Biology subjects

Pinto, A. J.

Publications and source records attributed to Pinto, A. J..

26 records · Page 2Linked to original sources

bioRxiv2021

Characterization of new recombinant Immunoglobulins type-M binding to C1q by Biolayer Interferometry

The Immunoglobulins type-M (IgMs) are one of the first antibody classes mobilized during immune responses against pathogens and tumor cells. Binding to specific target antigens enables the interaction with the C1 complex which strongly activates the classical complement pathway. This biological function is the basis for the huge therapeutic potential of IgMs but due to their high oligomeric complexity, in vitro production, biochemical and biophysical characterizations are challenging. In the present study, we present recombinant production of two IgM models (IgM617 and IgM012) in pentameric and hexameric states and the evaluation of their polymer distribution using different biophysical methods (AUC, SEC-MALLS, Mass Photometry and Transmission Electron Microscopy). Each IgM oligomer has individual specific expression pattern and yield with different protein quality likely due to intrinsic IgM properties and patterning. Nevertheless, the purified recombinant IgMs retain their ability to activate complement in a C1q dependent manner. And more importantly, a new method to evaluate their functional quality attribute by characterizing the kinetics of C1q binding to recombinant IgM has been developed using BioLayer Interferometry (BLI). We show that recombinant IgMs possess similar C1q binding properties as IgMs purified from human plasma.

bioengineering↗

bioRxiv2021

Evaluating de novo assembly and binning strategies for time-series drinking water metagenomes.

Reconstructing microbial genomes from metagenomic short-read data can be challenging due to the unknown and uneven complexity of microbial communities. This complexity encompasses highly diverse populations which often includes strain variants. Reconstructing high-quality genomes is a crucial part of the metagenomic workflow as subsequent ecological and metabolic inferences depend on their accuracy, quality, and completeness. In contrast to microbial communities in other ecosystems, there has been no systematic assessment of genome-centric metagenomic workflows for drinking water microbiomes. In this study, we assessed the performance of a combination of assembly and binning strategies for time-series drinking water metagenomes that were collected over a period of 6 months. The goal of this study was to identify the combination of assembly and binning approaches that results in high quality and quantity metagenome-assembled genomes (MAGs), representing most of the sequenced metagenome. Our findings suggest that the metaSPAdes co-assembly strategies had the best performance as they resulted in larger and less fragmented assemblies with at least 85% of the sequence data mapping to contigs greater than 1kbp. Furthermore, a combination of metaSPAdes co-assembly strategies and MetaBAT2 produced the highest number of medium-quality MAGs while capturing at least 70% of the metagenomes based on read recruitment. Utilizing different assembly/binning approaches also assist in the reconstruction of unique MAGs from closely related species that would have otherwise collapsed into a single MAG using a single workflow. Overall, our study suggests that leveraging multiple binning approaches with different metaSPAdes co-assembly strategies may be required to maximize the recovery of good-quality MAGs, which more accurately capture the microbial diversity of drinking water samples.

genomics↗

bioRxiv2021

Spatial-temporal targeted and non-targeted surveys to assess microbiological composition of drinking water in Puerto Rico following Hurricane Maria.

Loss of basic utilities, such as drinking water and electricity distribution, were sustained for months in the aftermath of Hurricane Marias (HM) landfall in Puerto Rico (PR) in September 2017. The goal of this study was to assess if there was deterioration in biological quality of drinking water due to these disruptions. This study characterized the microbial composition of drinking water following HM across nine drinking water systems (DWSs) in PR and utilized an extended temporal sampling campaign to determine if changes in the drinking water microbiome were indicative of HM associated disturbance followed by recovery. In addition to monitoring water chemistry, the samples were subjected to culture independent targeted and non-targeted microbial analysis including quantitative PCR (qPCR) and genome-resolved metagenomics. The qPCR results showed that residual disinfectant was the major driver of bacterial concentrations in tap water with marked decrease in concentrations from early to late sampling timepoints. While Mycobacterium avium and Pseudomonas aeruginosa were not detected in any sampling locations and timepoints, genetic material from Leptospira and Legionella pneumophila were transiently detected in a few sampling locations. The majority of metagenome assembled genomes (MAGs) recovered from these samples were not associated with pathogens and were consistent with bacterial community members routinely detected in DWSs. Further, whole metagenome-level comparisons between drinking water samples collected in this study with samples from other full-scale DWS indicated no significant deviation from expected community membership of the drinking water microbiome. Overall, our results suggest that disruptions due to HM did not result in significant and sustained deterioration of biological quality of drinking water at our study sites. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=86 SRC="FIGDIR/small/442998v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@100c1e9org.highwire.dtl.DTLVardef@bae402org.highwire.dtl.DTLVardef@1cf41bborg.highwire.dtl.DTLVardef@1e46105_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2019

Disinfection exhibits systematic impacts on the drinking water microbiome.

Limiting microbial growth during drinking water distribution is achieved either by maintaining a disinfectant residual or through nutrient limitation without the use of a disinfectant. The impact of these contrasting approaches on the drinking water microbiome is not systematically understood. We utilized genome-resolved metagenomics to compare the structure, metabolic traits, and population genomes of drinking water microbiomes across multiple full-scale drinking water systems utilizing these two-distinct microbial growth control strategies. Microbial communities cluster together at the structural- and functional potential-level based on the presence or absence of a disinfectant residual. Disinfectant residual concentrations alone explained 17 and 6.5% of the variance in structure and functional potential of the drinking water microbiome, respectively, despite including samples from multiple drinking water systems with variable source waters and source water communities, treatment strategies, and chemical compositions. The drinking water microbiome is structurally and functionally less diverse and less variable across disinfected systems as compared to non-disinfected systems. While bacteria were the most abundant domain, archaea and eukaryota were more abundant in non-disinfected and disinfected systems, respectively. Community-level differences in functional potential were driven by enrichment of genes associated with carbon and nitrogen fixation in non-disinfected systems and {gamma}-aminobutyrate metabolism in disinfected systems which may be associated with the recycling of amino acids. Metagenome-assembled genome-level analyses for a subset of phylogenetically related microorganisms suggests that disinfection may select for microorganisms capable of using fatty acids, presumably from microbial decay products, via the glyoxylate cycle. Overall, we find that disinfection exhibits systematic and consistent selective pressures on the drinking water microbiome and may select for microorganisms able to utilize microbial decay products originating from disinfection inactivated microorganisms.

microbiology↗

bioRxiv2019

Reproducible microbial community dynamics of two drinking water systems treating similar source waters.

Understanding whether the spatial-temporal dynamics of the drinking water microbiome are reproducible in full-scale drinking water systems is an important step towards devising engineering strategies to manipulate it. Yet, direct comparisons across full-scale drinking water systems are challenging because multiple factors, from source water to treatment process choice and configuration, can be unique to each system. This study compared the spatial-temporal dynamics of the drinking water microbiome in two drinking water treatment plants (DWTPs) with identical sequence of treatment strategies treating source waters from the same river system and with treated drinking water distributed in same large-scale (but independent) distribution system (DWDS) with similar disinfectant residual regiment. Dissimilarities in source water communities were tempered by the pre-disinfection treatments, resulting in highly similar post-filtration microbial communities between the two systems. However, high community turnover due to disinfection resulted in highly dissimilar microbial communities in the finished water between the two systems. Interestingly however, the microbial communities in the two systems increased in similarity during transit through the DWDS despite presence of a disinfectant residual. Overall our study finds that the drinking water microbiome demonstrated reproducible spatial and temporal dynamics within both independent but nearly identical DWTPs and their corresponding DWDSs. Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

microbiology↗

bioRxiv2019

Microbial Nitrogen Metabolism in Chloraminated Drinking Water Reservoirs

Nitrification is a common concern in chloraminated drinking water distribution systems. The addition of ammonia promotes the growth of nitrifying organisms, causing the depletion of chloramine residuals and resulting in operational problems for many drinking water utilities. Therefore, a comprehensive understanding of the microbially mediated processes behind nitrogen metabolism together with chemical water quality data, may allow water utilities to better address the undesirable effects caused by nitrification. In this study, a metagenomic approach was applied to characterise the microbial nitrogen metabolism within chloraminated drinking water reservoirs. Samples from two geographically separated but connected chloraminated reservoirs within the same drinking water distribution system (DWDS) were collected within a 2-year sampling campaign. Spatial changes in the nitrogen compounds (ammonium (NH4+), nitrites (NO2-) and nitrates (NO3-)) across the DWDS were observed, where nitrate concentrations increased as the distance from the site of chloramination increased. The observed dominance of Nitrosomonas and Nitrospira-like bacteria, together with the changes in the concentration of nitrogen species, suggests that these bacteria play a significant role in contributing to varying stages of nitrification in both reservoirs. Functionally annotated protein sequences were mined for the genes associated with nitrogen metabolism and the community gene catalogue contained mostly genes involved in nitrification, nitrate and nitrite reduction and nitric oxide reduction. Furthermore, based on the construction of Metagenome Assembled Genomes (MAGs), a highly diverse assemblage of bacteria (i.e., predominately Alpha- and Betaproteobacteria in this study) was observed among the draft genomes. Specifically, 5 MAGs showed high coverage across all samples including two Nitrosomonas, Nitrospira, Sphingomonas and a Rhizobiales-like MAGs. The role of these MAGs in nitrogen metabolism revealed that the fate nitrate may be linked to changes in ammonia concentrations, that is, when ammonia concentrations are low, nitrate may be assimilated back to ammonia for growth. Alternatively, nitrate may be reduced to nitric oxide and potentially used in the regulation of biofilm formation. Therefore, this study provides insight into the genetic network behind microbially mediated nitrogen metabolism and together with the water chemistry data improves our understanding nitrification in chloraminated DWDSs.

microbiology↗

bioRxiv2019

Oligotyping and Genome-Resolved Metagenomics Reveal Distinct Candidatus Accumulibacter Communities in Full-Scale Side-Stream versus Conventional Enhanced Biological Phosphorus Removal (EBPR) Configurations

Candidatus Accumulibacter phosphatis (CAP) and its sub-clades-level diversity has been associated and implicated in successful phosphorus removal performance in enhanced biological phosphorus removal (EBPR). Development of high-throughput untargeted methods to characterize clades of CAP in EBPR communities can enable a better understanding of Accumulibacter ecology at a higher-resolution beyond OTU-level in wastewater resource recovery facilities (WRRFs). In this study, for the first time, using integrated 16S rRNA gene sequencing, oligotyping and genome-resolved metagenomics, we were able to reveal clade-level differences in Accumulibacter communities and associate the differences with two different full-scale EBPR configurations. The results led to the identification and characterization of a distinct and dominant Accumulibacter oligotype - Oligotype 2 (belonging to Clade IIC) and its matching MAG (RC14) associated with side-stream EBPR configuration. We are also able to extract MAGs belonging to CAP clades IIB (RCAB4-2) and II (RC18) which did not have representative genomes before. This study demonstrates and validates the use of a high-throughput approach of oligotyping analysis of 16S rRNA gene sequences to elucidate CAP clade-level diversity. We also show the existence of a previously uncharacterized diversity of CAP clades in full-scale EBPR communities through extraction of MAGs, for the first time from full-scale facilities.

microbiology↗

bioRxiv2018

NanoAmpli-Seq: A workflow for amplicon sequencing from mixed microbial communities on the nanopore sequencing platform

BackgroundAmplicon sequencing on Illumina sequencing platforms leverages their deep sequencing and multiplexing capacity, but is limited in genetic resolution due to short read lengths. While Oxford Nanopore or Pacific Biosciences platforms overcome this limitation, their application has been limited due to higher error rates or smaller data output.\n\nResultsIn this study, we introduce an amplicon sequencing workflow, i.e., NanoAmpli-Seq, that builds on Intramolecular-ligated Nanopore Consensus Sequencing (INC-Seq) approach and demonstrate its application for full-length 16S rRNA gene sequencing. NanoAmpli-Seq includes vital improvements to the aforementioned protocol that reduces sample-processing time while significantly improving sequence accuracy. The developed protocol includes chopSeq software for fragmentation and read orientation correction of INC-Seq consensus reads while nanoClust algorithm was designed for read partitioning-based de novo clustering and within cluster consensus calling to obtain full-length 16S rRNA gene sequences.\n\nConclusionsNanoAmpli-Seq accurately estimates the diversity of tested mock communities with average sequence accuracy of 99.5% for 2D and 1D2 sequencing on the nanopore sequencing platform. Nearly all residual errors in NanoAmpli-Seq sequences originate from deletions in homopolymer regions, indicating that homopolymer aware basecalling or error correction may allow for sequencing accuracy comparable to short-read sequencing platforms.

microbiology↗