposure conditions, ENM transformation, dose, and body burden be used in interpreting biological and computational findings for assessing risks? These questions were addressed in the context of this critical review. As a result, three main recommendations emerged. First, researchers should improve ecotoxicology of ENMs by choosing test end points, duration, and study conditions-including ENM test concentrations-that align with realistic exposure scenarios. Second, testing should proceed via tiers with iterative feedback that informs experiments at other levels of biological organization. Finally, environmental realism in ENM hazard assessments should involve greater coordination among ENM quantitative analysts, exposure modelers, and ecotoxicologists, across government, industry, and academia.

Sustainable production and use of carbon nanotube (CNT)-enabled materials require efficient assessment of CNT environmental hazards, including the potential for CNT bioaccumulation and biomagnification in environmental receptors. Microbes, as abundant organisms responsible for nutrient cycling in soil and water, are important ecological receptors for studying the effects of CNTs. Quantification of CNT association with microbial cells requires efficient separation of CNT-associated cells from individually dispersed CNTs and CNT agglomerates. Here, we designed, optimized, and demonstrated procedures for separating bacteria (Pseudomonas aeruginosa) from unbound multiwall carbon nanotubes (MWCNTs) and MWCNT agglomerates using sucrose density gradient centrifugation. We demonstrate separation of protozoa (Tetrahymena thermophila) from MWCNTs, bacterial agglomerates, and protozoan fecal pellets by centrifugation in an iodixanol solution. The presence of MWCNTs in the density gradients after centrifugation was determined by quantification of 14C-labeled MWCNTs; the recovery of microbes from the density gradient media was confirmed by optical microscopy. Protozoan intracellular contents of MWCNTs and of bacteria were also unaffected by the designed separation process. The optimized methods contribute to improved efficiency and accuracy in quantifying MWCNT association with bacteria and MWCNT accumulation in protozoan cells, thus supporting improved assessment of CNT bioaccumulation.