Variability and bias in microbiome metagenomic sequencing: an interlaboratory study comparing experimental protocols.

Several studies have documented the significant impact of methodological choices in microbiome analyses. The myriad of methodological options available complicate the replication of results and generally limit the comparability of findings between independent studies that use differing techniques and measurement pipelines. Here we describe the Mosaic Standards Challenge (MSC), an international interlaboratory study designed to assess the impact of methodological variables on the results. The MSC did not prescribe methods but rather asked participating labs to analyze 7 shared reference samples (5 × human stool samples and 2 × mock communities) using their standard laboratory methods. To capture the array of methodological variables, each participating lab completed a metadata reporting sheet that included 100 different questions regarding the details of their protocol. The goal of this study was to survey the methodological landscape for microbiome metagenomic sequencing (MGS) analyses and the impact of methodological decisions on metagenomic sequencing results. A total of 44 labs participated in the MSC by submitting results (16S or WGS) along with accompanying metadata; thirty 16S rRNA gene amplicon datasets and 14 WGS datasets were collected. The inclusion of two types of reference materials (human stool and mock communities) enabled analysis of both MGS measurement variability between different protocols using the biologically-relevant stool samples, and MGS bias with respect to ground truth values using the DNA mixtures. Owing to the compositional nature of MGS measurements, analyses were conducted on the ratio of Firmicutes: Bacteroidetes allowing us to directly apply common statistical methods. The resulting analysis demonstrated that protocol choices have significant effects, including both bias of the MGS measurement associated with a particular methodological choices, as well as effects on measurement robustness as observed through the spread of results between labs making similar methodological choices. In the analysis of the DNA mock communities, MGS measurement bias was observed even when there was general consensus among the participating laboratories. This study was the result of a collaborative effort that included academic, commercial, and government labs. In addition to highlighting the impact of different methodological decisions on MGS result comparability, this work also provides insights for consideration in future microbiome measurement study design.

SARS-CoV-2 variant of concern fitness and adaptation in primary human airway epithelia.

The severe acute respiratory syndrome coronavirus 2 pandemic is characterized by the emergence of novel variants of concern (VOCs) that replace ancestral strains. Here, we dissect the complex selective pressures by evaluating variant fitness and adaptation in human respiratory tissues. We evaluate viral properties and host responses to reconstruct forces behind D614G through Omicron (BA.1) emergence. We observe differential replication in airway epithelia, differences in cellular tropism, and virus-induced cytotoxicity. D614G accumulates the most mutations after infection, supporting zoonosis and adaptation to the human airway. We perform head-to-head competitions and observe the highest fitness for Gamma and Delta. Under these conditions, RNA recombination favors variants encoding the B.1.617.1 lineage 3' end. Based on viral growth kinetics, Alpha, Gamma, and Delta exhibit increased fitness compared to D614G. In contrast, the global success of Omicron likely derives from increased transmission and antigenic variation. Our data provide molecular evidence to support epidemiological observations of VOC emergence.

Saliva sampling method influences oral microbiome composition and taxa distribution associated with oral diseases.

Saliva is a readily accessible and inexpensive biological specimen that enables investigation of the oral microbiome, which can serve as a biomarker of oral and systemic health. There are two routine approaches to collect saliva, stimulated and unstimulated; however, there is no consensus on how sampling method influences oral microbiome metrics. In this study, we analyzed paired saliva samples (unstimulated and stimulated) from 88 individuals, aged 7-18 years. Using 16S rRNA gene sequencing, we investigated the differences in bacterial microbiome composition between sample types and determined how sampling method affects the distribution of taxa associated with untreated dental caries and gingivitis. Our analyses indicated significant differences in microbiome composition between the sample types. Both sampling methods were able to detect significant differences in microbiome composition between healthy subjects and subjects with untreated caries. However, only stimulated saliva revealed a significant association between microbiome diversity and composition in individuals with diagnosed gingivitis. Furthermore, taxa previously associated with dental caries and gingivitis were preferentially enriched in individuals with each respective disease only in stimulated saliva. Our study suggests that stimulated saliva provides a more nuanced readout of microbiome composition and taxa distribution associated with untreated dental caries and gingivitis compared to unstimulated saliva.

Food budget ratio as an equitable metric for food affordability and insecurity: a population-based cohort study of 121 remote Indigenous communities in Canada.

BACKGROUND: Food insecurity is a public health issue for many regions globally, and especially Indigenous communities. We propose food budget ratio (FBR)-the ratio of food spending to after-tax income-as an affordability metric that better aligns with health equity over traditional price-focused metrics. Existing census and inflation monitoring programs render FBR an accessible tool for future affordability research. METHODS: Public census and food pricing datasets from 2011 to 2021 were analyzed to evaluate food affordability for a cohort of 121 remote Indigenous communities in Canada (n = 80,354 persons as of March 2021). Trends in population-weighted versus community-weighted averages, inflation-adjusted mean price of the Revised Northern Food Basket (RNFB), and distributions of FBR, per-capita price of food, and per-capita after-tax income were calculated and compared to Canada at large. RESULTS: Population-weighted versus community-weighted mean price of the RNFB differed by < 5% for most points in time, peaking at 17%. Mean raw price of the RNFB was relatively stable, while mean inflation-adjusted price of the RNFB decreased 19%. Mean and standard deviation in FBR trended downwards from (0.40; 0.21) in 2011 to (0.25; 0.10) in 2021, while the mean for Canada held stable at 0.10 ± 0.01. Mean and standard deviation in inflation-adjusted per-capita price of food fell from ($5,621; $493) to ($4,510; $243), while the Canada-wide mean rose from $2,189 to $2,567; values for per-capita after-tax income increased from ($17,384; $7,816) to ($21,661; $9,707), while the Canada-wide mean remained between $24,443 and $26,006. Current Nutrition North Canada (NNC) subsidy rates correlate closely with distance to nearest transportation hub (σXY = 0.68 to 0.70) whereas food pricing, after-tax income, and FBR correlate poorly with distance (σXY = -0.22 to 0.03). CONCLUSIONS: The FBR approach yields greater insights on food affordability compared to price-based results, while using readily available public datasets. Whereas 19% reductions in RNFB per-capita food price were observed, FBR decreased 63% yet remained 2.5 times the Canada-wide FBR. The reduction in FBR was driven both by the reduced price of food and a 25% increase in after-tax income. It is recommended that NNC consider FBR for performance measurement and setting subsidy rates.

An engineered immunocytokine with collagen affinity improves the tumor bioavailability, tolerability, and therapeutic efficacy of IL-2.

The clinical utility of human interleukin-2 (hIL-2) is limited by its short serum half-life, preferential activation of regulatory T (TReg) over immune effector cells, and dose-limiting toxicities. We previously engineered F10 immunocytokine (IC), an intramolecularly assembled cytokine/antibody fusion protein that linked hIL-2 to an anti-IL-2 antibody (denoted F10) that extended IL-2 half-life and augmented the immune effector to TReg ratio. Here, we leveraged molecular engineering to improve the anti-tumor therapeutic efficacy and tolerability of F10 IC by developing an iteration, denoted F10 IC-CBD (collagen binding domain), designed for intratumoral administration and in situ retention based on collagen affinity. F10 IC-CBD retained IL-2 bioactivity exclusively in the tumor and eliminated IL-2-associated toxicities. Furthermore, F10 IC exhibited potent single-agent therapeutic efficacy and synergy with systemic immune checkpoint blockade and elicited an abscopal response in mouse tumors models. This engineered fusion protein presents a prototype for the design of intratumoral therapies.

Medical Mistrust Mediates the Relationship Between Nonconsensual Intersex Surgery and Healthcare Avoidance Among Intersex Adults.

BACKGROUND: Intersex individuals experience poor health due, in part, to healthcare avoidance. Nonconsensual intersex surgery may contribute to medical mistrust and avoidance among intersex populations. PURPOSE: The purpose of this study was to explore the relationship between nonconsensual surgery and healthcare avoidance among intersex populations and to examine if medical mistrust mediates this relationship. METHODS: Data for this cross-sectional study were collected in 2018 and analyzed in 2022. Participants completed a survey collecting information on demographics, medical mistrust, history of nonconsensual surgery, and history of postponing healthcare. One hundred nine participants with valid responses to all regression model variables were included in the study. Multivariable logistic regression models controlling for age, race, and income, examined the relationship between nonconsensual surgery and postponing preventive and emergency healthcare. Mediation analyses of cross-sectional data examined whether medical mistrust mediated the relationship between nonconsensual surgery and postponing preventive and emergency healthcare. RESULTS: Mean medical mistrust score was 2.8 (range = 1-4; standard deviation = 0.8), 49.7% of participants had nonconsensual surgery in their lifetime, 45.9% postponed emergency healthcare, and 61.5% postponed preventive healthcare in their lifetime. Nonconsensual surgery was associated with increased odds of delaying preventive (adjusted odds ratio [AOR] = 4.17; confidence interval [CI] = 1.76-9.88; p = .016) and emergency healthcare (AOR = 4.26; CI = 1.71-10.59; p = .002). Medical mistrust mediated the relationship between nonconsensual surgery and delaying preventive (indirect effect = 1.78; CI = 1.16-3.67) and emergency healthcare (indirect effect = 1.66; CI = 1.04-3.30). CONCLUSIONS: Nonconsensual surgery contributed to healthcare avoidance in this intersex population by increasing medical mistrust. To decrease healthcare avoidance, intersex health promotion interventions should restrict nonconsensual surgery and build trust through trauma-informed care.

Many intersex people experience nonconsensual surgery during childhood to alter their genitalia and other anatomy. Some intersex people who have experienced nonconsensual surgery develop subsequent mistrust in medical providers and avoidance of healthcare. The purpose of this study was to understand the relationship between nonconsensual surgery and delay in emergency and preventive healthcare among intersex adults. Additionally, this study aimed to understand whether mistrust in medical providers mediates the relationship between nonconsensual surgery and delaying emergency and preventive healthcare. This study found that ever having nonconsensual surgery was positively associated with delaying both emergency and preventive healthcare among intersex adults. Additionally, this study found that increased mistrust in medical providers mediated the relationship between nonconsensual surgery and delaying emergency and preventive healthcare. Interventions aimed at improving the healthcare engagement of intersex adults may focus on building trust between intersex patients and healthcare providers and restricting nonconsensual intersex surgeries.

The association between religiosity and resilience among young trans women.

INTRODUCTION: Young transgender women (trans women) experience poor health in part due to discrimination. Factors that promote resilience may help young trans women positively adapt to discrimination, resulting in attenuation of poor health outcomes. While religion is sometimes a source of stigma and transphobia, qualitative studies have identified religiosity as an important resilience resource for young trans women. The goals of this study were to quantitatively measure religiosity and resilience among young trans women and to assess whether they are associated. METHODS: From 2012-2013, 300 young trans women between the ages of 16-24 years were enrolled in a longitudinal study; we examined the cross-sectional baseline data on demographics, religiosity, and resilience. Bivariate and multivariable logistic regression analysis examined the correlation between demographics (age, gender, race/ethnicity, education, income) and religiosity among young trans women. Additionally, bivariate and multivariable logistic regression analysis examined the association between religiosity and resilience among young trans women, controlling for age, gender, race/ethnicity, education, and income. RESULTS: Participants who reported high religiosity had significantly greater odds (aOR 1.78, 95% CI 1.05-3.01, p = .03) of reporting high resilience compared to those reporting low religiosity. Black/African American participants had significantly higher odds (aOR 6.16, 95% CI 2.34-16.20, p = < .001) of reporting high religiosity compared to those who identified as White. CONCLUSION: Religiosity may be an important resilience resource for young trans women. Gender affirming religious and spiritual interventions may promote resilience among some young trans women.

Tailoring Interactions of Random Copolymer Polyelectrolyte Complexes to Remove Nanoplastic Contaminants from Water.

We investigate the usage of polyelectrolyte complex materials for water remediation purposes, specifically their ability to remove nanoplastics from water, on which there is currently little to no prior research. We demonstrate that oppositely charged random copolymers are effective at quantitatively removing nanoplastic contamination from aqueous solution. The mechanisms underlying this remediation ability are explored through computational simulations, with corroborating quartz crystal microbalance adsorption experiments. We find that hydrophobic nanostructures and interactions likely play an important role.

Amplicon Sequencing Reveals Complex Infection in Infants Congenitally Infected With Trypanosoma Cruzi and Informs the Dynamics of Parasite Transmission.

Congenital transmission of Trypanosoma cruzi is an important source of new Chagas infections worldwide. The mechanisms of congenital transmission remain poorly understood, but there is evidence that parasite factors are involved. Investigating changes in parasite strain diversity during transmission could provide insight into the parasite factors that influence the process. Here we use amplicon sequencing of a single copy T. cruzi gene to evaluate the diversity of infection in clinical samples from Chagas positive mothers and their infected infants. Several infants and mothers were infected with multiple parasite strains, mostly of the same TcV lineage, and parasite strain diversity was higher in infants than mothers. Two parasite haplotypes were detected exclusively in infant samples, while one haplotype was never found in infants. Together, these data suggest multiple parasites initiate a congenital infection and that parasite factors influence the probability of vertical transmission.

Polishing De Novo Nanopore Assemblies of Bacteria and Eukaryotes With FMLRC2.

As the accuracy and throughput of nanopore sequencing improve, it is increasingly common to perform long-read first de novo genome assemblies followed by polishing with accurate short reads. We briefly introduce FMLRC2, the successor to the original FM-index Long Read Corrector (FMLRC), and illustrate its performance as a fast and accurate de novo assembly polisher for both bacterial and eukaryotic genomes.

Plastid Genome Assembly Using Long-read data.

Although plastid genome (plastome) structure is highly conserved across most seed plants, investigations during the past two decades have revealed several disparately related lineages that experienced substantial rearrangements. Most plastomes contain a large inverted repeat and two single-copy regions, and a few dispersed repeats; however, the plastomes of some taxa harbour long repeat sequences (>300 bp). These long repeats make it challenging to assemble complete plastomes using short-read data, leading to misassemblies and consensus sequences with spurious rearrangements. Single-molecule, long-read sequencing has the potential to overcome these challenges, yet there is no consensus on the most effective method for accurately assembling plastomes using long-read data. We generated a pipeline, plastid Genome Assembly Using Long-read data (ptGAUL), to address the problem of plastome assembly using long-read data from Oxford Nanopore Technologies (ONT) or Pacific Biosciences platforms. We demonstrated the efficacy of the ptGAUL pipeline using 16 published long-read data sets. We showed that ptGAUL quickly produces accurate and unbiased assemblies using only ~50× coverage of plastome data. Additionally, we deployed ptGAUL to assemble four new Juncus (Juncaceae) plastomes using ONT long reads. Our results revealed many long repeats and rearrangements in Juncus plastomes compared with basal lineages of Poales. The ptGAUL pipeline is available on GitHub: https://github.com/Bean061/ptgaul.

Factors Associated with Unsatisfactory Pap Tests Among Sexually Active Trans Masculine Adults.

Purpose: Unsatisfactory collection of cells during Papanicolaou (Pap) tests prevents the detection of cervical cancer and dysplasia. Prior research found that trans masculine (TM) individuals are significantly more likely than cisgender women to have an unsatisfactory Pap test. The purpose of this study was to identify factors that place some TM individuals at greater risk for an unsatisfactory Pap test than others. Methods: Between 2015 and 2016, 150 TM adults were enrolled in a cross-sectional survey assessing demographics, health characteristics, health care experiences, trauma history, and unsatisfactory Pap test history. Bivariate and multivariable logistic regression analyses conducted in 2020 examined associations between age, length of time on testosterone, smoking history, having to educate a provider about transgender people to receive appropriate care, anticipated health care stigma, post-traumatic stress disorder (PTSD) symptoms, and lifetime history of unsatisfactory Pap tests. Results: Of all participants, 20.2% had an unsatisfactory test in their lifetime, age ranged from 21 to 50 years, 55.1% used testosterone for 1 year or more, and 41.3% had PTSD symptoms. In the multivariable model, older age (adjusted odds ratio [AOR] = 1.15; 95% confidence interval (CI) = 1.04-1.27; p < 0.01), 1 year or more lifetime testosterone use (AOR = 3.51; 95% CI = 1.02-12.08; p = 0.046), and PTSD symptoms (AOR = 3.48; 95% CI = 1.10-11.00, p = 0.03) were significantly associated with increased odds of having an unsatisfactory Pap test. Conclusions: Older age, testosterone use, and PTSD symptoms are associated with lifetime unsatisfactory Pap tests among TM adults. Clinicians should assess TM patients' trauma and testosterone use history before Pap tests and utilize trauma-informed practices that facilitate the collection of adequate Pap samples.

Protective and aggressive bacterial subsets and metabolites modify hepatobiliary inflammation and fibrosis in a murine model of PSC.

OBJECTIVE: Conflicting microbiota data exist for primary sclerosing cholangitis (PSC) and experimental models. GOAL: define the function of complex resident microbes and their association relevant to PSC patients by studying germ-free (GF) and antibiotic-treated specific pathogen-free (SPF) multidrug-resistant 2 deficient (mdr2-/- ) mice and microbial profiles in PSC patient cohorts. DESIGN: We measured weights, liver enzymes, RNA expression, histological, immunohistochemical and fibrotic biochemical parameters, faecal 16S rRNA gene profiling and metabolomic endpoints in gnotobiotic and antibiotic-treated SPF mdr2-/- mice and targeted metagenomic analysis in PSC patients. RESULTS: GF mdr2-/- mice had 100% mortality by 8 weeks with increasing hepatic bile acid (BA) accumulation and cholestasis. Early SPF autologous stool transplantation rescued liver-related mortality. Inhibition of ileal BA transport attenuated antibiotic-accelerated liver disease and decreased total serum and hepatic BAs. Depletion of vancomycin-sensitive microbiota exaggerated hepatobiliary disease. Vancomycin selectively decreased Lachnospiraceae and short-chain fatty acids (SCFAs) but expanded Enterococcus and Enterobacteriaceae. Antibiotics increased Enterococcus faecalis and Escherichia coli liver translocation. Colonisation of GF mdr2-/- mice with translocated E. faecalis and E. coli strains accelerated hepatobiliary inflammation and mortality. Lachnospiraceae colonisation of antibiotic pretreated mdr2-/- mice reduced liver fibrosis, inflammation and translocation of pathobionts, and SCFA-producing Lachnospiraceae and purified SCFA decreased fibrosis. Faecal Lachnospiraceae negatively associated, and E. faecalis/ Enterobacteriaceae positively associated, with PSC patients' clinical severity by Mayo risk scores. CONCLUSIONS: We identified novel functionally protective and detrimental resident bacterial species in mdr2-/- mice and PSC patients with associated clinical risk score. These insights may guide personalised targeted therapeutic interventions in PSC patients.

A meta-analysis of extended ECG monitoring in detection of atrial fibrillation in patients with cryptogenic stroke.

OBJECTIVE: The aim of this systematic review is to evaluate the various modalities available for extended ECG monitoring in the detection of atrial fibrillation (AF) following a cryptogenic stroke. METHODS: MEDLINE (Ovid), EMBASE (Ovid), Cochrane Central Register of Controlled Trials (CENTRAL) were searched from January 2011 to November 2021. All randomised controlled trials and prospective cohort studies including the use of extended ECG monitoring >24 hours with a minimum duration of AF of 30 s in patients with either cryptogenic strokes or transient ischaemic attacks were included. A random-effects model was used to pool effect estimates of AF detection rates from different ECG modalities. RESULTS: 3924 studies were identified, of which 47 were included reporting on a pooled population of 6448 patients with cryptogenic stroke. The pooled AF rate for implantable loop recorders (ILRs) increased from 4.9% (3.0%-7.9%) at 1 month to 38.4% (20.4%-60.2%) at 36 months. Mobile cardiac outpatient telemetry (MCOT) had a significantly higher pooled AF detection rate of 12.8% (8.9%-17.9%) versus 4.9% (3.0%-7.9%) for ILR at 1 month (p<0.0001). Predictors for AF detection include duration of monitoring (p<0.0001) and age (p<0.0001) for ILRs, but only age for MCOTs (p<0.020). CONCLUSION: MCOT has a higher rate of detection at 1 month and is less invasive. Beyond 1 month, compliance becomes a significant limitation for MCOT. MCOT may be a reasonable alternative AF screening tool for patients with cryptogenic stroke if ILR is not available. PROSPERO REGISTRATION NUMBER: CRD42022297782.

Acute Leukemia Classification Using Transcriptional Profiles From Low-Cost Nanopore mRNA Sequencing.

PURPOSE: Most cases of pediatric acute leukemia occur in low- and middle-income countries, where health centers lack the tools required for accurate diagnosis and disease classification. Recent research shows the robustness of using unbiased short-read RNA sequencing to classify genomic subtypes of acute leukemia. Compared with short-read sequencing, nanopore sequencing has low capital and consumable costs, making it suitable for use in locations with limited health infrastructure. MATERIALS AND METHODS: We show the feasibility of nanopore mRNA sequencing on 134 cryopreserved acute leukemia specimens (26 acute myeloid leukemia [AML], 73 B-lineage acute lymphoblastic leukemia [B-ALL], 34 T-lineage acute lymphoblastic leukemia, and one acute undifferentiated leukemia). Using multiple library preparation approaches, we generated long-read transcripts for each sample. We developed a novel composite classification approach to predict acute leukemia lineage and major B-ALL and AML molecular subtypes directly from gene expression profiles. RESULTS: We demonstrate accurate classification of acute leukemia samples into AML, B-ALL, or T-lineage acute lymphoblastic leukemia (96.2% of cases are classifiable with a probability of > 0.8, with 100% accuracy) and further classification into clinically actionable genomic subtypes using shallow RNA nanopore sequencing, with 96.2% accuracy for major AML subtypes and 94.1% accuracy for major B-lineage acute lymphoblastic leukemia subtypes. CONCLUSION: Transcriptional profiling of acute leukemia samples using nanopore technology for diagnostic classification is feasible and accurate, which has the potential to improve the accuracy of cancer diagnosis in low-resource settings.

Functional enzyme-polymer complexes.

SignificanceThe use of biological enzyme catalysts could have huge ramifications for chemical industries. However, these enzymes are often inactive in nonbiological conditions, such as high temperatures, present in industrial settings. Here, we show that the enzyme PETase (polyethylene terephthalate [PET]), with potential application in plastic recycling, is stabilized at elevated temperature through complexation with random copolymers. We demonstrate this through simulations and experiments on different types of substrates. Our simulations also provide strategies for designing more enzymatically active complexes by altering polymer composition and enzyme charge distribution.