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Biotechnology has the potential to deliver solutions to many global problems in medicine, materials science, nutrition, agriculture, natural resource preservation, and energy. Engineered cells and enzymes can perform chemical transformations that are rare or unknown in nature, and even catalyze reactions not accessible to traditional synthetic chemistry while also operating at gentler, more environmentally friendly conditions. Decreases in the price of DNA sequencing and synthesis has led to generation of vast databases that can be screened for any imaginable function. These sequence databases are even more powerful now due to the development of software to enable rapid generation of 3D protein structures, like AlphaFold2. However, tools to predict the function of these proteins or their performance in engineered cells are not yet robust, leading to long development times and limited successful applications to date. New tools and methods must be developed for the true potential of biotechnology to be unlocked.For my thesis, I explore metabolic pathway construction and screening, protein design, and protein sequence-structure-function relationships across broad contexts with the objective of tool and knowledge development for future efforts in biotechnology. My first chapter discusses the introduction of the triazine degradation pathway, of interest for remediation of contaminated sites, into E. coli and methods for characterizing pathway flux and identifying bottlenecks to guide engineering efforts and limit accumulation of metabolic intermediates. My second chapter focuses on methods for the design of supercharged proteins, which have many interesting potential applications, and parameters that increase the likelihood of successful design of these proteins. My third chapter regards the generation and characterization of a library of single point mutations in the enzyme B-glucosidase B and use of kinetic data to predict the effects of changes in sequence on enzyme function. These seemingly disparate topics all serve to improve tools for protein screening, production, functional prediction, and application, addressing several gaps toward improved development timelines and success rates for biocatalysts.

Cancer stem cells (CSCs) are responsible for cancer initiation, drug resistance, and aggressive tumor phenotypes. Our lab has established a novel method to induce CSCs from induced pluripotent stem (iPS) cells in a microenvironment mimicking chronic inflammation. The converted cells acquired CSC characteristics and developed malignant tumors. Recently, we demonstrated that nonmutagenic chemical inhibitors accelerated the conversion of mouse iPS (miPS) cells into CSCs. Here, we investigated the effects of AZD-6244, a MEK1/2-specific inhibitor, on the conversion of iPS cells into CSCs. The miPS cells were cultured for one week in the presence of the conditioned medium (CM) of Lewis lung carcinoma (LLC) cells and AZD-6244, PD0325901, a pan-MEK inhibitor, or GDC-0879, a B-Raf inhibitor. As a result, AZD-6244 enhanced the conversion of iPS cells into CSCs and upregulated AKT phosphorylation as same as GDC-0879 and PD0325901. The converted cells maintained their self-renewal ability and stemness gene expression. The expression of the CSC markers CD24, CD44 and CD133 was higher in the cells cultured with MAPK inhibitors than in those cultured without MAPK inhibitors. Moreover, converted cells gained migration and invasion abilities assessed by in vitro assays. Therefore, the inhibition of MEK1/2 was found to be critical for the conversion of normal stem cells into CSCs in the tumor-inducing microenvironment.

High-throughput mapping of latent heat flux (λET) is critical to efforts to optimize water resources management and to accelerate forest tree breeding for improved drought tolerance. Ideally, investigation of the energy response at the tree level may promote tailored irrigation strategies and, thus, maximize crop biomass productivity. However, data availability is limited and planning experimental campaigns in the field can be highly operationally complex. To this end, a multi-platform multi-sensor observational approach is herein developed to dissect the λET signature of a black poplar (Populus nigra) breeding population (“POP6”) at the canopy level. POP6 comprised more than 4600 trees representing 503 replicated genotypes, whose parents were derived from contrasting environmental conditions. Trees were trialed in two adjacent plots where different irrigation treatments (moderate drought [mDr] and well-watered [WW]) were applied. Data collected from satellite and unmanned aerial vehicles (UAVs) remote sensing as well as from ground-based proximal sensors were integrated at consistent spatial aggregation and combined to compute the surface energy balance of the trees through a modified Priestley-Taylor method. Here, we demonstrated that λET response was significantly different between WW and mDr trees, whereby genotypes in mDr conditions exhibited larger standard deviations. Importantly, genotypes classified as drought tolerant based on the stress susceptibility index (SSI) presented λET values significantly higher than the rest of the population. This study confirmed that water limitation in mDr settings led to reduced soil moisture in the tree root zone and, thus, to lower λET. These results pave the way to breeding poplar and other bioenergy crops with this underexploited trait for higher λET. Most notably, the illustrated work demonstrates a multi-platform multi-sensor data fusion approach to tackle the global challenge of monitoring landscape-scale ecosystem processes at fine resolution.

Several species of Cichla successfully colonized lakes and reservoirs of Brazil, since the 1960’s, causing serious damage to local wildlife. In this study, 135 peacock bass were collected in a reservoir complex in order to identify if they represented a single dominant species or multiple ones, as several Cichla species have been reported in the basin. Specimens were identified by color pattern, morphometric and meristic data, and using mitochondrial markers COI, 16S rDNA and Control Region (CR). Overlapping morphological data and similar coloration patterns prevented their identification using the taxonomic keys to species identification available in the literature. However, Bayesian and maximum likelihood from sequencing data demonstrated the occurrence of a single species, Cichla kelberi. A single haplotype was observed for the 16S and CR, while three were detected for COI, with a dominant haplotype present in 98.5% of the samples. The extreme low diversity of the transplanted C. kelberi evidenced a limited number of founding maternal lineages. The success of this colonization seems to rely mainly on abiotic factors, such as increased water transparency of lentic environments that favor visual predators that along with the absence of predators, have made C. kelberi a successful invader of these reservoirs. Resumo Muitas espécies de Cichla colonizaram com sucesso lagos e reservatórios do Brasil desde os anos 1960, causando graves prejuízos à vida selvagem nesses locais. Neste estudo, 135 tucunarés foram coletados em um complexo de reservatórios a fim de identificar se representavam uma espécie dominante ou múltiplas espécies, uma vez que diversas espécies de Cichla foram registradas na bacia. Os espécimes foram identificados com base na coloração, dados morfométricos e merísticos, e por marcadores mitocondriais COI, 16S rDNA e Região Controle (RC). A sobreposição dos dados morfométricos e o padrão similar de coloração impediram a identificação utilizando as chaves de identificação disponíveis na literatura. Entretanto, as análises bayesiana e de máxima verossimilhança de dados moleculares demonstraram a ocorrência de uma única espécie, Cichla kelberi. Um único haplótipo foi observado para o 16S e RC, enquanto três foram detectados para o COI, com um haplótipo dominante presente em 98,5% das amostras. A baixa diversidade nos exemplares introduzidos de C. kelberi evidenciou um número limitado de linhagens maternas fundadoras. O sucesso da invasão parece depender de fatores abióticos, como a maior transparência da água de ambientes lênticos que favorece predadores visuais que, atrelado à ausência de predadores, fez do C. kelberi um invasor bem-sucedido nesses reservatórios.

Population bottlenecks can have dramatic consequences for the health and long-term survival of a species. Understanding of historic population size and standing genetic variation prior to a contraction allows estimating the impact of a bottleneck on the species' genetic diversity. Although historic population sizes can be modelled based on extant genomics, uncertainty is high for the last 10-20 millenia. Hence, integrating ancient genomes provides a powerful complement to retrace the evolution of genetic diversity through population fluctuations. Here, we recover 15 high-quality mitogenomes of the once nearly extinct Alpine ibex spanning 8601 BP to 1919 CE and combine these with 60 published modern whole genomes. Coalescent demography simulations based on modern whole genomes indicate population fluctuations coinciding with the last major glaciation period. Using our ancient and historic mitogenomes, we investigate the more recent demographic history of the species and show that mitochondrial haplotype diversity was reduced to a fifth of the prebottleneck diversity with several highly differentiated mitochondrial lineages having coexisted historically. The main collapse of mitochondrial diversity coincides with elevated human population growth during the last 1-2 kya. After recovery, one lineage was spread and nearly fixed across the Alps due to recolonization efforts. Our study highlights that a combined approach integrating genomic data of ancient, historic and extant populations unravels major long-term population fluctuations from the emergence of a species through its near extinction up to the recent past.

Rhododendron meddianum is a critically endangered species with important ornamental value and is also a plant species with extremely small populations. In this study, we used double digest restriction-site-associated DNA sequencing (ddRAD) technology to assess the genetic diversity, genetic structure and demographic history of the three extant populations of R. meddianum. Analysis of SNPs indicated that R. meddianum populations have a high genetic diversity (π = 0.0772 ± 0.0024, HE = 0.0742 ± 0.002). Both FST values (0.1582–0.2388) and AMOVA showed a moderate genetic differentiation among the R. meddianum populations. Meanwhile, STRUCTURE, PCoA and NJ trees indicated that the R. meddianum samples were clustered into three distinct genetic groups. Using the stairway plot, we found that R. meddianum underwent a population bottleneck about 70,000 years ago. Furthermore, demographic models of R. meddianum and its relative, Rhododendron cyanocarpum, revealed that these species diverged about 3.05 (2.21–5.03) million years ago. This divergence may have been caused by environmental changes that occurred after the late Pliocene, e.g., the Asian winter monsoon intensified, leading to a drier climate. Based on these findings, we recommend that R. meddianum be conserved through in situ, ex situ approaches and that its seeds be collected for germplasm.

The more recent a bottleneck occurs, the more likely it is that its harmful effects will be avoided or minimized by management procedures. Even very large populations are likely to experience a severe bottleneck. The spread of artificial insemination and the global sperm trade have provided worldwide use of the sperm from the high-yielding bulls. In Holstein cows, which are the most common dairy breeds, the sperm from bulls raised by especially some countries are used globally. In addition, intensive selection programs applied to obtain high yield often result in allele losses. In this study, it was investigated whether breeding bulls used to inseminate Holstein cows in Türkiye were under any bottleneck effect or not. For this purpose, sperm from 300 high-yielding Holstein bulls produced in and imported to Türkiye was used as the material of the study. The bottleneck assessment was carried out by using Sign and Wilcoxon Tests under Stepwise Mutation Model and Two-Phase Mutation Model using Bottleneck v.1.2.02 program based on ten microsatellite loci. In addition, Mode-Shift analysis has also been interpreted to understand whether the bottleneck is recent or not. Based on this, it was concluded that a total of 300 Holstein bulls bred by 13 countries in 3 continents (Asia, Europe, Americas) were under pressure of a bottleneck effect in the past, but this bottleneck did not occurred in the recent time., Bir darboğaz ne kadar yakın zamanda olursa yönetim prosedürleriyle zararlı etkilerinden kaçınılması veya bu etkilerin en aza indirilme olasılığı o kadar yüksek olur. Sayıca çok büyük popülasyonların bile ciddi bir genetik darboğaz yaşaması muhtemeldir. Suni tohumlamanın yaygınlaşması ve küresel sperm ticareti yüksek verimli boğaların spermlerinin tüm dünyada baskın olarak kullanılmasına alt yapı oluşturmuştur. Dünyada sütçü ırk olarak en çok yetiştirilen Holstein ineklerin tohumlanmasında özellikle bazı ülkelerin yetiştirdiği boğalara ait spermler küresel çapta sıklıkla kullanılmaktadır. Yüksek verim elde etmek için uygulanan yoğun seleksiyon programları çoğunlukla allel kayıplarıyla sonuçlanmaktadır. Bu çalışmada Türkiye’de Holstein inek popülasyonşarını tohumlamak için kullanılan damızlık boğaların herhangi bir darboğaz etkisi altında kalıp kalmadığı araştırılmıştır. Bunun için Türkiye’de üretilen ve Türkiye’ye ithal edilen yüksek verimli 300 baş Holstein boğaya ait sperma materyal olarak kullanılmıştır. On mikrosatellit lokus temel alınarak Bottleneck v.1.2.02 programı aracılığıyla Aşamalı Mutasyon Modeli ve İki Fazlı Mutasyon Modeli altında İşaret ve Wilcoxon Testlerine tabi tutularak darboğaz değerlendirmesi yapılmıştır. Ayrıca Mod-Kayması analizi ile darboğazın yakın zamanda olup olmadığı da yorumlanmıştır. Buna istinaden 3 kıtada (Asya, Avrupa, Amerika) 13 ülke tarafından yetiştirilen toplam 300 baş Holstein boğanın geçmişte bir darboğaz etkisi altında kaldığı ancak bu darboğazın yakın zamanda gerçekleşmiş bir darboğaz olmadığı sonucuna varılmıştır.

The Carthusian horse is a Pura Raza Español (PRE) strain (CS), bred as a closed population since its creation more than 500 years ago. The aim of this study was to analyse for the first time its population structure and situation of variability combining both genealogical (GEL) and genomic (GEN) data. The GEL data comprised 348,429 pedigree records (56,105 CS horses), while the GEN analysis included the high-density genotypes (670,804 SNPs) of 287 horses. Pedigree completeness demonstrated its accuracy, showing a good correlation of GEL (F) and GEN (FROH) inbreeding coefficient in the case of PRE subpopulations partially related and non-related to Carthusian strain (0.68) but a lower value in the 100% Carthusian horses (0.42), due to the high weight of founders not detected by GEL analysis. GEN (PCA, AMOVA, and Admixture) and GEL analysis showed a good differentiation of subpopulations, but also a high level of introgression of the CS in the breed during past decades. A recent change in this trend was noteworthy, with a considerable reduction in CS variability and a genetic bottleneck (effective population sizes of 31.57 and 30.20 in GEL and GEN analysis, respectively, in last generation). The PRE has maintained its variability, and a considerable difference in estimated Ne by GEL (60.77) and GEN (188.0) data was observed. Using two sources of complementary information, it was found the existence of an ancient PRE strain with a unique genetic landmark, practically free from the influence of other equine populations. Fil: Poyato Bonilla, Julia. Universidad de Sevilla; España Fil: Laseca, Nora. Universidad de Córdoba; España Fil: Demyda Peyrás, Sebastián. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Clínica. Cátedra de Reproducción Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina Fil: Molina Alcalá, Antonio. Universidad de Córdoba; España Fil: Valera Córdoba, Mercedes. Universidad de Sevilla; España

International audience; Abstract The domestication of plant and animal species lead to repeatable morphological evolution, often referred to as the phenotypic domestication syndrome . Domestication is also associated with important genomic changes, such as the loss of genetic diversity and modifications of gene expression patterns. Here, we explored theoretically the effect of domestication at the genomic level by characterizing the impact of a domestication-like scenario on gene regulatory networks. We ran population genetics simulations in which individuals were featured by their genotype (an interaction matrix encoding a gene regulatory network) and their gene expressions, representing the phenotypic level. Our domestication scenario included a population bottleneck and a selection switch (change in the optimal gene expression level) mimicking canalizing selection, i.e. evolution towards more stable expression to parallel enhanced environmental stability in man-made habitat. We showed that domestication profoundly alters genetic architectures. Based on the well-documented example of the maize ( Zea mays ssp. mays ) domestication, our simulations predicted (i) a drop in neutral allelic diversity, (ii) a change in gene expression variance that depended upon the domestication scenario, (iii) transient maladaptive plasticity, (iv) a deep rewiring of the gene regulatory networks, with a trend towards gain of regulatory interactions between genes, and (v) a global increase in the genetic correlations among gene expressions, with a loss of modularity in the resulting coexpression patterns and in the underlying networks. Extending the range of parameters, we provide empirically testable predictions on the differences of genetic architectures between wild and domesticated and forms. The characterization of such systematic evolutionary changes in the genetic architecture of traits contributes to define a molecular domestication syndrome .

Southern sea otter (Enhydra lutris nereis) population recovery is influenced by a variety of factors, including predation, biotoxin exposure, infectious disease, oil spills, habitat degradation, and resource limitation. This population has also experienced a significant genetic bottleneck, resulting in low genetic diversity. We investigated how two metrics, familial relatedness and genetic diversity, are correlated with common causes of mortality in southern sea otters, including cardiomyopathy, acanthocephalan (Profilicollis spp.) peritonitis, systemic protozoal infection (Toxoplasma gondii and Sarcocystis neurona), domoic acid intoxication, end-lactation syndrome, and shark bite. Microsatellite genetic markers were used to examine this association in 356 southern sea otters necropsied from 1998 to 2012. Significant associations with genetic diversity or familial relatedness (P

Anthropogenic activities such as industrialization and urbanization has resulted in environmental crisis. The excessive use of the chemical fertilizers, and release of the heavy metal contaminants and drug waste from the industries and medical settings, respectively, has become a bottleneck to environmental sustainability. This situation demands the introduction of modern yet eco-friendly solutions that are cost effective, widely acceptable, and easily applicable. Phytoremediation has emerged as a feasible option owing to the ubiquitous nature of plants and their role in the bringing sustainability. Although few studies are underway to use plants for remediating contaminants and pollutants, their genetic make-up limits their capacity to remediate beyond a certain threshold. Therefore, here we tried to shed light on how new molecular editing toolboxes such as CRISPR-Cas can play a vital role in the editing of the plant genome to increase their capacity for remediation without harming the overall plant growth. Also, we discuss about how CRISPR-Cas can be used for gene knockout, gene replacement, and the multiplexing as well as regulating transcriptome and epigenome in the phytoremediators. Lastly, we have summarized the transgenic plants developed to enhance of the phytoremediation capacity and the tolerance to their toxic environment.

We present the Cuckoo Trie, a fast, memory-efficient ordered index structure. The Cuckoo Trie is designed to have memory-level parallelism -- which a modern out-of-order processor can exploit to execute DRAM accesses in parallel -- without sacrificing memory efficiency. The Cuckoo Trie thus breaks a fundamental performance barrier faced by current indexes, whose bottleneck is a series of dependent pointer-chasing DRAM accesses -- e.g., traversing a search tree path -- which the processor cannot parallelize. Our evaluation shows that the Cuckoo Trie outperforms state-of-the-art-indexes by up to 20%--360% on a variety of datasets and workloads, typically with a smaller or comparable memory footprint., Comment: Published in SOSP 2021

Prokaryotic genomes are generally gene dense and encode relatively few pseudogenes, or nonfunctional/inactivated remnants of genes. However, in certain contexts, such as recent ecological shifts or extreme population bottlenecks (such as those experienced by symbionts and pathogens), pseudogenes can quickly accumulate and form a substantial fraction of the genome. Identification of pseudogenes is, thus, a critical step for understanding the evolutionary forces acting upon, and the functional potential encoded within, prokaryotic genomes. Here, we present Pseudofinder, an open-source software dedicated to pseudogene identification and analysis. With Pseudofinder’s multi-pronged, reference-based approach, we demonstrate its capacity to detect a wide variety of pseudogenes, including those that are highly degraded and typically missed by gene-calling pipelines, as well newly formed pseudogenes, which can have only one or a few inactivating mutations. Additionally, Pseudofinder can detect intact genes undergoing relaxed selection, which may indicate incipient pseudogene formation. Implementation of Pseudofinder in annotation pipelines will not only clarify the functional potential of sequenced microbes, but will also generate novel insights and hypotheses regarding the evolutionary dynamics of bacterial and archaeal genomes.

To overcome pandemics, such as COVID-19, vaccines are urgently needed at very high volumes. Here we assess the techno-economic feasibility of producing RNA vaccines for the demand associated with a global vaccination campaign. Production process performance is assessed for three messenger RNA (mRNA) and one self-amplifying RNA (saRNA) vaccines, all currently under clinical development, as well as for a hypothetical next-generation saRNA vaccine. The impact of key process design and operation uncertainties on the performance of the production process was assessed. The RNA vaccine drug substance (DS) production rates, volumes and costs are mostly impacted by the RNA amount per vaccine dose and to a lesser extent by the scale and titre in the production process. The resources, production scale and speed required to meet global demand vary substantially in function of the RNA amount per dose. For lower dose saRNA vaccines, global demand can be met using a production process at a scale of below 10 L bioreactor working volume. Consequently, these small-scale processes require a low amount of resources to set up and operate. RNA DS production can be faster than fill-to-finish into multidose vials, hence the latter may constitute a bottleneck.

Genetic diversity represents a population’s evolutionary potential, as well as its demographic and evolutionary history. Advances in DNA sequencing have allowed the development of new and potentially powerful methods to quantify this diversity. However, when using these methods best practices for sampling populations and analyzing data are still being developed. Furthermore, while effects of the landscape on spatial patterns of genetic variation have received considerable attention, we have a poorer understanding of how genetic diversity changes as a result of temporal variation in environmental and demographic variables. Here, I take advantage of advances in DNA sequencing to investigate genetic diversity at single nucleotide polymorphisms (SNPs) across space and time in a model system of the butterfly, Parnassius smintheus. I used double digest restriction site associated DNA sequencing to genotype SNPs in P. smintheus from populations in Alberta, Canada. To develop recommendations for analyzing data, I tested the effect of varying the maximum amount of missing data (and therefore the number of SNPs) on common population genetic analyses. Most analyses were robust to varying amounts of missing data, except for population assignment tests where larger datasets (with more missing data) revealed higher-resolution population structure. I also examined the effect of sample size on the same set of analyses, finding that some (e.g., estimation of genetic differentiation) required as few as five individuals per population, while others (e.g., population assignment) required at least 15. I used the SNP dataset to investigate factors shaping patterns of genetic diversity at different spatial scales and across time. At a larger spatial scale but a single time point, both weather (snow depth and mean minimum temperatures) and land cover (the distance between meadow patches) predicted genetic diversity and differentiation. At a smaller spatial but longer temporal scale, I used a smaller SNP dataset to show that genetic diversity is lost over repeated demographic bottlenecks driven by winter weather, and subsequently recovered through gene flow. My work contributes to understanding how genetic diversity is shaped in natural populations, and points to the importance of both land cover and weather (and specifically, variability in weather) to this process.

We analyze the commuting patterns and parking modes for integrated daily commuting under different regimes. Based on a bi-direction bottleneck network with a spatial pattern of parking, the daily commuting patterns with parking location choices are analyzed. Without road toll and parking fee, there exist two equilibrium flow patterns and one parking mode. Increasing the parking density would postpone the earliest departure time and shorten the length of departure time period. A linear location-dependent parking fee regime without road toll is then proposed. We find that three parking modes occur across the parking fee rate and the morning efficiency loss always exists no matter how the fee rate is changed. Furthermore, a combined regime of linear location-dependent parking fee and time-varying road toll is developed to minimize the total daily social cost (TDSC). Two parking modes occur as the parking fee rate increases and there exist four equilibrium flow patterns. We show that the linear location-dependent parking fees could either advance or postpone the earliest morning departure time. An inappropriate parking fee rate may increase both individual and social costs. The best system performance can be achieved with joint consideration of both the TDSC and parking supply cost.

This dissertation outlines research quantifying the entering and exiting the quinone reaction chamber in NADH dehydrogenase or respiratory complex I. Respiratory complex I, the first complex in the respiratory electron transport chain. The respiratory electron transport is essential for all aerobic life. The methods used to quantify the entrance and exit process are geometric modeling, steered molecular dynamics, and singular value decomposition of the process. Five structures were analyzed: bacterial, yeast, ovine mt, mice mt, and human complex I. The structures reveal an almost 30 angstrom tunnel-like chamber for quinone binding in the core part of the enzyme, at the joint between the membrane and hydrophilic arms of the enzyme. The entrance of this quinone chamber located in ND1 subunit and has an apparent bottleneck of quinone/quinol passage. The first chapter introduces complex I and how transition state theory using diffusion kinetics gives an approximate maximum for the energy of crossing the bottleneck for quinone/quinol passage. Chapter 2 introduces the techniques used to quantify the difficulty of passage as well as methods to identify modes for collective confirmational changes for bottleneck opening. Chapters 3 and 4 are reproductions of the published papers based on this work. The appendices are reproductions of the supplemental information for those two papers.

Arabidopsis halleri is a hyperaccumulating pseudo-metallophyte and an emerging model to explore molecular basis of metal tolerance and hyperaccumulation. In this regard, understanding of interacting genes can be a crucial aspect as these interactions regulate several biological functions at molecular level in response to multiple signals. In this current study, we applied a weighted gene co-expression network analysis (WGCNA) on root and shoot RNA-seq data of A. halleri to predict the related scale-free organ specific co-expression networks, for the first time. A total of 19,653 genes of root and 18,081 genes of shoot were grouped into 14 modules and subjected to GO and KEGG enrichment analysis. "Photosynthesis" and "photosynthesis-antenna proteins" were identified as the most enriched and common pathway to both root and shoot. Whereas "glucosinolate biosynthesis," "autophagy," and "SNARE interactions in vesicular transport" were specific to root, and "circadian rhythm" was found to be enriched only in shoot. Later, hub and bottleneck genes were identified in each module by using cytoHubba plugin based on Cytoscape and scoring the relevance of each gene to the topology of the network. The modules with the most significant differential expression pattern across control and treatment (Cd-Zn treatment) were selected and their hub and bottleneck genes were screened to validate their possible involvement in heavy metal stress. Moreover, we combined the analysis of co-expression modules together with protein-protein interactions (PPIs), confirming some genes as potential candidates in plant heavy metal stress and as biomarkers. The results from this analysis shed the light on the pivotal functions to the hyperaccumulative trait of A. halleri, giving perspective to new paths for future research on this species.

The recent elucidation of atomistic structures of Cl− channel CFTR provides opportunities for understanding the molecular basis of cystic fibrosis. Despite having been activated through phosphorylation and provided with ATP ligands, several near-atomistic cryo-EM structures of CFTR are in a closed state, as inferred from the lack of a continuous passage through a hydrophobic bottleneck region located in the extracellular portion of the pore. Here, we present repeated, microsecond-long molecular dynamics simulations of human CFTR solvated in a lipid bilayer and aqueous NaCl. At equilibrium, Cl− ions enter the channel through a lateral intracellular portal and bind to two distinct cationic sites inside the channel pore but do not traverse the narrow, de-wetted bottleneck. Simulations conducted in the presence of a strong hyperpolarizing electric field led to spontaneous Cl− translocation events through the bottleneck region of the channel, suggesting that the protein relaxed to a functionally open state. Conformational changes of small magnitude involving transmembrane helices 1 and 6 preceded ion permeation through diverging exit routes at the extracellular end of the pore. The pore bottleneck undergoes wetting prior to Cl− translocation, suggesting that it acts as a hydrophobic gate. Although permeating Cl− ions remain mostly hydrated, partial dehydration occurs at the binding sites and in the bottleneck. The observed Cl− pathway is largely consistent with the loci of mutations that alter channel conductance, anion binding, and ion selectivity, supporting the model of the open state of CFTR obtained in the present study.

As current shotgun proteomics experiments can produce gigabytes of mass spectrometry data per hour, processing these massive data volumes has become progressively more challenging. Spectral clustering is an effective approach to speed up downstream data processing by merging highly similar spectra to minimize data redundancy. However, because state-of-the-art spectral clustering tools fail to achieve optimal runtimes, this simply moves the processing bottleneck. In this work, we present a fast spectral clustering tool, HyperSpec, based on hyperdimensional computing (HDC). HDC shows promising clustering capability while only requiring lightweight binary operations with high parallelism that can be optimized using low-level hardware architectures, making it possible to run HyperSpec on graphics processing units to achieve extremely efficient spectral clustering performance. Additionally, HyperSpec includes optimized data preprocessing modules to reduce the spectrum preprocessing time, which is a critical bottleneck during spectral clustering. Based on experiments using various mass spectrometry data sets, HyperSpec produces results with comparable clustering quality as state-of-the-art spectral clustering tools while achieving speedups by orders of magnitude, shortening the clustering runtime of over 21 million spectra from 4 h to only 24 min.

Molecular barcoding techniques have emerged as powerful tools to understand microbial pathogenesis. However, barcoding strategies have not been extended to protozoan parasites, which have unique genomic structures and virulence strategies compared to viral and bacterial pathogens. Here, we present a versatile CRISPR-based method to barcode protozoa, which we successfully apply to Toxoplasma gondii and Trypanosoma brucei. The murine brain is an important transmission niche for T. gondii, and brain persistence is a clinically untreatable feature of infection. The blood-brain barrier is expected to physically restrict parasite colonization of this niche, resulting in a selection bottleneck. Using libraries of barcoded T. gondii we evaluate shifts in the population structure from acute to chronic infection of mice. Contrary to expectation, most barcodes were present in the brain one-month post-intraperitoneal infection in both inbred CBA/J and outbred Swiss mice. Although parasite cyst number and barcode diversity declined over time, barcodes that represented a minor fraction of the inoculum could become a dominant population in the brain by three months post-infection. Together, these data establish the first, robust molecular barcoding approach for protozoa and evidence that the blood-brain barrier does not represent a major bottleneck to colonization by T. gondii.

Deletion-containing viral genomes (DelVGs) are commonly produced during influenza A virus infection and have been implicated in influencing clinical infection outcomes. Despite their ubiquity, the specific molecular mechanisms that govern DelVG formation and their packaging into defective interfering particles (DIPs) remain poorly understood. Here, we utilized next-generation sequencing to analyze DelVGs that form de novo early during infection, prior to packaging. Analysis of these early DelVGs revealed that deletion formation occurs in clearly defined hot spots and is significantly associated with both direct sequence repeats and enrichment of adenosine and uridine bases. By comparing intracellular DelVGs with those packaged into extracellular virions, we discovered that DelVGs face a significant bottleneck during genome packaging relative to wild-type genomic RNAs. Interestingly, packaged DelVGs exhibited signs of enrichment for larger DelVGs suggesting that size is an important determinant of packaging efficiency. Our data provide the first unbiased, high-resolution portrait of the diversity of DelVGs that are generated by the influenza A virus replication machinery and shed light on the mechanisms that underly DelVG formation and packaging. IMPORTANCE Defective interfering particles (DIPs) are commonly produced by RNA viruses and have been implicated in modulating clinical infection outcomes; hence, there is increasing interest in the potential of DIPs as antiviral therapeutics. For influenza viruses, DIPs are formed by the packaging of genomic RNAs harboring internal deletions. Despite decades of study, the mechanisms that drive the formation of these deletion-containing viral genomes (DelVGs) remain elusive. Here, we used a specialized sequencing pipeline to characterize the first wave of DelVGs that form during influenza virus infection. This data set provides an unbiased profile of the deletion-forming preferences of the influenza virus replicase. In addition, by comparing the early intracellular DelVGs to those that get packaged into extracellular virions, we described a significant segment-specific bottleneck that limits DelVG packaging relative to wild-type viral RNAs. Altogether, these findings reveal factors that govern the production of both DelVGs and DIPs during influenza virus infection.