Multiple Replicates Research Articles

Hidden diversity: DNA metabarcoding reveals hyper-diverse benthic invertebrate communities

BackgroundFreshwater ecosystems, such as streams, are facing increasing pressures from agricultural land use and recent literature stresses the importance of robust biomonitoring to detect trends in insect decline globally. Aquatic insects and other macroinvertebrates are often used as indicators of ecological condition in freshwater biomonitoring programs; however, these diverse groups can present challenges to morphological identification and coarse-level taxonomic resolution can mask patterns in community composition. Here, we incorporate molecular identification (DNA metabarcoding) into a stream biomonitoring sampling design to explore the diversity and variability of aquatic macroinvertebrate communities at small spatial scales. While individual stream reaches can be very heterogenous, most community ecology studies focus on larger, landscape-level patterns of community composition. A high degree of community variability at the local scale has important implications for both biomonitoring and ecological research, and the incorporation of DNA metabarcoding into local biodiversity assessments will inform future sampling protocols.ResultsWe sampled twenty streams in southern Ontario, Canada, for aquatic macroinvertebrates across multiple time points and assessed local community variability by comparing field replicates taken ten meters apart within the same stream. Using bulk-tissue DNA metabarcoding, we revealed that aquatic macroinvertebrate communities are highly diverse at small spatial scales with unprecedented levels of local taxonomic turnover. We detected over 1600 Operational Taxonomic Units (OTUs) from 149 families, and a single insect family, the Chironomidae, contained over one third of the total number of OTUs detected in our study. Benthic communities were largely comprised of rare taxa detected only once per stream despite multiple biological replicates (24–94% rare taxa per site). In addition to numerous rare taxa, our species pool estimates indicated that there was a large proportion of taxa that remained undetected by our sampling regime (14–94% per site). Our sites were located across a gradient of agricultural activity, and while we predicted that increased land use would homogenize benthic communities, this was not supported as within-stream dissimilarity was unrelated to land use. Within-stream dissimilarity estimates were consistently high for all levels of taxonomic resolution (invertebrate families, invertebrate OTUs, chironomid OTUs), indicating stream communities are very dissimilar at small spatial scales.

Coral reef fish communities of natural habitats and man-made coastal structures in Bora-Bora (French Polynesia)

Coastal habitats have long been recognised to be nurseries and growing grounds for many marine organisms. Worldwide, coastal hardening and urbanisation are leading to the removal of natural ecosystems. The tropical island of Bora-Bora in the South Pacific has undergone extensive coastal changes, with the construction of seawalls along more than half of its coastline since the 1950s. The daytime and night-time juvenile and adult fish communities were surveyed with multiple temporal replicates on a range of lagoon and coastal habitats on Bora-Bora. Over 47% of all fish on coastal habitats were juveniles. Mangroves, traditionally viewed as nurseries, had a high daytime and night-time abundance of juveniles, but less than 1% of the coastline of Bora-Bora consists of mangroves. The manmade seawalls, which are the most common type of coastal habitat on the island, were associated with lower juvenile densities during the day and promoted the presence of predators. The comparison of coastal and lagoon sites also highlighted contrasting life history strategies depending on coral reef fish species: although many favour coastal habitats as juveniles, others do not undergo ontogenetic shifts and thus other habitats must be considered when designing management plans to protect juvenile fish. Overall, our surveys show the importance of natural coastal zones in the lifecycle of numerous coral reef fish species in the lagoon of Bora-Bora and highlight the potential long-term impacts of coastal hardening on fish communities.

Abstract 2109: Analytical characterization of a multiplex panel measuring 45 cytokines by proximity extension assay in plasma from subjects with NSCLC

Abstract Introduction Cytokines are fundamental regulators of immune system processes and are responsible for the differentiation, proliferation, and function of immune cells. With many anti-cancer therapeutic strategies focused on targeting or harnessing the immune system, or components thereof, quantification of circulating cytokines has become important to describe the pharmacodynamic and mechanistic effects of these investigational therapies. To support these efforts, new multiplexed immunoassay panels have been developed to expand the available options for broad spectrum characterization of cytokine profiles from biospecimens. Here we characterize the analytical performance of one such panel based on proximity extension assay technology to measure 45 cytokines in plasma. Experimental Procedures Plasma samples (n = 27) from patients with non-small cell lung cancer (NSCLC), along with age-matched plasma samples (n = 6) from healthy normal subjects were analyzed using the Olink Cytokine 48 kit. Samples were thawed and processed according to manufacturer instructions for the incubation, extension, and detection on the Olink Signature Q100 instrument. A subset of the screened samples was run in quadruplicate across multiple runs and analyzed on two separate instruments. Results Of the 45 cytokines assayed, 41 had values above the assay limit of detection in ≥75% of samples analyzed. When compared to the normal human plasma, 26 cytokines were significantly elevated (p-value ≤ 0.05) in plasma from NSCLC subjects, including those known to be associated both with poor (e.g., IL-1β, IL-6) and good prognosis (e.g., IFN-γ). Furthermore, many of the cytokines with significant differences were of low abundance; 13 of the 26 cytokines had mean values ≤ 20.0 pg/mL. For two of the incurred samples which were run across multiple replicates and runs, with mid- to high- levels of cytokines, the median intra-assay CV across all assays was 6.2% with 90% of the assays demonstrating CVs of less than 12%. This only increased to a 7.7% median CV in inter-run comparison with 90% of the assays remaining below 12%. Additional results from the incurred sample analysis will be presented. Conclusions These results demonstrate that multiplex cytokine analysis, based on proximity extension assays, can be effectively deployed to screen a large number of targets and can uncover clinically relevant changes in low abundant cytokines. Citation Format: Anne Jang, Daniel Feingold, Mark H. Watson, Gwenaël Pottiez, Rudolf Guilbaud, Nicholas Francois Dupuis. Analytical characterization of a multiplex panel measuring 45 cytokines by proximity extension assay in plasma from subjects with NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2109.

Abstract 2690: In vivo CRISPR screen identifies SOAT1 as a chemotherapy chemosensitizing target for non-small cell lung cancer (NSCLC)

Abstract Purpose: To identify druggable gene targets that would sensitize NSCLCs to available chemotherapy agents. Experimental Procedures: We developed a CRISPR-Cas9 lentivirus library of 12,474 sgRNAs targeting 660 FDA proved ‘druggable’ putative proteins (18 guides/gene) and investigated their potential to chemosensitize (“drop out” in the presence of taxane) the NSCLC lung adenocarcinoma (LUAD) line NCI-H2009 (TP53 and KRAS mutant) in parallel studies in vitro (tissue culture) & in vivo (xenografts). Key elements of our experiments were: the use low doses of paclitaxel (IC10 values for in vitro and in vivo doses that barely affected tumor growth) compared to control treatment; treatment schedules in vitro and in vivo that mirrored those used in patients; multiple biologic replicates for each transfection and drug selection; and large representation (2,000 cells/sgRNA) for each guide. At the end time point (1 month) we harvested multiple replicates of the taxane and control treatments and sequenced each to identify each guide that “remained” or “dropped out.” Data Summary: We were looking for guides that selectively dropped out in the setting of low dose taxane exposure comparing taxane to control treatment and in vitro to in vivo results. Of importance, we found little overlap between guides that dropped out during taxane exposure in tissue culture vs those that dropped out in xenografts. From the top 10 gene “sensitizers” that selectively dropped out only with taxane treatment and selectively dropped out in xenografts compared to 2D mass tissue culture, we focused on SOAT1 (Sterol O-Acyltransferase 1). SOAT1 is a key enzyme for lipid metabolism which mediates conversion of intracellular free cholesterol to cholesteryl esters which are then stored as lipid droplets. SOAT1 is a potential cancer therapeutic target with a clinically available inhibitor, avasimibe. H2009 cells with SOAT1 hemizygously removed (CRISPR) grew well in vitro and in vivo in the absence of chemotherapy treatment, but were dramatically sensitized to taxanes compared to parental H2009 cells. While avasimibe treatment, at concentrations achievable in patients, sensitized NSCLC to taxanes, this sensitization was not as dramatic as hemizygous removal by CRISPR. Of equal importance, SOAT1 H2009 hemizygous knockout (KO) cells were also dramatically sensitized to etoposide, pemetrexed, and gemcitabine, other chemotherapy agents used in NSCLC treatment. Mechanistically, RNAseq analysis identified G2/M cell cycle related gene sets as enriched in the H2009 SOAT1 KO xenografts compared to SOAT1 wildtype xenografts with paclitaxel treatment. Conclusions: Our results using CRISPR screening in vivo, identified SOAT1 as a critical therapeutic chemosensitizing target requiring only 50% inhibition of activity for sensitizing NSCLC to several chemotherapy agents routinely used in the clinic. Citation Format: Long Shan Li, Kenneth Huffman, Huiyu Li, Michael Peyton, Hyunsil Park, Kimberley Avila, Luc Girard, Mathew Augustine, Joshua T. Mendell, John D. Minna. In vivo CRISPR screen identifies SOAT1 as a chemotherapy chemosensitizing target for non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2690.

Interdisciplinary approaches to predicting evolutionary biology.

Interdisciplinary approaches to predicting evolutionary biology.

Characterizing Mosquito Biting Behavior Using the BiteOscope.

The biteOscope enables the high-resolution monitoring and video recording of blood-feeding mosquitoes. Mosquito biting is induced by combining host cues, an artificial bloodmeal, a membrane, and a transparent heater in a transparent behavioral arena. Machine vision techniques enable the tracking and pose estimation of individual mosquitoes to discern behavior and resolve individual feeding events. The workflow allows multiple replicates and large amounts of imaging data to be generated rapidly. These data are suitable for downstream analysis using machine learning tools for behavioral analysis, allowing subtle behavioral effects to be characterized.

Application of Mixed-Solvent Molecular Dynamics Simulations for Prediction of Allosteric Sites on G Protein-Coupled Receptors.

The development of small molecule allosteric modulators acting at G protein-coupled receptors (GPCRs) is becoming increasingly attractive. Such compounds have advantages over traditional drugs acting at orthosteric sites on these receptors, in particular target specificity. However, the number and locations of druggable allosteric sites within most clinically relevant GPCRs are unknown. In the present study, we describe the development and application of a mixed-solvent molecular dynamics (MixMD)-based method for the identification of allosteric sites on GPCRs. The method employs small organic probes with druglike qualities to identify druggable hotspots in multiple replicate short-timescale simulations. As proof of principle, we first applied the method retrospectively to a test set of five GPCRs (cannabinoid receptor type 1, C-C chemokine receptor type 2, M2 muscarinic receptor, P2Y purinoceptor 1, and protease-activated receptor 2) with known allosteric sites in diverse locations. This resulted in the identification of the known allosteric sites on these receptors. We then applied the method to the μ-opioid receptor. Several allosteric modulators for this receptor are known, although the binding sites for these modulators are not known. The MixMD-based method revealed several potential allosteric sites on the mu-opioid receptor. Implementation of the MixMD-based method should aid future efforts in the structure-based drug design of drugs targeting allosteric sites on GPCRs. SIGNIFICANCE STATEMENT: Allosteric modulation of G protein-coupled receptors (GPCRs) has the potential to provide more selective drugs. However, there are limited structures of GPCRs bound to allosteric modulators, and obtaining such structures is problematic. Current computational methods utilize static structures and therefore may not identify hidden or cryptic sites. Here we describe the use of small organic probes and molecular dynamics to identify druggable allosteric hotspots on GPCRs. The results reinforce the importance of protein dynamics in allosteric site identification.

The Lysin Exebacase Has a Low Propensity for Resistance Development in Staphylococcus aureus and Suppresses the Emergence of Resistance to Antistaphylococcal Antibiotics.

Exebacase (CF-301) belongs to a novel class of protein-based antibacterial agents, called lysins (peptidoglycan hydrolases). Exebacase exhibits potent antistaphylococcal activity and is the first lysin to initiate clinical trials in the United States. To support clinical development, the potential for resistance development to exebacase was assessed over 28 days of serial daily subculture in the presence of increasing concentrations of the lysin performed in its reference broth medium. Exebacase MICs remained unchanged over serial subculture for three replicates each of methicillin-susceptible Staphylococcus aureus (MSSA) strain ATCC 29213 and methicillin-resistant S. aureus (MRSA) strain MW2. For comparator antibiotics also tested, oxacillin MICs increased by 32-fold with ATCC 29213 and daptomycin and vancomycin MICs increased by 16- and 8-fold, respectively, with MW2. Serial passage was also used to examine the capacity of exebacase to suppress selection for increased oxacillin, daptomycin, and vancomycin MICs when used together in combination, wherein daily exposures to increasing concentrations of antibiotic were performed over 28 days with the added presence of fixed sub-MIC amounts of exebacase. Exebacase suppressed increases in antibiotic MICs over this period. These findings are consistent with a low propensity for resistance to exebacase and an added benefit of reducing the potential for development of antibiotic resistance. IMPORTANCE To guide development of an investigational new antibacterial drug, microbiological data are required to understand the potential for development of resistance to the drug in the target organism(s). Exebacase is a lysin (peptidoglycan hydrolase) that represents a novel antimicrobial modality based on degradation of the cell wall of Staphylococcus aureus. Exebacase resistance was examined here using an in vitro serial passage method that assesses the impact of daily exposures to increasing concentrations of exebacase over 28 days in medium approved for use in exebacase antimicrobial susceptibility testing (AST) by the Clinical and Laboratory Standards Institute (CLSI). No changes in susceptibility to exebacase were observed over the 28-day period for multiple replicates of two S. aureus strains, indicating a low propensity for resistance development. Interestingly, while high-level resistance to commonly used antistaphylococcal antibiotics was readily obtained using the same method, the added presence of exebacase acted to suppress antibiotic resistance development.

Variation with In Vitro Analysis of Volatile Profiles among Aspergillus flavus Strains from Louisiana

Volatile organic compounds (VOCs) produced by A. flavus strains were first captured and identified to discern between non-aflatoxigenic and toxigenic phenotypes, and more recently to help with detecting fungal infection, but not with the goal of using VOCs produced by non-aflatoxigenic strains to inhibit growth and/or production of one or more mycotoxins (e.g., aflatoxin and cyclopiazonic acid) by toxigenic aspergilli. In this study, four Aspergillus strains from Louisiana (one non-aflatoxigenic and three toxigenic) were grown on various substrates and had their headspaces captured and analyzed by solid-phase microextraction/gas chromatography/mass spectroscopy (SPME/GC/MS), to find biocontrol and biomarker compounds. Here, we present a collection of nearly 100 fungus-related VOCs, many of which were substrate dependent. Thirty-one were produced across multiple replicates and the rest were observed in a single replicate. At least three VOCs unique to non-aflatoxigenic strain LA1 can be tested for biocontrol properties (e.g., euparone, 4-nonyne), and at least four VOCs unique to toxigenic strains LA2-LA4 can be explored as biomarkers (e.g., 2-heptanone, glycocyamidine) to detect their presence while infecting crops in the field or in storage.

De Novo SNP Discovery and Genotyping of Masson Pine (Pinus massoniana Lamb.) via Genotyping-by-Sequencing

Masson pine (Pinus massoniana Lamb.) is an important tree species in China, but its genomic research has been hindered due to a large genome size. Genotyping-by-sequencing (GBS) has been a powerful approach to revolutionize the field of genomic research by facilitating the discovery of thousands of single nucleotide polymorphisms (SNPs) and genotyping in non-model organisms, at relatively low cost. Here, we performed de novo SNP discovery and genotyping in 299 trees via the genotyping-by-sequencing (GBS) approach. The effort produced 9.33 × 109 sequence reads, 265,525 SNP-associated contigs, and 6,739,240 raw SNPs. Further filtering and validation of the SNP-associated contigs for reliable SNPs were performed using blasting against the Pinus tabuliformis reference genome, functional annotation, technical replicates, and custom parameter settings for the optimization. The 159,372 SNP-associated contigs were aligned and validated for SNP prediction, in which 60,038 contigs were searched with hits in the NCBI nr database. We further improved the SNP discovery and genotyping with multiple technical replicates and custom parameter settings filtering. It was found that the use of blasting, annotation, technical replicates, and specific parameter settings removed many unreliable SNPs and identified 20,055 more precise and reliable SNPs from the 10,712 filtered contigs. We further demonstrated the informativeness of the identified SNPs in the inference of some genetic diversity and structure. These findings should be useful to stimulate genomic research and genomics-assisted breeding of Masson pine.

Shear stress shifts clathrin-coated vesicle formation to favor the flat-to-curved model in endothelial cells.

Endothelial cells (ECs) experience a variety of highly dynamic mechanical stresses. Among others, cyclic stretch and increased plasma membrane tension inhibit clathrin-mediated endocytosis (CME) in non-ECs cells. How ECs overcome such unfavorable conditions and maintain CME remains elusive. Previously, we have used simultaneous two-wavelength axial ratiometry (STAR) microscopy to resolve how clathrin-coated vesicles (CCVs) from in fibroblast-like cells (Cos-7) and human umbilical vein endothelial cells (HUVECs). This revealed multiple productive ways of CCV formation, supporting the flexible model of CME. We next asked whether biophysical stresses generated by blood flow could favor one mechanism of CCV formation to overcome unfavorable environment present in vasculature. Addressing this question required development of an automated MATLAB-based accelerated STAR data processing pipeline (DrSTAR) to enable the processing of multiple experimental conditions and biological replicates in a robust and reproducible environment. Moreover, DrSTAR employs a dynamic local referencing algorithm, which resolves the curvature of long-lasting CCSs. We used this new platform to examine if clathrin dynamics are altered in HUVECs grown under sheer stress. We found sheer stress led to an increase in clathrin dynamics. Surprisingly, we found this was due to an increase of flat clathrin accumulations in flow-stimulated cells, while the number of curved events remained consistent between groups. The curvature-positive events had significantly delayed curvature initiation in flow-stimulated cells, highlighting a shift toward flat-to-curved clathrin transitions in vesicle formation. Moreover, CME was more resistant to increased osmotic pressure in HUVECs grown under shear stress. Overall, our findings indicate that clathrin dynamics and CCV formation can be modulated by the local environment and represents an important regulatory mechanism.

ScHi-CSim: a flexible simulator that generates high-fidelity single-cell Hi-C data for benchmarking.

Single-cell Hi-C technology provides an unprecedented opportunity to reveal chromatin structure in individual cells. However, high sequencing cost impedes the generation of biological Hi-C data with high sequencing depths and multiple replicates for downstream analysis. Here, we developed a single-cell Hi-C simulator (scHi-CSim) that generates high-fidelity data for benchmarking. scHi-CSim merges neighboring cells to overcome the sparseness of data, samples interactions in distance-stratified chromosomes to maintain the heterogeneity of single cells, and estimates the empirical distribution of restriction fragments to generate simulated data. We demonstrated that scHi-CSim can generate high-fidelity data by comparing the performance of single-cell clustering and detection of chromosomal high-order structures with raw data. Furthermore, scHi-CSim is flexible to change sequencing depth and the number of simulated replicates. We showed that increasing sequencing depth could improve the accuracy of detecting topologically associating domains. We also used scHi-CSim to generate a series of simulated datasets with different sequencing depths to benchmark scHi-C clustering methods.

A comparison of four sampling techniques for assessing species richness of adult odonates at riverbanks

Members of the insect order Odonata are known as good ecological indicators. Many are sensitive to habitat modifications and are easily monitored for use in environmental assessment studies. Rapid assessments rely on efficient sampling techniques. However, there is limited information available on sampling techniques for adult odonates, and protocols require evaluation. To do this, we standardized counting methods during sampling of odonates from August to November 2016 at the Mula River, Pune, India. We used four counting techniques; full-width belt transect (FWBT), full-circle point count (FCPC), half-width belt transect (HWBT), and half-circle point count (HCPC). For HWBT and HCPC areas facing the river were sampled, and for each technique we took multiple temporal replicates. We compared species detected per unit time, species detected per unit area, new species detected per unit time, and new species detected per unit area. Additionally, we compared species estimates. With HCPC we detected the maximum number of species and new species per unit area, whereas FWBT returned maximum coverage of recorded species. We recommend our proposed techniques be considered in the future across various habitats to decide the most suitable sampling strategy for the different habitats or situations.

DrSTAR: Tracking real-time nanometer axial changes

Protein interactions with the plasma membrane mediate processes critical for cell viability such as migration and endocytosis, yet our understanding of how recruitment of key proteins correlates with their ability to sense or induce energetically unfavorable plasma membrane shapes remains limited. Simultaneous two-wavelength axial ratiometry (STAR) microscopy provides millisecond time resolution and nanometer axial resolution of protein dynamics at the basal plasma membrane. However, STAR microscopy requires extensive and time-consuming quantitative data processing to access axial (Δz) information. Therefore, addressing questions about the influence of biological and biophysical factors on the interaction between the plasma membrane and protein of interest remains challenging. Here, we overcome the limitations in STAR data processing and present dynamic reference STAR (DrSTAR): a user-friendly, automated, open-source MATLAB-based package. DrSTAR enables processing multiple experimental conditions and biological replicates, employs a novel local background referencing algorithm, and accelerates processing time to facilitate broad adaptation of STAR for studying nanometer axial changes in protein distribution.

Direct wastewater extraction as a simple and effective method for SARS-CoV-2 surveillance and COVID-19 community-level monitoring.

Wastewater surveillance has proven to be an effective tool to monitor the transmission and emergence of infectious agents at a community scale. Workflows for wastewater surveillance generally rely on concentration steps to increase the probability of detection of low-abundance targets, but preconcentration can substantially increase the time and cost of analyses while also introducing additional loss of target during processing. To address some of these issues, we conducted a longitudinal study implementing a simplified workflow for SARS-CoV-2 detection from wastewater, using a direct column-based extraction approach. Composite influent wastewater samples were collected weekly for 1 year between June 2020 and June 2021 in Athens-Clarke County, Georgia, USA. Bypassing any concentration step, low volumes (280µl) of influent wastewater were extracted using a commercial kit, and immediately analyzed by RT-qPCR for the SARS-CoV-2 N1 and N2 gene targets. SARS-CoV-2 viral RNA was detected in 76% (193/254) of influent samples, and the recovery of the surrogate bovine coronavirus was 42% (IQR: 28%, 59%). N1 and N2 assay positivity, viral concentration, and flow-adjusted daily viral load correlated significantly with per-capita case reports of COVID-19 at the county-level (ρ = 0.69-0.82). To compensate for the method's high limit of detection (approximately 106-107 copies l-1 in wastewater), we extracted multiple small-volume replicates of each wastewater sample. With this approach, we detected as few as five cases of COVID-19 per 100000 individuals. These results indicate that a direct-extraction-based workflow for SARS-CoV-2 wastewater surveillance can provide informative and actionable results.

Highly sensitive detection of okadaic acid in seawater by magnetic solid-phase extraction based on low-cost metal/nitrogen-doped carbon nanotubes

Algae toxins pose a severe threat to human health all over the world. In this study, magnetic metal/nitrogen-doped carbon nanotubes (M-NCNTs) were facilely synthesized based on one-step carbonization and applied for magnetic solid-phase extraction of okadaic acid (OA) from seawater followed by high performance liquid chromatographic tandem mass spectrometry (HPLC-MS/MS) analyses. Differences in the physicochemical properties of the three prepared materials (Fe/Co/Ni-NCNTs) were investigated to confirm the best extraction material. Among them, Ni-NCNTs demonstrated a faster extraction rate (10 min) and higher adsorption capacity (223.5 mg g−1), mainly due to the higher specific surface area, suitable pore structure and more abundant pyridine nitrogen ring. Under the optimal conditions, the calibration curve was linear over the range (1.0–800.0 pg mL−1) with good determination coefficients (R) of 0.9992. The limit of detection (LOD) obtained in multiple replicates was 0.4 pg mL−1. Three seawater samples were measured by the developed method, 12.3 pg mL−1 of OA was detected with a satisfying recovery (88.6%-106.7%) and acceptable repeatability (RSD ≤ 4.8%, n = 6). The results demonstrate that M-NCNTs materials are a promising candidate for magnetic solid-phase extraction. Benefiting from its high extraction and interference resistance, the established analytical method is expected to be extended to detect other marine environmental pollutions.

Uncertainty in Composite Membranes: From Defect Engineering to Film Processing.

Composite membranes featuring metal-organic framework (MOF)-dispersed polymers have attracted tremendous attention in recent years. However, evaluating commercial viability is oftentimes obscured by the irreproducibility in both MOF synthesis and film manufacturing protocols. Variability in MOF property sets are typically ascribed to crystal defects resulting from subtle variations in synthesis, but quantitative studies investigating the role of defects on transport properties are exceedingly rare. Likewise, controlled film formation protocols are rarely reported in the open literature, making it difficult to provide substantial and informative structure-property correlations. This study aims to address these uncertainties. To this end, two samples of a prototypical MOF, UiO-66-NH2, were synthesized to feature similar particle size, morphology, and colloidal stability. However, defect engineering protocols coupled with careful screening experiments were developed to synthesize the two MOFs with maximally different porosities. Composite membranes were prepared for each MOF and a high-performance polymer, 6FDA-Durene, and then tested for light gas permeation measurements, revealing a small and unexpected enhancement in CO2/CH4 performance for samples containing low-porosity UiO-66-NH2. Mechanistic studies on sorption revealed a surprising 50% decrease in sorption capacity for high-porosity UiO-66-NH2, completely offsetting enhancements from increased gas diffusion. By using multiple replicate experiments, the sample-to-sample variation was large enough to obscure any differences in permeability and selectivity between the two types of MOF composites at low volume fractions. Application of the Maxwell model to extrapolate pure-MOF performance led to significant variations in predicted values, demonstrating the importance of collecting and reporting replicate experiments for membrane preparation and testing.

Impact of Internal Fragments on Top-Down Analysis of Intact Proteins by 193 nm UVPD

193 nm ultraviolet photodissociation (UVPD) allows high sequence coverage to be obtained for intact proteins using terminal fragments alone. However, internal fragments, those that contain neither N- nor C- terminus, are typically ignored, neglecting their potential to bolster characterization of intact proteins. Here, we explore internal fragments generated by 193 nm UVPD for proteins ranging in size from 17-47 kDa and using the ClipsMS algorithm to facilitate searches for internal fragments. Internal fragments were only retained if identified in multiple replicates in order to reduce spurious assignments and to explore the reproducibility of internal fragments generated by UVPD. Inclusion of internal fragment improved sequence coverage by an average of 18% and 32% for UVPD and HCD, respectively, across all proteins and charge states studied. However, only an average of 18% of UVPD internal fragments were identified in two out of three replicates relative to the average number identified across all replicates for all proteins studied. Conversely, for HCD, an average of 63% of internal fragments were retained across replicates. These trends reflect an increased risk of false-positive identifications and a need for caution when considering internal fragments for UVPD. Additionally, proton-transfer charge reduction (PTCR) reactions were performed following UVPD or HCD to assess the impact on internal fragment identifications, allowing up to 20% more fragment ions to be retained across multiple replicates. At this time, it is difficult to recommend the inclusion of the internal fragment when searching UVPD spectra without further work to develop strategies for reducing the possibilities of false-positive identifications. All mass spectra are available in the public repository jPOST with the accession number JPST001885.

Geographic variability of hybridization between red-breasted and red-naped sapsuckers.

Hybrid zones reveal the strength of reproductive isolation between populations undergoing speciation and are a key tool in evolutionary biology research. Multiple replicate transects across the same hybrid zone offer insight into the dynamics of hybridization in different environments, clarifying the role of extrinsic forces on the speciation process. Red-breasted and red-naped sapsuckers (Sphyrapicus ruber and Sphyrapicus nuchalis) have a long zone of contact over approximately 1,600 km from central British Columbia, Canada to central California, USA. We used Genotyping-by-Sequencing data from three independent sapsucker hybrid zone transects to compare hybridization dynamics between these species under variable geoclimatic conditions. We generated geographic clines of the genomic data to compare hybrid zone widths and used random forests models and linear regression to assess the relationship between climate and sapsucker ancestry along each transect. Our results show variation in the directionality of backcrossing, often indicative of moving hybrid zones. We note variable cline widths among transects, indicating differences in selection maintaining hybrid zone dynamics. Furthermore, random forests models identified different variables in close association with sapsucker ancestry across each transect. These results indicate a lack of repeatability across replicate transects and a strong influence of the local environment on hybrid zone dynamics.

ScRNA-seq for Microcephaly Research [IV]: Dirichlet Regression for Single-Cell Population Differences.

Analysis of single-cell RNA sequencing typically includes the clustering of cells and subsequent determination of the population size of each cluster, relative to the whole. In an experimental setting, two or more conditions are compared to assess changes in cellular composition of the sampled tissue. Cluster populations are frequently normalized to the total number of cells from each replicate in order to facilitate comparisons. After normalization, they become interdependent fractions and therefore cannot be compared using individual t-tests. Here we describe the use of Dirichlet regression to compare changes in cellular composition between two or more conditions when multiple biological replicates (three or more) are sampled under each condition. We provide an example of R code to conduct a similar analysis and interpret the results.