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Mothers living with HIV are faced with managing their own complex healthcare and wellness needs while caring for their children. Understanding the lived experiences of mothers living with HIV, including grandmothers and mothers with older children - who are less explicitly represented in existing literature, may guide the development of interventions that best support them and their families. This study sought to explore the role of motherhood and related social/structural factors on engagement with HIV care, treatment-seeking behaviour, and overall HIV management among mothers living with HIV in the USA to inform such efforts. Semi-structured interviews were conducted between June and December 2015 with 52 mothers living with HIV, recruited from the Women's Interagency HIV Study (WIHS) sites in four US cities. Five broad themes were identified from the interviews: children as a motivation for optimal HIV management; children as providing logistical support for HIV care and treatment; the importance of social support for mothers; stressors tied to responsibilities of motherhood; and stigma about being a mother living with HIV. Findings underscore the importance of considering the demands of motherhood when developing more effective strategies to support mothers in managing HIV and promoting the overall health and well-being of their families.

Bacteria take up environmental DNA using dynamic appendages called type IV pili (T4P) to elicit horizontal gene transfer in a process called natural transformation. Natural transformation is widespread amongst bacteria yet determining how different factors universally contribute to or limit this process across species has remained challenging. Here we show that Acinetobacter baylyi, the most naturally transformable species, is highly transformable due to its ability to robustly bind nonspecific DNA via a dedicated orphan minor pilin, FimT. We show that, compared to its homologues, A. baylyi FimT contains multiple positively charged residues that additively promote DNA binding efficiency. Expression of A. baylyi FimT in a closely related Acinetobacter pathogen is sufficient to substantially improve its capacity for natural transformation, demonstrating that T4P-DNA binding is a rate-limiting step in this process. These results demonstrate the importance of T4P-DNA binding efficiency in driving natural transformation, establishing a key factor limiting horizontal gene transfer.

Diverse bacterial species use type IVa pili (T4aP) to interact with their environments. The dynamic extension and retraction of T4aP is critical for their function, but the mechanisms that regulate this dynamic activity remain poorly understood. T4aP are typically extended via the activity of a dedicated extension motor ATPase and retracted via the action of an antagonistic retraction motor ATPase called PilT. These motors are generally functionally independent, and loss of PilT commonly results in T4aP hyperpiliation due to undeterred pilus extension. However, for the mannose-sensitive hemagglutinin (MSHA) T4aP of Vibrio cholerae, the loss of PilT unexpectedly results in a loss of surface piliation. Here, we employ a combination of genetic and cell biological approaches to dissect the underlying mechanism. Our results demonstrate that PilT is necessary for MSHA pilus extension in addition to its well-established role in promoting MSHA pilus retraction. Through a suppressor screen, we also provide genetic evidence that the MshA major pilin impacts pilus extension. Together, these findings contribute to our understanding of the factors that regulate pilus extension and describe a previously uncharacterized function for the PilT motor ATPase., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Hughes et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

In mammals, subcellular protein localization of factors like planar cell polarity proteins is a key driver of the multicellular organization of tissues. Bacteria also form organized multicellular communities, but these patterns are largely thought to emerge from regulation of whole-cell processes like growth, motility, cell shape, and differentiation. Here we show that a unique intracellular patterning of appendages known as type IV pili (T4P) can drive multicellular development of complex bacterial communities. Specifically, dynamic T4P appendages localize in a line along the long axis of the cell in the bacterium Acinetobacter baylyi. This long-axis localization is regulated by a functionally divergent chemosensory Pil-Chp system, and an atypical T4P protein homologue (FimV) bridges Pil-Chp signaling and T4P positioning. We further demonstrate through modeling and empirical approaches that subcellular T4P localization controls how individual cells interact with one another, independently of T4P dynamics, with different patterns of localization giving rise to distinct multicellular architectures. Our results reveal how subcellular patterning of single cells regulates the development of multicellular bacterial communities., (© 2022. The Author(s).)

The conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) is a key step in DNA demethylation that is mediated by ten-eleven translocation (TET) enzymes, which require ascorbate/vitamin C. Here, we report the 5hmC landscape of normal hematopoiesis and identify cell type-specific 5hmC profiles associated with active transcription and chromatin accessibility of key hematopoietic regulators. We utilized CRISPR/Cas9 to model TET2 loss-of-function mutations in primary human hematopoietic stem and progenitor cells (HSPC). Disrupted cells exhibited increased colonies in serial replating, defective erythroid/megakaryocytic differentiation, and in vivo competitive advantage and myeloid skewing coupled with reduction of 5hmC at erythroid-associated gene loci. Azacitidine and ascorbate restored 5hmC abundance and slowed or reverted the expansion of TET2-mutant clones in vivo. These results demonstrate the key role of 5hmC in normal hematopoiesis and TET2-mutant phenotypes and raise the possibility of utilizing these agents to further our understanding of preleukemia and clonal hematopoiesis., Significance: We show that 5-hydroxymethylation profiles are cell type-specific and associated with transcriptional abundance and chromatin accessibility across human hematopoiesis. TET2 loss caused aberrant growth and differentiation phenotypes and disrupted 5hmC and transcriptional landscapes. Treatment of TET2 KO HSPCs with ascorbate or azacitidine reverted 5hmC profiles and restored aberrant phenotypes. This article is highlighted in the In This Issue feature, p. 265., (©2022 American Association for Cancer Research.)

Bacteria and archaea rely on appendages called type IV pili (T4P) to participate in diverse behaviors including surface sensing, biofilm formation, virulence, protein secretion and motility across surfaces. T4P are broadly distributed fibers that dynamically extend and retract, and this dynamic activity is essential for their function in broad processes. Despite the essentiality of dynamics in T4P function, little is known about the role of these dynamics and molecular mechanisms controlling them. Recent advances in microscopy have yielded insight into the role of T4P dynamics in their diverse functions and recent structural work has expanded what is known about the inner workings of the T4P motor. This review discusses recent progress in understanding the function, regulation, and mechanisms of T4P dynamics., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS.)

When tissue biopsy is not medically prudent or tissue is insufficient for molecular testing, alternative methods are needed. Because cell-free DNA (cfDNA) has been shown to provide a representative surrogate for tumor tissue, we sought to evaluate its utility in this clinical scenario. cfDNA was isolated from the plasma of patients and assayed with low-coverage (∼0.3×), genome-wide sequencing. Copy-number alterations (CNA) were identified and characterized using analytic methods originally developed for noninvasive prenatal testing (NIPT) and quantified using the genomic instability number (GIN), a metric that reflects the quantity and magnitude of CNAs across the genome. The technical variability of the GIN was first evaluated in an independent cohort comprising genome-wide sequencing results from 27,754 women who consented to have their samples used for research and whose NIPT results yielded no detected CNAs to establish a detection threshold. Subsequently, cfDNA sequencing data from 96 patients with known cancers but for whom a tissue biopsy could not be obtained are presented. An elevated GIN was detected in 35% of patients and detection rates varied by tumor origin. Collectively, CNAs covered 96.6% of all autosomes. Survival was significantly reduced in patients with an elevated GIN relative to those without. Overall, these data provide a proof of concept for the use of low-coverage, genome-wide sequencing of cfDNA from patients with cancer to obtain relevant molecular information in instances where tissue is difficult to access. These data may ultimately serve as an informative complement to other molecular tests., (©2021 The Authors; Published by the American Association for Cancer Research.)

The competence pili of transformable Gram-positive species are phylogenetically related to the diverse and widespread class of extracellular filamentous organelles known as type IV pili. In Gram-negative bacteria, type IV pili act through dynamic cycles of extension and retraction to carry out diverse activities including attachment, motility, protein secretion, and DNA uptake. It remains unclear whether competence pili in Gram-positive species exhibit similar dynamic activity, and their mechanism of action for DNA uptake remains unclear. They are hypothesized to either (1) leave transient cavities in the cell wall that facilitate DNA passage, (2) form static adhesins to enrich DNA near the cell surface for subsequent uptake by membrane-embedded transporters, or (3) play an active role in translocating bound DNA via dynamic activity. Here, we use a recently described pilus labeling approach to demonstrate that competence pili in Streptococcus pneumoniae are highly dynamic structures that rapidly extend and retract from the cell surface. By labeling the principal pilus monomer, ComGC, with bulky adducts, we further demonstrate that pilus retraction is essential for natural transformation. Together, our results suggest that Gram-positive competence pili in other species may also be dynamic and retractile structures that play an active role in DNA uptake., (© 2021 John Wiley & Sons Ltd.)

Bacteria use extracellular appendages called type IV pili (T4P) for diverse behaviors including DNA uptake, surface sensing, virulence, protein secretion, and twitching motility. Dynamic extension and retraction of T4P is essential for their function, and T4P extension is thought to occur through the action of a single, highly conserved motor, PilB. Here, we develop Acinetobacter baylyi as a model to study T4P by employing a recently developed pilus labeling method. By contrast to previous studies of other bacterial species, we find that T4P synthesis in A. baylyi is dependent not only on PilB but also on an additional, phylogenetically distinct motor, TfpB. Furthermore, we identify a protein (CpiA) that inhibits T4P extension by specifically binding and inhibiting PilB but not TfpB. These results expand our understanding of T4P regulation and highlight how inhibitors might be exploited to disrupt T4P synthesis.

Pseudomonas aeruginosa is a significant threat in both healthcare and industrial biofouling. Surface attachment of P. aeruginosa is particularly problematic as surface association induces virulence and is necessary for the ensuing process of biofilm formation, which hampers antibiotic treatments. Previous efforts have searched for dispersal agents of mature biofilm collectives, but there are no known factors that specifically disperse individual surface-attached P. aeruginosa. In this study, we develop a quantitative single-cell surface-dispersal assay and use it to show that P. aeruginosa itself produces factors that can stimulate its dispersal. Through bioactivity-guided fractionation, mass spectrometry, and nuclear magnetic resonance, we elucidated the structure of one such factor, 2-methyl-4-hydroxyquinoline (MHQ). MHQ is an alkyl quinolone with a previously unknown activity and is synthesized by the PqsABC enzymes. Pure MHQ is sufficient to disperse P. aeruginosa, but the dispersal activity of natural P. aeruginosa conditioned media requires additional factors. Whereas other alkyl quinolones have been shown to act as antibiotics or membrane depolarizers, MHQ lacks these activities and known antibiotics do not induce dispersal. In contrast, we show that MHQ inhibits the activity of Type IV Pili (TFP) and that TFP targeting can explain its dispersal activity. Our work thus identifies single-cell surface dispersal as a new activity of P. aeruginosa-produced small molecules, characterizes MHQ as a promising dispersal agent, and establishes TFP inhibition as a viable mechanism for P. aeruginosa dispersal., Competing Interests: Conflict of interest A provisional patent describing MHQ as a potential therapeutic in surface dispersal has been filed., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

DNA 5-hydroxymethylcytosine (5hmC) modification is known to be associated with gene transcription and frequently used as a mark to investigate dynamic DNA methylation conversion during mammalian development and in human diseases. However, the lack of genome-wide 5hmC profiles in different human tissue types impedes drawing generalized conclusions about how 5hmC is implicated in transcription activity and tissue specificity. To meet this need, we describe the development of a 5hmC tissue map by characterizing the genomic distributions of 5hmC in 19 human tissues derived from ten organ systems. Subsequent sequencing results enabled the identification of genome-wide 5hmC distributions that uniquely separates samples by tissue type. Further comparison of the 5hmC profiles with transcriptomes and histone modifications revealed that 5hmC is preferentially enriched on tissue-specific gene bodies and enhancers. Taken together, the results provide an extensive 5hmC map across diverse human tissue types that suggests a potential role of 5hmC in tissue-specific development; as well as a resource to facilitate future studies of DNA demethylation in pathogenesis and the development of 5hmC as biomarkers.

Pancreatic cancer is often detected late, when curative therapies are no longer possible. Here, we present non-invasive detection of pancreatic ductal adenocarcinoma (PDAC) by 5-hydroxymethylcytosine (5hmC) changes in circulating cell free DNA from a PDAC cohort (n = 64) in comparison with a non-cancer cohort (n = 243). Differential hydroxymethylation is found in thousands of genes, most significantly in genes related to pancreas development or function (GATA4, GATA6, PROX1, ONECUT1, MEIS2), and cancer pathogenesis (YAP1, TEAD1, PROX1, IGF1). cfDNA hydroxymethylome in PDAC cohort is differentially enriched for genes that are commonly de-regulated in PDAC tumors upon activation of KRAS and inactivation of TP53. Regularized regression models built using 5hmC densities in genes perform with AUC of 0.92 (discovery dataset, n = 79) and 0.92-0.94 (two independent test sets, n = 228). Furthermore, tissue-derived 5hmC features can be used to classify PDAC cfDNA (AUC = 0.88). These findings suggest that 5hmC changes enable classification of PDAC even during early stage disease.

Cellular differentiation is a fundamental strategy used by cells to generate specialized functions at specific stages of development. The bacterium Caulobacter crescentus employs a specialized dimorphic life cycle consisting of two differentiated cell types. How environmental cues, including mechanical inputs such as contact with a surface, regulate this cell cycle remain unclear. Here, we find that surface sensing by the physical perturbation of retracting extracellular pilus filaments accelerates cell-cycle progression and cellular differentiation. We show that physical obstruction of dynamic pilus activity by chemical perturbation or by a mutation in the outer-membrane pilus secretin CpaC stimulates early initiation of chromosome replication. In addition, we find that surface contact stimulates cell-cycle progression by demonstrating that surface-stimulated cells initiate early chromosome replication to the same extent as planktonic cells with obstructed pilus activity. Finally, we show that obstruction of pilus retraction stimulates the synthesis of the cell-cycle regulator cyclic diguanylate monophosphate (c-di-GMP) through changes in the activity and localization of two key regulatory histidine kinases that control cell fate and differentiation. Together, these results demonstrate that surface contact and sensing by alterations in pilus activity stimulate C. crescentus to bypass its developmentally programmed temporal delay in cell differentiation to more quickly adapt to a surface-associated lifestyle., Competing Interests: The authors declare no competing interest.

Biofilm formation by Vibrio cholerae facilitates environmental persistence, and hyperinfectivity within the host. Biofilm formation is regulated by 3',5'-cyclic diguanylate (c-di-GMP) and requires production of the type IV mannose-sensitive hemagglutinin (MSHA) pilus. Here, we show that the MSHA pilus is a dynamic extendable and retractable system, and its activity is directly controlled by c-di-GMP. The interaction between c-di-GMP and the ATPase MshE promotes pilus extension, whereas low levels of c-di-GMP correlate with enhanced retraction. Loss of retraction facilitated by the ATPase PilT increases near-surface roaming motility, and impairs initial surface attachment. However, prolonged retraction upon surface attachment results in reduced MSHA-mediated surface anchoring and increased levels of detachment. Our results indicate that c-di-GMP directly controls MshE activity, thus regulating MSHA pilus extension and retraction dynamics, and modulating V. cholerae surface attachment and colonization.

A widespread class of prokaryotic motors powered by secretion motor adenosine triphosphatases (ATPases) drives the dynamic extension and retraction of extracellular fibers, such as type IV pili (T4P). Among these, the tight adherence (tad) pili are critical for surface sensing and biofilm formation. As for most other motors belonging to this class, how tad pili retract despite lacking a dedicated retraction motor ATPase has remained a mystery. Here, we find that a bifunctional pilus motor ATPase, CpaF, drives both activities through adenosine 5'-triphosphate (ATP) hydrolysis. We show that mutations within CpaF result in a correlated reduction in the rates of extension and retraction that directly scales with decreased ATP hydrolysis and retraction force. Thus, a single motor ATPase drives the bidirectional processes of pilus fiber extension and retraction., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Surface appendages, such as flagella and type IV pili, mediate a broad range of bacterial behaviors, including motility, attachment, and surface sensing. While many species harbor both flagella and type IV pili, little is known about how or if their syntheses are coupled. Here, we show that deletions of genes encoding different flagellum machinery components result in a reduction of pilus synthesis in Caulobacter crescentus First, we show that different flagellar mutants exhibit different levels of sensitivity to a pilus-dependent phage and that fewer cells within populations of flagellar mutants make pili. Furthermore, we find that single cells within flagellar mutant populations produce fewer pili per cell. We demonstrate that these gene deletions result in reduced transcription of pilus-associated genes and have a slight but significant effect on general transcription profiles. Finally, we show that the decrease in pilus production is due to a reduction in the pool of pilin subunits that are polymerized into pilus fibers. These data demonstrate that mutations in flagellar gene components not only affect motility but also can have considerable and unexpected consequences for other aspects of cell biology. IMPORTANCE Most bacterial species synthesize surface-exposed appendages that are important for environmental interactions and survival under diverse conditions. It is often assumed that these appendages act independently of each other and that mutations in either system can be used to assess functionality in specific processes. However, we show that mutations in flagellar genes can impact the production of type IV pili, as well as alter general RNA transcriptional profiles compared to a wild-type strain. These data demonstrate that seemingly simple mutations can broadly affect cell-regulatory networks., (Copyright © 2019 American Society for Microbiology.)

Bacteria use surface-exposed, proteinaceous fibers called pili for diverse behaviors, including horizontal gene transfer, surface sensing, motility, and pathogenicity. Visualization of these filamentous nanomachines and their activity in live cells has proven challenging, largely due to their small size. Here, we describe a broadly applicable method for labeling and imaging pili and other surface-exposed nanomachines in live cells. This technique uses a combination of genetics and maleimide-based click chemistry in which a cysteine substitution is made in the major pilin subunit for subsequent labeling with thiol-reactive maleimide dyes. Large maleimide-conjugated molecules can also be used to physically interfere with the dynamic activity of filamentous nanomachines. We describe parameters for selecting cysteine substitution positions, optimized labeling conditions for epifluorescence imaging of pilus fibers, and methods for impeding pilus activity. After cysteine knock-in strains have been generated, this protocol can be completed within 30 min to a few hours, depending on the species and the experiment of choice. Visualization of extracellular nanomachines such as pili using this approach can provide a more comprehensive understanding of the role played by these structures in distinct bacterial behaviors.

Inhibitors of the PD-1/PD-L1/CTLA-4 immune checkpoint pathway have revolutionized cancer treatment. Indeed, some patients with advanced, refractory malignancies achieve durable responses; however, only a subset of patients benefit, necessitating new biomarkers to predict outcome. Interrogating cell-free DNA (cfDNA) isolated from plasma (liquid biopsy) provides a promising method for monitoring response. We describe the use of low-coverage, genome-wide sequencing of cfDNA, validated extensively for noninvasive prenatal testing, to detect tumor-specific copy-number alterations, and the development of a new metric-the genome instability number (GIN)-to monitor response to these drugs. We demonstrate how the GIN can be used to discriminate clinical response from progression, differentiate progression from pseudoprogression, and identify hyperprogressive disease. Finally, we provide evidence for delayed kinetics in responses to checkpoint inhibitors relative to molecularly targeted therapies. Overall, these data demonstrate a proof of concept for using this method for monitoring treatment outcome in patients with cancer receiving immunotherapy., (©2018 American Association for Cancer Research.)

To permanently attach to surfaces, Caulobacter crescentusproduces a strong adhesive, the holdfast. The timing of holdfast synthesis is developmentally regulated by cell cycle cues. When C. crescentusis grown in a complex medium, holdfast synthesis can also be stimulated by surface sensing, in which swarmer cells rapidly synthesize holdfast in direct response to surface contact. In contrast to growth in complex medium, here we show that when cells are grown in a defined medium, surface contact does not trigger holdfast synthesis. Moreover, we show that in a defined medium, flagellum synthesis and regulation of holdfast production are linked. In these conditions, mutants lacking a flagellum attach to surfaces over time more efficiently than either wild-type strains or strains harboring a paralyzed flagellum. Enhanced adhesion in mutants lacking flagellar components is due to premature holdfast synthesis during the cell cycle and is regulated by the holdfast synthesis inhibitor HfiA. hfiA transcription is reduced in flagellar mutants and this reduction is modulated by the diguanylate cyclase developmental regulator PleD. We also show that, in contrast to previous predictions, flagella are not necessarily required for C. crescentus surface sensing in the absence of flow, and that arrest of flagellar rotation does not stimulate holdfast synthesis. Rather, our data support a model in which flagellum assembly feeds back to control holdfast synthesis via HfiA expression in a c-di-GMP-dependent manner under defined nutrient conditions., (© 2018 John Wiley & Sons Ltd.)

The formation of multicellular microbial communities, called biofilms, starts from the adhesion of a few planktonic cells to the surface. The transition from a free-living planktonic lifestyle to a sessile, attached state is a multifactorial process that is determined by biological, chemical and physical properties of the environment, the surface and the bacterial cell. The initial weak, reversible interactions between a bacterium and a surface strengthen to yield irreversible adhesion. In this Review, we summarize our understanding of the mechanisms governing bacterial adhesion at the single-cell level, including the physical forces experienced by a cell before reaching the surface, the first contact with a surface and the transition from reversible to permanent adhesion.

Natural transformation is a broadly conserved mechanism of horizontal gene transfer in bacterial species that can shape evolution and foster the spread of antibiotic resistance determinants, promote antigenic variation and lead to the acquisition of novel virulence factors. Surface appendages called competence pili promote DNA uptake during the first step of natural transformation 1 ; however, their mechanism of action has remained unclear owing to an absence of methods to visualize these structures in live cells. Here, using the model naturally transformable species Vibrio cholerae and a pilus-labelling method, we define the mechanism for type IV competence pilus-mediated DNA uptake during natural transformation. First, we show that type IV competence pili bind to extracellular double-stranded DNA via their tip and demonstrate that this binding is critical for DNA uptake. Next, we show that type IV competence pili are dynamic structures and that pilus retraction brings tip-bound DNA to the cell surface. Finally, we show that pilus retraction is spatiotemporally coupled to DNA internalization and that sterically obstructing pilus retraction prevents DNA uptake. Together, these results indicate that type IV competence pili directly bind to DNA via their tip and mediate DNA internalization through retraction during this conserved mechanism of horizontal gene transfer.

It is critical for bacteria to recognize surface contact and initiate physiological changes required for surface-associated lifestyles. Ubiquitous microbial appendages called pili are involved in sensing surfaces and facilitating downstream behaviors, but the mechanism by which pili mediate surface sensing has been unclear. We visualized Caulobacter crescentus pili undergoing dynamic cycles of extension and retraction. Within seconds of surface contact, these cycles ceased, which coincided with synthesis of the adhesive holdfast required for attachment. Physically blocking pili imposed resistance to pilus retraction, which was sufficient to stimulate holdfast synthesis without surface contact. Thus, to sense surfaces, bacteria use the resistance on retracting, surface-bound pili that occurs upon surface contact., (Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Background: Current methods for noninvasive prenatal testing (NIPT) ascertain fetal aneuploidies using either direct counting measures of DNA fragments from specific genomic regions or relative measures of single nucleotide polymorphism frequencies. Alternatively, the ratios of paralogous sequence pairs were predicted to reflect fetal aneuploidy. We developed a NIPT assay that uses paralog sequences to enable noninvasive detection of fetal trisomy 21 (T21) and trisomy 18 (T18) using cell-free DNA (cfDNA) from maternal plasma., Methods: A total of 1060 primer pairs were designed to determine fetal aneuploidy status, fetal sex, and fetal fraction. Each library was prepared from cfDNA by coamplifying all 1060 target pairs together in a single reaction well. Products were measured using massively parallel sequencing and deviations from expected paralog ratios were determined based on the read depth from each paralog., Results: We evaluated this assay in a blinded set of 480 cfDNA samples with fetal aneuploidy status determined by the MaterniT21 ® PLUS assay. Samples were sequenced (mean = 2.3 million reads) with 432 samples returning a result. Using the MaterniT21 PLUS assay for paired plasma aliquots from the same individuals as a reference, all 385 euploid samples, all 31 T21 samples, and 14 of 16 T18 samples were detected with no false positive results observed., Conclusions: This study introduces a novel NIPT aneuploidy detection approach using targeted sequencing of paralog motifs and establishes proof-of-concept for a potentially low-cost, highly scalable method for the identification of selected fetal aneuploidies with performance and nonreportable rate similar to other published methods., (© 2016 American Association for Clinical Chemistry.)

Complex, quantitative traits are often the function of the coordinated action of many physically independent genetic factors. Interactive properties of multilocus genotypes, such as epistasis, are thought to be pervasive components of the genetic architecture of complex phenotypes. Here, we utilize a panel of interspecific backcross introgression lines to evaluate the genetic architecture of song variation, a quantitative sexual signaling phenotype, in the Hawaiian swordtail cricket genus Laupala. Allelic effects across five quantitative trait loci are consistent with a purely additive model of gene action, where alleles at multiple loci are found to have fully independent and discrete effects with respect to the sexual signaling phenotype. Whereas a more complex genetic architecture featuring non-additive dominance and epistasis components may constrain potential evolutionary trajectories and reduce the rate of evolutionary change, the polygenic, additive genetic architecture observed for sexual signaling in Laupala should respond rapidly to directional selection pressures and freely move throughout phenotypic space. This classic type I genetic architecture may facilitate the explosive radiation of song variation observed across the Laupala genus.

Coenzyme M is an essential coenzyme for methanogenesis. The proposed biosynthetic pathway consists of five steps, of which the fourth step is catalyzed by sulfopyruvate decarboxylase (ComDE). Disruption of the gene comE by transposon mutagenesis resulted in a partial coenzyme M auxotroph, which grew poorly in the absence of coenzyme M and retained less than 3% of the wild type level of coenzyme M biosynthesis. Upon coenzyme M addition, normal growth of the mutant was restored. Moreover, complementation of the mutation with the wild type comE gene in trans restored full growth in the absence of coenzyme M. These results confirm that ComE plays an important role in coenzyme M biosynthesis. The inability to yield a complete CoM auxotroph suggests that either the transposon insertion failed to completely inactivate the gene or M. maripaludis possesses a promiscuous activity that partially complemented the mutation.

The evolution of novel sexual communication systems is integral to the process of speciation, as it discourages gene flow between incipient species. Physical linkage between genes underlying male-female communication (i.e. sexual signals and preferences for them) facilitates both rapid and coordinated divergence of sexual communication systems between populations and reduces recombination in the face of occasional hybridization between diverging populations. Despite these ramifications of the genetic architecture of sexual communication for sexual selection and speciation, few studies have examined this relationship empirically. Previous studies of the closely related Hawaiian crickets Laupala paranigra and Laupala kohalensis have indirectly suggested that many of the genes underlying the difference in pulse rate of male song are physically linked with genes underlying the difference in female preference for pulse rate. Using marker-assisted introgression, we moved 'slow pulse rate' alleles from L. paranigra at five known quantitative trait loci (QTL) underlying male pulse rate into the 'fast pulse rate' genetic background of L. kohalensis and assessed the effect of these loci on female preference. An astounding four out of five song QTL predicted the preferences of female fourth-generation backcrosses, providing direct evidence for the extensive genetic linkage of song and preference in one of the fastest diversifying genera currently known.

Sexual behaviours often evolve rapidly and are critical for sexual isolation. We suggest that coordinated sexual signals and preferences generate stabilizing selection, favouring the accumulation of many small-effect mutations in sexual communication traits. Rapid radiation of a sexual behaviour used in signalling, song pulse rate, has been observed in the Hawaiian cricket genus Laupala. Using marker-assisted introgression, we isolated five known quantitative trait loci (QTL) influencing species-level differences in pulse rate from one species, L. paranigra, into a closely related species, L. kohalensis. All five QTL were found to have a significant effect on song and appear to be largely additive in backcross introgression lines. Furthermore, all effect sizes were small in magnitude. Our data provide support for the hypothesis that stabilizing selection on sexual signals in Laupala creates genetic conditions favourable to incremental divergence during speciation, through the evolution of alleles of minor rather than major phenotypic effects., (© 2011 The Authors. Journal of Evolutionary Biology © 2011 European Society For Evolutionary Biology.)

Background: Simple sequence repeats (SSRs) are tandemly repeated sequence motifs common in genomic nucleotide sequence that often harbor significant variation in repeat number. Frequently used as molecular markers, SSRs are increasingly identified via in silico approaches. Two common classes of genomic resources that can be mined are bacterial artificial chromosome (BAC) libraries and expressed sequence tag (EST) libraries., Results: 288 SSR loci were screened in the rapidly radiating Hawaiian swordtail cricket genus Laupala. SSRs were more densely distributed and contained longer repeat structures in BAC library-derived sequence than in EST library-derived sequence, although neither repeat density nor length was exceptionally elevated despite the relatively large genome size of Laupala. A non-random distribution favoring AT-rich SSRs was observed. Allelic diversity of SSRs was positively correlated with repeat length and was generally higher in AT-rich repeat motifs., Conclusion: The first large-scale survey of Orthopteran SSR allelic diversity is presented. Selection contributes more strongly to the size and density distributions of SSR loci derived from EST library sequence than from BAC library sequence, although all SSRs likely are subject to similar physical and structural constraints, such as slippage of DNA replication machinery, that may generate increased allelic diversity in AT-rich sequence motifs. Although in silico approaches work well for SSR locus identification in both EST and BAC libraries, BAC library sequence and AT-rich repeat motifs are generally superior SSR development resources for most applications.

Organismal fitness requires functional integration of nuclear and mitochondrial genomes. Structural and regulatory elements coevolve within lineages and several studies have found that interpopulation hybridization disrupts mitonuclear interactions. Because mitochondrial RNA polymerase (mtRPOL) plays key roles in both mitochondrial DNA (mtDNA) replication and transcription, the interaction between mtRPOL and coevolved regulatory sites in the mtDNA may be central to mitonuclear integration. Here, we generate interpopulation hybrids between divergent populations of the copepod Tigriopus californicus to obtain lines having different combinations of mtRPOL and mtDNA. Lines were scored for mtDNA copy number and ATP6 (mtDNA) gene expression. We find that there is a genotype-dependent negative association between mitochondrial transcriptional response and mtDNA copy number. We argue that an observed increase in mtDNA copy number and reduced mtDNA transcription in hybrids reflects the regulatory role of mtRPOL; depending on the mitonuclear genotype, hybridization may disrupt the normal balance between transcription and replication of the mitochondrial genome.

Male F(2) hybrids of the wasps Nasonia giraulti and Nasonia vitripennis suffer increased mortality during development. Previous studies suggested that the mitochondria may play an important role in this pattern of hybrid breakdown. The mitochondrial genome encodes 13 polypeptides, which are integral subunits of the oxidative phosphorylation enzyme complexes I, III, IV and V. We show that the mitochondrial ATP production rate and the efficacy of the enzyme complexes I, III and IV, but not that of the completely nuclear-encoded complex II, are reduced in F(2) hybrid males of N. giraulti and N. vitripennis. We hypothesize that nuclear-mitochondrial protein interactions in the oxidative phosphorylation pathway are disrupted in these hybrids, reducing energy generation capacity and potentially reducing hybrid fitness. Our results suggest that dysfunctional cytonuclear interactions could represent an under-appreciated post-zygotic isolation mechanism that, due to elevated evolutionary rates of mitochondrial genes, evolves very early in the speciation process.

Hybridization between populations can disrupt gene expression, frequently resulting in deleterious hybrid phenotypes. Reduced fitness in interpopulation hybrids of the marine copepod Tigriopus californicus has been traced to interactions between the nuclear and mitochondrial genomes. Here, we determine transcript levels of four to six genes involved in the mitochondrial oxidative phosphorylation pathway for a series of parental and inbred hybrid lines using RT-qPCR. Both nuclear and mitochondrial-encoded genes are included in the analysis. Although all genes studied are up-regulated under salinity stress, only expression of genes located on the mtDNA differed among lines. Because mitochondrial genes are transcribed by a dedicated RNA polymerase encoded in the nuclear genome, we compare transcript levels among hybrid lines with different combinations of mitochondrial RNA polymerase and mtDNA genotypes. Lines bearing certain mtDNA-mitochondrial RNA polymerase genotypic combinations show a diminished capacity to up-regulate mitochondrial genes in response to hypoosmotic stress. Effects on the transcriptional profile depend on the specific interpopulation cross and are correlated with viability effects. We hypothesize that disruption of the mitochondrial transcriptional system in F(2) hybrids may play a central role in hybrid breakdown.

Hybrid breakdown, or outbreeding depression, is the loss of fitness observed in crosses between genetically divergent populations. The role of maternally inherited mitochondrial genomes in hybrid breakdown has not been widely examined. Using laboratory crosses of the marine copepod Tigriopus californicus, we report that the low fitness of F(3) hybrids is completely restored in the offspring of maternal backcrosses, where parental mitochondrial and nuclear genomic combinations are reassembled. Paternal backcrosses, which result in mismatched mitochondrial and nuclear genomes, fail to restore hybrid fitness. These results suggest that fitness loss in T. californicus hybrids is completely attributable to nuclear-mitochondrial genomic interactions. Analyses of ATP synthetic capacity in isolated mitochondria from hybrid and backcross animals found that reduced ATP synthesis in hybrids was also largely restored in backcrosses, again with maternal backcrosses outperforming paternal backcrosses. The strong fitness consequences of nuclear-mitochondrial interactions have important, and often overlooked, implications for evolutionary and conservation biology.

Through the processes of natural selection and genetic drift, allopatric populations diverge genetically and may ultimately become reproductively incompatible. In cases of prezygotic reproductive isolation, candidate systems for speciation genes logically include genes involved in mate or gamete recognition. However, where only postzygotic isolation exists, candidate speciation genes could include any genes that affect hybrid performance. We hypothesize that because mitochondrial genes frequently evolve more rapidly than the nuclear genes with which they interact, interpopulation hybridization might be particularly disruptive to mitochondrial function. Understanding the potential impact of intergenomic (nuclear and mitochondrial) coadaptation on the evolution of allopatric populations of the intertidal copepod Tigriopus californicus has required a broadly integrative research program; here we present the results of experiments spanning the spectrum of biological organization in order to demonstrate the consequences of molecular evolution on physiological performance and organismal fitness. We suggest that disruption of mitochondrial function, known to result in a diverse set of human diseases, may frequently underlie reduced fitness in interpopulation and interspecies hybrids in animals.

Electron transport system (ETS) function in mitochondria is essential for the aerobic production of energy. Because ETS function requires extensive interactions between mitochondrial and nuclear gene products, coadaptation between mitochondrial and nuclear genomes may evolve within populations. Hybridization between allopatric populations may then expose functional incompatibilities between genomes that have not coevolved. The intertidal copepod Tigriopus californicus has high levels of nucleotide divergence among populations at mitochondrial loci and suffers F2 hybrid breakdown in interpopulation hybrids. We hypothesize that hybridization results in incompatibilities among subunits in ETS enzyme complexes and that these incompatibilities result in diminished mitochondrial function and fitness. To test this hypothesis, we measured fitness, mitochondrial function, and ETS enzyme activity in inbred recombinant hybrid lines of Tigriopus californicus. We found that (1) both fitness and mitochondrial function are reduced in hybrid lines, (2) only those ETS enzymes with both nuclear and mitochondrial subunits show a loss of activity in hybrid lines, and (3) positive relationships exist between ETS enzyme activity and mitochondrial function and between mitochondrial function and fitness. We also present evidence that hybrid lines harboring mitochondrial DNA (mtDNA) and mitochondrial RNA polymerase (mtRPOL) from the same parental source population have higher fitness than those with mtDNA and mtRPOL from different populations, suggesting that mitochondrial gene regulation may play a role in disruption of mitochondrial performance and fitness of hybrids. These results suggest that disruption of coadaptation between nuclear and mitochondrial genes contributes to the phenomenon of hybrid breakdown.