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Eight first-time and eight second-time working class mothers were observed interacting with their newborn babies during the first half-hour of life. In all cases the father was present for at least some of this period. The interactions were notable for their passivity, mothers looked for long periods at their babies and engaged in some finger-tip touching of the babies face and head, but they engaged in very little exploration of the babies, few vocalizations and few expressive acts such as kissing. However, the presence of the father was associated with more intense mother-newborn interaction. First-time but not second-time fathers tended to be excluded from interaction with either the mother or the baby at this time. These results are discussed in the light of current thinking on mother-newborn bonding and the role of midwifery staff in promoting that bonding process. [ABSTRACT FROM AUTHOR]

The serine/threonine protein phosphatase 5 (PP5) regulates hormone and stress-induced signaling networks. Unlike other phosphoprotein phosphatases, PP5 contains both regulatory and catalytic domains and is further regulated through post-translational modifications (PTMs). Here we identify that SUMOylation of K430 in the catalytic domain of PP5 regulates phosphatase activity. Additionally, phosphorylation of PP5-T362 is pre-requisite for SUMOylation, suggesting the ordered addition of PTMs regulates PP5 function in cells. Using the glucocorticoid receptor, a well known substrate for PP5, we demonstrate that SUMOylation results in substrate release from PP5. We harness this information to create a non-SUMOylatable K430R mutant as a 'substrate trap' and globally identified novel PP5 substrate candidates. Lastly, we generated a consensus dephosphorylation motif using known substrates, and verified its presence in the new candidate substrates. This study unravels the impact of cross talk of SUMOylation and phosphorylation on PP5 phosphatase activity and substrate release in cells., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Background/aim: This study examined the effects of tocotrienols (TT) in conjunction with statin on glucose homeostasis, bone microstructure, gut microbiome, and systemic and liver inflammatory markers in obese C57BL/6J mice., Materials and Methods: Forty male C57BL/6J mice were fed a high-fat diet (HFD) and assigned into four groups in a 2 (no statin vs. 120 mg statin/kg diet)×2 (no TT vs. 400 mg TT/kg diet) factorial design for 14 weeks., Results: Statin and TT improved glucose tolerance only when each was given alone, and only statin supplementation decreased insulin resistance. Consistently, only statin supplementation decreased serum insulin levels and HOMA-IR. Pancreatic insulin was also increased with statin treatment. Statin and TT, alone or in combination, reduced the levels of serum IL-6, but only TT attenuated the increased serum leptin levels induced by a HFD. Statin supplementation increased bone area/total area and connectivity density at LV-4, while TT supplementation increased bone area/total area and trabecular number, but decreased trabecular separation at the distal femur. Statin supplementation, but not TT, reduced hepatic inflammatory cytokine gene expression. Neither TT supplementation nor statin supplementation statistically altered microbiome species evenness or richness. However, they altered the relative abundance of certain microbiome species. Most notably, both TT and statin supplementation increased the relative abundance of Lachnospiraceae UCG-006., Conclusion: TT and statin collectively benefit bone microstructure, glucose homeostasis, and microbial ecology in obese mice. Such changes may be, in part, associated with suppression of inflammation in the host., (Copyright © 2024, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Facioscapulohumeral dystrophy (FSHD) has a unique genetic aetiology resulting in partial chromatin relaxation of the D4Z4 macrosatellite repeat array on 4qter. This D4Z4 chromatin relaxation facilitates inappropriate expression of the transcription factor DUX4 in skeletal muscle. DUX4 is encoded by a retrogene that is embedded within the distal region of the D4Z4 repeat array. In the European population, the D4Z4 repeat array is usually organized in a single array that ranges between 8 and 100 units. D4Z4 chromatin relaxation and DUX4 derepression in FSHD is most often caused by repeat array contraction to 1-10 units (FSHD1) or by a digenic mechanism requiring pathogenic variants in a D4Z4 chromatin repressor like SMCHD1, combined with a repeat array between 8 and 20 units (FSHD2). With a prevalence of 1.5% in the European population, in cis duplications of the D4Z4 repeat array, where two adjacent D4Z4 arrays are interrupted by a spacer sequence, are relatively common but their relationship to FSHD is not well understood. In cis duplication alleles were shown to be pathogenic in FSHD2 patients; however, there is inconsistent evidence for the necessity of an SMCHD1 mutation for disease development. To explore the pathogenic nature of these alleles we compared in cis duplication alleles in FSHD patients with or without pathogenic SMCHD1 variant. For both groups we showed duplication-allele-specific DUX4 expression. We studied these alleles in detail using pulsed-field gel electrophoresis-based Southern blotting and molecular combing, emphasizing the challenges in the characterization of these rearrangements. Nanopore sequencing was instrumental to study the composition and methylation of the duplicated D4Z4 repeat arrays and to identify the breakpoints and the spacer sequence between the arrays. By comparing the composition of the D4Z4 repeat array of in cis duplication alleles in both groups, we found that specific combinations of proximal and distal repeat array sizes determine their pathogenicity. Supported by our algorithm to predict pathogenicity, diagnostic laboratories should now be furnished to accurately interpret these in cis D4Z4 repeat array duplications, alleles that can easily be missed in routine settings., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Guarantors of Brain.)

Serine/threonine protein phosphatase-5 (PP5) is involved in tumor progression and survival, making it an attractive therapeutic target. Specific inhibition of protein phosphatases has remained challenging because of their conserved catalytic sites. PP5 contains its regulatory domains within a single polypeptide chain, making it a more desirable target. Here we used an in silico approach to screen and develop a selective inhibitor of PP5. Compound P053 is a competitive inhibitor of PP5 that binds to its catalytic domain and causes apoptosis in renal cancer. We further demonstrated that PP5 interacts with FADD, RIPK1, and caspase 8, components of the extrinsic apoptotic pathway complex II. Specifically, PP5 dephosphorylates and inactivates the death effector protein FADD, preserving complex II integrity and regulating extrinsic apoptosis. Our data suggests that PP5 promotes renal cancer survival by suppressing the extrinsic apoptotic pathway. Pharmacologic inhibition of PP5 activates this pathway, presenting a viable therapeutic strategy for renal cancer., Competing Interests: Declaration of interests The authors declare no competing financial interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Fascioscapulohumeral muscular dystrophy (FSHD) is caused by a unique genetic mechanism that relies on contraction and hypomethylation of the D4Z4 macrosatellite array on the Chromosome 4q telomere allowing ectopic expression of the DUX4 gene in skeletal muscle. Genetic analysis is difficult because of the large size and repetitive nature of the array, a nearly identical array on the 10q telomere, and the presence of divergent D4Z4 arrays scattered throughout the genome. Here, we combine nanopore long-read sequencing with Cas9-targeted enrichment of 4q and 10q D4Z4 arrays for comprehensive genetic analysis including determination of the length of the 4q and 10q D4Z4 arrays with base-pair resolution. In the same assay, we differentiate 4q from 10q telomeric sequences, determine A/B haplotype, identify paralogous D4Z4 sequences elsewhere in the genome, and estimate methylation for all CpGs in the array. Asymmetric, length-dependent methylation gradients were observed in the 4q and 10q D4Z4 arrays that reach a hypermethylation point at approximately 10 D4Z4 repeat units, consistent with the known threshold of pathogenic D4Z4 contractions. High resolution analysis of individual D4Z4 repeat methylation revealed areas of low methylation near the CTCF/insulator region and areas of high methylation immediately preceding the DUX4 transcriptional start site. Within the DUX4 exons, we observed a waxing/waning methylation pattern with a 180-nucleotide periodicity, consistent with phased nucleosomes. Targeted nanopore sequencing complements recently developed molecular combing and optical mapping approaches to genetic analysis for FSHD by adding precision of the length measurement, base-pair resolution sequencing, and quantitative methylation analysis., (© 2023 Butterfield et al.; Published by Cold Spring Harbor Laboratory Press.)

The major determinant of disease severity in Duchenne muscular dystrophy (DMD) or milder Becker muscular dystrophy (BMD) is whether the dystrophin gene (DMD) mutation truncates the mRNA reading frame or allows expression of a partially functional protein. However, even in the complete absence of dystrophin, variability in disease severity is observed, and candidate gene studies have implicated several genes as modifiers. Here we present the largest genome-wide search to date for loci influencing severity in N = 419 DMD patients. Availability of subjects for such studies is quite limited, leading to modest sample sizes, which present a challenge for GWAS design. We have therefore taken special steps to minimize heterogeneity within our dataset at the DMD locus itself, taking a novel approach to mutation classification to effectively exclude the possibility of residual dystrophin expression, and utilized statistical methods that are well adapted to smaller sample sizes, including the use of a novel linear regression-like residual for time to ambulatory loss and the application of evidential statistics for the GWAS approach. Finally, we applied an unbiased in silico pipeline, utilizing functional genomic datasets to explore the potential impact of the best supported SNPs. In all, we obtained eight SNPs (out of 1,385,356 total) with posterior probability of trait-marker association (PPLD) ≥ 0.4, representing six distinct loci. Our analysis prioritized likely non-coding SNP regulatory effects on six genes (ETAA1, PARD6G, GALNTL6, MAN1A1, ADAMTS19, and NCALD), each with plausibility as a DMD modifier. These results support both recurrent and potentially new pathways for intervention in the dystrophinopathies., (© 2023. The Author(s), under exclusive licence to European Society of Human Genetics.)

DMD pathogenic variants for Duchenne and Becker muscular dystrophy are detectable with high sensitivity by standard clinical exome analyses of genomic DNA. However, up to 7% of DMD mutations are deep intronic and analysis of muscle-derived RNA is an important diagnostic step for patients who have negative genomic testing but abnormal dystrophin expression in muscle. In this study, muscle biopsies were evaluated from 19 patients with clinical features of a dystrophinopathy, but negative clinical DMD mutation analysis. Reverse transcription-polymerase chain reaction or high-throughput RNA sequencing methods identified 19 mutations with one of three pathogenic pseudoexon types: deep intronic point mutations, deletions or insertions, and translocations. In association with point mutations creating intronic splice acceptor sites, we observed the first examples of DMD pseudo 3'-terminal exon mutations causing high efficiency transcription termination within introns. This connection between splicing and premature transcription termination is reminiscent of U1 snRNP-mediating telescripting in sustaining RNA polymerase II elongation across large genes, such as DMD. We propose a novel classification of three distinct types of mutations identifiable by muscle RNA analysis, each of which differ in potential treatment approaches. Recognition and appropriate characterization may lead to therapies directed toward full-length dystrophin expression for some patients., (© 2022 Wiley Periodicals LLC.)

Fibromyalgia (FM) is a prevalent, chronic condition without a cure or reliable therapy. The etiopathogenesis of this syndrome is ambiguous, which has heightened the challenge of discovering treatments to minimize patients' painful symptoms. FM is characterized by diffuse musculoskeletal pain usually accompanied by functional pain syndromes, such as fatigue, sleep disturbances, cognitive difficulties, and mood issues. Currently available treatment options for FM are limited. Recent studies have suggested a potential role for dietary bioactive compounds in FM management. We performed a narrative review to evaluate the existing evidence regarding the dietary bioactive compounds for FM, and we proposed molecular mechanisms on this topic. The inclusion criteria were (i) human, in vivo, or in vitro studies, (ii) studies related to the effect of bioactive compounds on FM-like symptoms, (iii) peer-reviewed literature, and (iv) publications until February 2022 in PubMed and Google Scholar. Exclusion criteria were (i) study designs using CCI, SNI, or SNL models because they are more NP models rather than FM models, and (ii) studies published in a language other than English. Keywords were dietary bioactive compounds, fibromyalgia, cell, animals, humans. Here, we report the effects of commonly consumed bioactive compounds (capsaicin, ginger, curcumin, n-3 PUFA, grape seed extract, naringin, and genistein) on FM-like symptoms in cellular, animal, and human studies. Cellular studies demonstrated that these bioactive compounds reduce pro-inflammatory production and increase antioxidant capacity of neurons or myoblasts that regulate apoptosis/cell survival. Animal studies showed that these regularly consumed bioactive compounds have an effect on FM-like symptoms, as evidenced by decreased pain hypersensitivity and fatigue as well as improved social behaviors. Further studies are warranted to allow meaningful comparison and quantification of the efficacy of these bioactive compounds on FM-like symptoms across studies, in terms of actual changes in antioxidant capacity, pain hypersensitivity, fatigue, and social behaviors. To date, human studies regarding the efficacy of these bioactive compounds on FM-like symptoms are limited and inconclusive. Our review identifies this important knowledge gap and proposes that the development and use of improved preclinical FM models are needed, particularly concerning the usage of female animals to better mimic FM pathophysiology and symptomatology.

Osteoporosis is a major health problem in postmenopausal women. Herein we evaluated the effects of 12-week tocotrienols (TT) supplementation on serum metabolites in postmenopausal, osteopenic women. Eighty-nine participants (59.7 ± 6.8 yr, BMI 28.7 ± 5.7 kg/m 2 ) were assigned to 3 treatments: placebo (860 mg olive oil/day), 300mg TT (300 mg TT/day), and 600mg TT (600 mg TT/day) for 12 weeks. TT consisted of 90% δ-TT and 10% γ-TT. In this metabolomic study, we evaluated the placebo and 600mgTT at baseline and 12 weeks. As expected, TT and its metabolite levels were higher in the supplemented group after 12 weeks. At baseline, there were no differences in demographic parameters or comprehensive metabolic panels (CMP). Metabolomics analysis of serum samples revealed that 48 biochemicals were higher and 65 were lower in the 600mg TT group at 12 weeks, compared to baseline. The results confirmed higher serum levels of tocotrienols and lysophospholipids, but lower acylcarnitines and catabolites of tryptophan and steroids in subjects given 600mg TT. In summary, 12-week TT supplementation altered many serum metabolite levels in postmenopausal women. The present study supports our previous findings that TT supplementation helps reduce bone loss in postmenopausal osteopenic women by suppressing inflammation and oxidative stress. Furthermore, the body incorporates TT which restructures biomembranes and modifies phospholipid metabolism, a response potentially linked to reduced inflammation and oxidative stress., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Shen, Mo, Dunn and Watkins.)

Heat shock protein-90 (Hsp90) is an essential molecular chaperone in eukaryotes that plays a vital role in protecting and maintaining the functional integrity of deregulated signaling proteins in tumors. We have previously reported that the stability and activity of the mitotic checkpoint kinase Mps1 depend on Hsp90. In turn, Mps1-mediated phosphorylation Hsp90 regulates its chaperone function and is essential for the mitotic arrest. Cdc14-assisted dephosphorylation of Hsp90 is vital for the mitotic exit. Post-translational regulation of Hsp90 function is also known as the Hsp90 "Chaperone Code." Here, we demonstrate that only the active Mps1 is ubiquitinated on K86, K827, and K848 by the tumor suppressor von Hippel-Lindau (VHL) containing E3 enzyme, in a prolyl hydroxylation-independent manner and degraded in the proteasome. Furthermore, we show that this process regulates cell exit from the mitotic checkpoint. Collectively, our data demonstrates an interplay between the Hsp90 chaperone and VHL degradation machinery in regulating mitosis., (© 2021. The Author(s).)

Exon skipping therapies for Duchenne muscular dystrophy that restore an open reading frame can be induced by the use of noncoding U7 small nuclear RNA (U7snRNA) modified by an antisense exon-targeting sequence delivered by an adeno-associated virus (AAV) vector. We have developed an AAV vector (AAV9.U7-ACCA) containing four U7snRNAs targeting the splice donor and acceptor sites of dystrophin exon 2, resulting in highly efficient exclusion of DMD exon 2. We assessed the specificity of splice variation induced by AAV9.U7-ACCA delivery in the Dmd exon 2 duplication (Dup2) mouse model through an unbiased RNA-seq approach. Treatment-related effects on pre-mRNA splicing were quantified using local splicing variation (LSV) analysis. Filtering the transcriptome for differences in treatment-related splicing resulted in only 16 candidate off-target LSVs. Only a single candidate off-target LSV was found in both skeletal and cardiac muscle tissue and occurred at a known variable cassette exon. In contrast, four LSVs represented significant on-target correction of Dmd exon 2 splicing and transcriptome analysis showed correction of known dystrophin-deficient gene dysregulation. We conclude that the absence of off-target splicing induced by treatment with the U7-ACCA vector supports the continued clinical development of this approach.

Gene networks for disorders of social behavior provide the mechanisms critical for identifying therapeutic targets and biomarkers. Large behavioral phenotypic effects of small human deletions make the positive sociality of Williams syndrome (WS) ideal for determining transcriptional networks for social dysfunction currently based on DNA variations for disorders such as autistic spectrum disorder (ASD) and schizophrenia (SCHZ). Consensus on WS networks has been elusive due to the need for larger cohort size, sensitive genome-wide detection and analytic tools. We report a core set of WS network perturbations in a cohort of 58 individuals (34 with typical, 6 atypical deletions and 18 controls). Genome-wide exon-level expression arrays robustly detected changes in differentially expressed gene (DEG) transcripts from WS deleted genes that ranked in the top 11 of 12 122 transcripts, validated by quantitative reverse transcription PCR, RNASeq and western blots. WS DEG's were strictly dosed in the full but not the atypical deletions that revealed a breakpoint position effect on non-deleted CLIP2, a caveat for current phenotypic mapping based on copy number variants. Network analyses tested the top WS DEG's role in the dendritic spine, employing GeneMANIA to harmonize WS DEGs with comparable query gene-sets. The results indicate perturbed actin cytoskeletal signaling analogous to the excitatory dendritic spines. Independent protein-protein interaction analyses of top WS DEGs generated a 100-node graph annotated topologically revealing three interacting pathways, MAPK, IGF1-PI3K-AKT-mTOR/insulin and actin signaling at the synapse. The results indicate striking similarity of WS transcriptional networks to genome-wide association study-based ASD and SCHZ risk suggesting common network dysfunction for these disorders of divergent sociality., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Genome erosion is a frequently observed result of relaxed selection in insect nutritional symbionts, but it has rarely been studied in defensive mutualisms. Solitary beewolf wasps harbor an actinobacterial symbiont of the genus Streptomyces that provides protection to the developing offspring against pathogenic microorganisms. Here, we characterized the genomic architecture and functional gene content of this culturable symbiont using genomics, transcriptomics, and proteomics in combination with in vitro assays. Despite retaining a large linear chromosome (7.3 Mb), the wasp symbiont accumulated frameshift mutations in more than a third of its protein-coding genes, indicative of incipient genome erosion. Although many of the frameshifted genes were still expressed, the encoded proteins were not detected, indicating post-transcriptional regulation. Most pseudogenization events affected accessory genes, regulators, and transporters, but " Streptomyces philanthi " also experienced mutations in central metabolic pathways, resulting in auxotrophies for biotin, proline, and arginine that were confirmed experimentally in axenic culture. In contrast to the strong A+T bias in the genomes of most obligate symbionts, we observed a significant G+C enrichment in regions likely experiencing reduced selection. Differential expression analyses revealed that-compared to in vitro symbiont cultures-" S. philanthi " in beewolf antennae showed overexpression of genes for antibiotic biosynthesis, the uptake of host-provided nutrients and the metabolism of building blocks required for antibiotic production. Our results show unusual traits in the early stage of genome erosion in a defensive symbiont and suggest tight integration of host-symbiont metabolic pathways that effectively grants the host control over the antimicrobial activity of its bacterial partner., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Background: Myotonic dystrophy type 1 (DM1) is caused by CTG repeat expansions in the DMPK gene and is the most common form of muscular dystrophy. Patients can have long delays from onset to diagnosis, since clinical signs and symptoms are often nonspecific and overlapping with other disorders. Clinical genetic testing by Southern blot or triplet-primed PCR (TP-PCR) is technically challenging and cost prohibitive for population surveys., Methods: Here, we present a high throughput, low-cost screening tool for CTG repeat expansions using TP-PCR followed by high resolution melt curve analysis with saturating concentrations of SYBR GreenER dye., Results: We determined that multimodal melt profiles from the TP-PCR assay are a proxy for amplicon length stoichiometry. In a screen of 10,097 newborn blood spots, melt profile analysis accurately reflected the tri-modal distribution of common alleles from 5 to 35 CTG repeats, and identified the premutation and full expansion alleles., Conclusion: We demonstrate that robust detection of expanded CTG repeats in a single tube can be achieved from samples derived from specimens with minimal template DNA such as dried blood spots (DBS). This technique is readily adaptable to large-scale testing programs such as population studies and newborn screening programs., (© 2021 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

Human Cytomegalovirus (HCMV) infection modulates cellular metabolism to support viral replication. Calcium/calmodulin-dependent kinase kinase (CaMKK) and AMP-activated protein kinase (AMPK) regulate metabolic activation and have been found to be important for successful HCMV infection. Here, we explored the contributions that specific CaMKK isoforms and AMPK subunit isoforms make toward HCMV infection. Our results indicate that various CaMKK and AMPK isoforms contribute to infection in unique ways. For example, CaMKK1 is important for HCMV infection at a low multiplicity of infection, but is dispensable for AMPK activation at the earliest times of infection, which our data suggest is more reliant on CaMKK2. Our results also indicate that HCMV specifically induces the expression of the non-ubiquitous AMPKa2 catalytic subunit, found to be important for both HCMV-mediated glycolytic activation and high titer infection. Further, we find that AMPK-mediated glycolytic activation is important for infection, as overexpression of GLUT4, the high capacity glucose transporter, partially rescues viral replication in the face of AMPK inhibition. Collectively, our data indicate that HCMV infection selectively induces the expression of specific metabolic regulatory kinases, relying on their activity to support glycolytic activation and productive infection. IMPORTANCE Viruses are obligate parasites that depend on the host cell to provide the energy and molecular building blocks to mass produce infectious viral progeny. The processes that govern viral modulation of cellular resources have emerged as critical for successful infection. Here, we find that HCMV depends on two kinase isoforms to support infection, CaMKK1 and AMPKa2. We find that HCMV specifically induces expression of the AMPKa2 subunit to induce metabolic activation and drive robust viral replication. These results suggest that HCMV has evolved mechanisms to target specific metabolic regulatory kinase subunits to support productive infection, thereby providing insight into how HCMV hijacks cellular metabolism for its replication, and sheds light on potential viral therapeutic vulnerabilities., (Copyright © 2020 American Society for Microbiology.)

Calcium signaling and the AMP-activated protein kinase (AMPK) signaling networks broadly regulate numerous aspects of cell biology. Human Cytomegalovirus (HCMV) infection has been found to actively manipulate the calcium-AMPK signaling axis to support infection. Many HCMV genes have been linked to modulating calcium signaling, and HCMV infection has been found to be reliant on calcium signaling and AMPK activation. Here, we focus on the cell biology of calcium and AMPK signaling and what is currently known about how HCMV modulates these pathways to support HCMV infection and potentially contribute to oncomodulation., (Copyright © 2020 Dunn and Munger.)

Eosinophilia (EOS) is an important component of airway inflammation and hyperresponsiveness in allergic reactions including those leading to asthma. Although cigarette smoking (CS) is a significant contributor to long-term adverse outcomes in these lung disorders, there are also the curious reports of its ability to produce acute suppression of inflammatory responses including EOS through poorly understood mechanisms. One possibility is that proinflammatory processes are suppressed by nicotine in CS acting through nicotinic receptor α7 (α7). Here we addressed the role of α7 in modulating EOS with two mouse models of an allergic response: house dust mites (HDM; Dermatophagoides sp.) and ovalbumin (OVA). The influence of α7 on EOS was experimentally resolved in wild-type mice or in mice in which a point mutation of the α7 receptor (α7 E260A:G ) selectively restricts normal signaling of cellular responses. RNA analysis of alveolar macrophages and the distal lung epithelium indicates that normal α7 function robustly impacts gene expression in the epithelium to HDM and OVA but to different degrees. Notable was allergen-specific α7 modulation of Ccl11 and Ccl24 (eotaxins) expression, which was enhanced in HDM but suppressed in OVA EOS. CS suppressed EOS induced by both OVA and HDM, as well as the inflammatory genes involved, regardless of α7 genotype. These results suggest that EOS in response to HDM or OVA is through signaling pathways that are modulated in a cell-specific manner by α7 and are distinct from CS suppression.

Objective: Duchenne muscular dystrophy (DMD) is a severe X-linked recessive disease caused by loss-of-function dystrophin (DMD) mutations in boys, who typically suffer loss of ambulation by age 12. Previously, we reported that coding variants in latent transforming growth factor beta (TGFβ)-binding protein 4 (LTBP4) were associated with reduced TGFβ signaling and prolonged ambulation (p = 1.0 × 10 -3 ) in DMD patients; this result was subsequently replicated by other groups. In this study, we evaluated whether additional DMD modifier genes are observed using whole-genome association in the original cohort., Methods: We performed a genome-wide association study (GWAS) for single-nucleotide polymorphisms (SNPs) influencing loss of ambulation (LOA) in the same cohort of 253 DMD patients used to detect the candidate association with LTBP4 coding variants. Gene expression and chromatin interaction databases were used to fine-map association signals above the threshold for genome-wide significance., Results: Despite the small sample size, two loci associated with prolonged ambulation met genome-wide significance and were tagged by rs2725797 (chr15, p = 6.6 × 10 -9 ) and rs710160 (chr19, p = 4.7 × 10 -8 ). Gene expression and chromatin interaction data indicated that the latter SNP tags regulatory variants of LTBP4, whereas the former SNP tags regulatory variants of thrombospondin-1 (THBS1): an activator of TGFβ signaling by direct binding to LTBP4 and an inhibitor of proangiogenic nitric oxide signaling., Interpretation: Together with previous evidence implicating LTBP4, the THBS1 modifier locus emphasizes the role that common regulatory variants in gene interaction networks can play in mitigating disease progression in muscular dystrophy. Ann Neurol 2018;84:234-245., (© 2018 American Neurological Association.)

The human MN blood group antigens are isoforms of glycophorin A (GPA) encoded by the gene, GYPA, and are the most abundant erythrocyte sialoglycoproteins. The distribution of MN antigens has been widely studied in human populations yet the evolutionary and/or demographic factors affecting population variation remain elusive. While the primary function of GPA is yet to be discovered, it serves as the major binding site for the 175-kD erythrocyte-binding antigen (EB-175) of the malarial parasite, Plasmodium falciparum, a major selective pressure in recent human history. More specifically, exon two of GYPA encodes the receptor-binding ligand to which P. falciparum binds. Accordingly, there has been keen interest in understanding what impact, if any, natural selection has had on the distribution of variation in GYPA and exon two in particular. To this end, we resequenced GYPA in individuals sampled from both P. falciparum endemic (sub-Saharan Africa and South India) and non-endemic (Europe and East Asia) regions of the world. Observed patterns of variation suggest that GYPA has been subject to balancing selection in populations living in malaria endemic areas and in Europeans, but no such evidence was found in samples from East Asia, Oceania, and the Americas. These results are consistent with malaria acting as a selective pressure on GYPA, but also suggest that another selective force has resulted in a similar pattern of variation in Europeans. Accordingly, GYPA has perhaps a more complex evolutionary history, wherein on a global scale, spatially varying selective pressures have governed its natural history.

Objectives: The collagen VI related muscular dystrophies (COL6-RD), Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy (BM) are among the most common congenital muscular dystrophies and are characterized by distal joint laxity and a combination of distal and proximal joint contractures. Inheritance can be dominant negative (DN) or recessive depending on the type and location of the mutation. DN mutations allow incorporation of abnormal chains into secreted tetramers and are the most commonly identified mutation type in COL6-RD. Null alleles (nonsense, frameshift, and large deletions) do not allow incorporation of abnormal chains and act recessively. To better define the pathways disrupted by mutations in collagen VI, we have used a transcriptional profiling approach with RNA-Seq to identify differentially expressed genes in COL6-RD individuals from controls., Methods: RNA-Seq allows precise detection of all expressed transcripts in a sample and provides a tool for quantification of expression data on a genomic scale. We have used RNA-Seq to identify differentially expressed genes in cultured dermal fibroblasts from 13 COL6-RD individuals (8 dominant negative and 5 null) and 6 controls. To better assess the transcriptional changes induced by abnormal collagen VI in the extracellular matrix (ECM); we compared transcriptional profiles from subjects with DN mutations and subjects with null mutations to transcriptional profiles from controls., Results: Differentially expressed transcripts between COL6-RD and control fibroblasts include upregulation of ECM components and downregulation of factors controlling matrix remodeling and repair. DN and null samples are differentiated by downregulation of genes involved with DNA replication and repair in null samples., Conclusions: Differentially expressed genes identified here may help identify new targets for development of therapies and biomarkers to assess the efficacy of treatments.

The tumor suppressors Tsc1 and Tsc2 form the tuberous sclerosis complex (TSC), a regulator of mTOR activity. Tsc1 stabilizes Tsc2; however, the precise mechanism involved remains elusive. The molecular chaperone heat-shock protein 90 (Hsp90) is an essential component of the cellular homeostatic machinery in eukaryotes. Here, we show that Tsc1 is a new co-chaperone for Hsp90 that inhibits its ATPase activity. The C-terminal domain of Tsc1 (998-1,164 aa) forms a homodimer and binds to both protomers of the Hsp90 middle domain. This ensures inhibition of both subunits of the Hsp90 dimer and prevents the activating co-chaperone Aha1 from binding the middle domain of Hsp90. Conversely, phosphorylation of Aha1-Y223 increases its affinity for Hsp90 and displaces Tsc1, thereby providing a mechanism for equilibrium between binding of these two co-chaperones to Hsp90. Our findings establish an active role for Tsc1 as a facilitator of Hsp90-mediated folding of kinase and non-kinase clients-including Tsc2-thereby preventing their ubiquitination and proteasomal degradation., (© 2017 The Authors. Published under the terms of the CC BY 4.0 license.)

The serine/threonine protein phosphatase 5 (PP5) regulates multiple cellular signaling networks. A number of cellular factors, including heat shock protein 90 (Hsp90), promote the activation of PP5. However, it is unclear whether post-translational modifications also influence PP5 phosphatase activity. Here, we show an "on/off switch" mechanism for PP5 regulation. The casein kinase 1δ (CK1δ) phosphorylates T362 in the catalytic domain of PP5, which activates and enhances phosphatase activity independent of Hsp90. Overexpression of the phosphomimetic T362E-PP5 mutant hyper-dephosphorylates substrates such as the co-chaperone Cdc37 and glucocorticoid receptor in cells. Our proteomic approach revealed that the tumor suppressor von Hippel-Lindau protein (VHL) interacts with and ubiquitinates K185/K199-PP5 for proteasomal degradation in a hypoxia- and prolyl-hydroxylation-independent manner. Finally, VHL-deficient clear cell renal cell carcinoma (ccRCC) cell lines and patient tumors exhibit elevated PP5 levels. Downregulation of PP5 causes ccRCC cells to undergo apoptosis, suggesting a prosurvival role for PP5 in kidney cancer., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Cigarette smoking (CS) is a principal contributor to a spectrum of devastating lung diseases whose occurrence and severity may vary between individuals and not appear for decades after prolonged use. One explanation for the variability and delay in disease onset is that nicotine, the addictive component of CS, acts through the ionotropic nicotinic acetylcholine receptor (nAChR) alpha7 (α7) to modulate anti-inflammatory protection. In this study we measured the impact α7 signaling has on the mouse distal lung response to side-stream CS exposure for mice of the control genotype (α7G) and those in which the α7-receptor signaling mechanisms are restricted by point mutation (α7E260A:G). Flow cytometry results show that after CS there is an increase in a subset of CD11c (CD11chi) alveolar macrophages (AMs) and histology reveals an increase in these cells within the alveolar space in both genotypes although the α7E260A:G AMs tend to accumulate into large aggregates rather than more widely distributed solitary cells common to the α7G lung after CS. Changes to lung morphology with CS in both genotypes included increased tissue cavitation due to alveolar expansion and bronchial epithelium dysplasia in part associated with altered club cell morphology. RNA-Seq analysis revealed changes in epithelium gene expression after CS are largely independent of the α7-genotype. However, the α7E260A:G genotype did reveal some unique variations to transcript expression of gene sets associated with immune responsiveness and macrophage recruitment, hypoxia, genes encoding mitochondrial respiration complex I and extracellular fibrillary matrix proteins (including alterations to fibrotic deposits in the α7G proximal airway bronchioles after CS). These results suggest α7 has a central role in modulating the response to chronic CS that could include altering susceptibility to associated lung diseases including fibrosis and cancer.

Purpose of Review: This review evaluates the state of the research regarding the effects of postpartum mental illness on the developing infant. It defines the scope of these disorders in the literature, and includes the impact of disorders referred to as perinatal mood and anxiety disorders (PMADs) on infants., Recent Findings: New research reveals that PMADs apply to not only mothers, but that fathers can also experience perinatal depression and anxiety. When untreated in a primary caregiver, PMADs adversely affect parental cognitions and beliefs, attachment to the infant, and the growing caregiver-infant relationship. PMADs affect early developmental outcomes of infants including neurosynaptic development, regulatory development, and developmental milestones. Early identification and treatment for PMADs are critical to ensure optimal infant development. Standardized and routine screening for PMADs, especially in the first 6 months postpartum, and cross-disciplinary communication among medical providers afford the best opportunity for early identification and treatment.

This work evaluates chronic safety in middle-aged ovariectomized rats supplemented with different dosages of green tea polyphenols (GTP) in drinking water. The experiment used 6-mo-old sham (n = 39) and ovariectomized (OVX, n = 143) female rats. All sham (n = 39) and 39 of the OVX animals received no GTP treatment and their samples were collected for outcome measures at baseline, 3 mo, and 6 mo (n = 13 per group for each). The remaining OVX animals were randomized into 4 groups receiving 0.15%, 0.5%, 1%, and 1.5% (n = 26 for each) of GTP (wt/vol), respectively, in drinking water for 3 and 6 mo. No mortality or abnormal treatment-related findings in clinical observations or ophthalmologic examinations were noted. No treatment-related macroscopic or microscopic findings were noted for animals administered 1.5% GTP supplementation. Throughout the study, there was no difference in the body weight among all OVX groups. In all OVX groups, feed intake and water consumption significantly decreased with GTP dose throughout the study period. At 6 mo, GTP intake did not affect hematology, clinical chemistry, and urinalysis, except for phosphorus and blood urea nitrogen (increased), total cholesterol, lactate dehydrogenase, and urine pH (decreased). This study reveals that the no-observed-adverse-effect level (NOAEL) of GTP is 1.5% (wt/vol) in drinking water, the highest dose used in this study., (© 2017 Institute of Food Technologists®.)

Nicotine modulates multiple inflammatory responses in the lung through the nicotinic acetylcholine receptor subtype alpha7 (α7). Previously we reported that α7 modulates both the hematopoietic and epithelium responses in the lung to the bacterial inflammogen, lipopolysaccharide (LPS). Here we apply immunohistochemistry, flow cytometry and RNA-Seq analysis of isolated distal lung epithelium to further define α7-expression and function in this tissue. Mouse lines were used that co-express a bicistronic tau-green fluorescent protein (tGFP) as a reporter of α7 (α7G) expression and that harbor an α7 with a specific point mutation (α7E260A:G) that selectively uncouples it from cell calcium-signaling mechanisms. The tGFP reporter reveals strong cell-specific α7-expression by alveolar macrophages (AM), Club cells and ATII cells. Ciliated cells do not express detectible tGFP, but their numbers decrease by one-third in the α7E260A:G lung compared to controls. Transcriptional comparisons (RNA-Seq) between α7G and α7E260A:G enriched lung epithelium 24 hours after challenge with either intra-nasal (i.n.) saline or LPS reveals a robust α7-genotype impact on both the stasis and inflammatory response of this tissue. Overall the α7E260A:G lung epithelium exhibits reduced inflammatory cytokine/chemokine expression to i.n. LPS. Transcripts specific to Club cells (e.g., CC10, secretoglobins and Muc5b) or to ATII cells (e.g., surfactant proteins) were constitutively decreased in in the α7E260A:G lung, but they were strongly induced in response to i.n. LPS. Protein analysis applying immunohistochemistry and ELISA also revealed α7-associated differences suggested by RNA-Seq including altered mucin protein 5b (Muc5b) accumulation in the α7E260A:G bronchia, that in some cases appeared to form airway plugs, and a substantial increase in extracellular matrix deposits around α7E260A:G airway bronchia linings that was not seen in controls. Our results show that α7 is an important modulator of normal gene expression stasis and the response to an inhaled inflammogen in the distal lung epithelium. Further, when normal α7 signaling is disrupted, changes in lung gene expression resemble those associated with long-term lung pathologies seen in humans who use inhaled nicotine products.

The serine/threonine phosphatase protein phosphatase 5 (PP5) regulates hormone- and stress-induced cellular signaling by association with the molecular chaperone heat shock protein 90 (Hsp90). PP5-mediated dephosphorylation of the cochaperone Cdc37 is essential for activation of Hsp90-dependent kinases. However, the details of this mechanism remain unknown. We determined the crystal structure of a Cdc37 phosphomimetic peptide bound to the catalytic domain of PP5. The structure reveals PP5 utilization of conserved elements of phosphoprotein phosphatase (PPP) structure to bind substrate and provides a template for many PPP-substrate interactions. Our data show that, despite a highly conserved structure, elements of substrate specificity are determined within the phosphatase catalytic domain itself. Structure-based mutations in vivo reveal that PP5-mediated dephosphorylation is required for kinase and steroid hormone receptor release from the chaperone complex. Finally, our data show that hyper- or hypoactivity of PP5 mutants increases Hsp90 binding to its inhibitor, suggesting a mechanism to enhance the efficacy of Hsp90 inhibitors by regulation of PP5 activity in tumors.

Heat shock protein-90 (Hsp90) is an essential molecular chaperone in eukaryotes involved in maintaining the stability and activity of numerous signalling proteins, also known as clients. Hsp90 ATPase activity is essential for its chaperone function and it is regulated by co-chaperones. Here we show that the tumour suppressor FLCN is an Hsp90 client protein and its binding partners FNIP1/FNIP2 function as co-chaperones. FNIPs decelerate the chaperone cycle, facilitating FLCN interaction with Hsp90, consequently ensuring FLCN stability. FNIPs compete with the activating co-chaperone Aha1 for binding to Hsp90, thereby providing a reciprocal regulatory mechanism for chaperoning of client proteins. Lastly, downregulation of FNIPs desensitizes cancer cells to Hsp90 inhibitors, whereas FNIPs overexpression in renal tumours compared with adjacent normal tissues correlates with enhanced binding of Hsp90 to its inhibitors. Our findings suggest that FNIPs expression can potentially serve as a predictive indicator of tumour response to Hsp90 inhibitors.

The molecular chaperone Hsp90 protects deregulated signaling proteins that are vital for tumor growth and survival. Tumors generally display sensitivity and selectivity toward Hsp90 inhibitors; however, the molecular mechanism underlying this phenotype remains undefined. We report that the mitotic checkpoint kinase Mps1 phosphorylates a conserved threonine residue in the amino-domain of Hsp90. This, in turn, regulates chaperone function by reducing Hsp90 ATPase activity while fostering Hsp90 association with kinase clients, including Mps1. Phosphorylation of Hsp90 is also essential for the mitotic checkpoint because it confers Mps1 stability and activity. We identified Cdc14 as the phosphatase that dephosphorylates Hsp90 and disrupts its interaction with Mps1. This causes Mps1 degradation, thus providing a mechanism for its inactivation. Finally, Hsp90 phosphorylation sensitizes cells to its inhibitors, and elevated Mps1 levels confer renal cell carcinoma selectivity to Hsp90 drugs. Mps1 expression level can potentially serve as a predictive indicator of tumor response to Hsp90 inhibitors., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Heat shock protein-90 (Hsp90) is a molecular chaperone critical to the folding, stability and activity of over 200 client proteins including many responsible for tumor initiation, progression and metastasis. Hsp90 chaperone function is linked to its ATPase activity and Hsp90 inhibitors interfere with this activity, thereby making Hsp90 an attractive target for cancer therapy. Also post-translational modification (PTM) and co-chaperone proteins modulate Hsp90 function, providing additional targets for secondary inhibition. Recent reports have shown that pathogens utilize both their own Hsp90 and that of their host for the propagation of infectious elements. In this review we will summarize our current knowledge of Hsp90 structure and function in both the pathogen and the host. We will focus on the role of Hsp90 in viral and parasitic diseases and the potential beneficial application of Hsp90 inhibitors alone and in combination with disease-specific inhibitors.

Heat Shock Protein 90 (Hsp90) is an essential chaperone that supports the function of a wide range of signaling molecules. Hsp90 binds to a suite of co-chaperone proteins that regulate Hsp90 function through alteration of intrinsic ATPase activity. Several studies have determined Aha1 to be an important co-chaperone whose binding to Hsp90 is modulated by phosphorylation, acetylation and SUMOylation of Hsp90 [1], [2]. In this study, we applied quantitative affinity-purification mass spectrometry (AP-MS) proteomics to understand how phosphorylation of hAha1 at Y223 altered global client/co-chaperone interaction [3]. Specifically, we characterized and compared the interactomes of Aha1-Y223F (phospho-mutant form) and Aha1-Y223E (phospho-mimic form). We identified 99 statistically significant interactors of hAha1, a high proportion of which (84%) demonstrated preferential binding to the phospho-mimic form of hAha1. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository [4] with the dataset identifier PXD001737.

The ability of Heat Shock Protein 90 (Hsp90) to hydrolyze ATP is essential for its chaperone function. The co-chaperone Aha1 stimulates Hsp90 ATPase activity, tailoring the chaperone function to specific "client" proteins. The intracellular signaling mechanisms directly regulating Aha1 association with Hsp90 remain unknown. Here, we show that c-Abl kinase phosphorylates Y223 in human Aha1 (hAha1), promoting its interaction with Hsp90. This, consequently, results in an increased Hsp90 ATPase activity, enhances Hsp90 interaction with kinase clients, and compromises the chaperoning of non-kinase clients such as glucocorticoid receptor and CFTR. Suggesting a regulatory paradigm, we also find that Y223 phosphorylation leads to ubiquitination and degradation of hAha1 in the proteasome. Finally, pharmacologic inhibition of c-Abl prevents hAha1 interaction with Hsp90, thereby hypersensitizing cancer cells to Hsp90 inhibitors both in vitro and ex vivo., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: Exon-skipping therapies aim to convert Duchenne muscular dystrophy (DMD) into less severe Becker muscular dystrophy (BMD) by altering pre-mRNA splicing to restore an open reading frame, allowing translation of an internally deleted and partially functional dystrophin protein. The most common single exon deletion-exon 45 (Δ45)-may theoretically be treated by skipping of either flanking exon (44 or 46). We sought to predict the impact of these by assessing the clinical severity in dystrophinopathy patients., Methods: Phenotypic data including clinical diagnosis, age at wheelchair use, age at loss of ambulation, and presence of cardiomyopathy were analyzed from 41 dystrophinopathy patients containing equivalent in-frame deletions., Results: As expected, deletions of either exons 45 to 47 (Δ45-47) or exons 45 to 48 (Δ45-48) result in BMD in 97% (36 of 37) of subjects. Unexpectedly, deletion of exons 45 to 46 (Δ45-46) is associated with the more severe DMD phenotype in 4 of 4 subjects despite an in-frame transcript. Notably, no patients with a deletion of exons 44 to 45 (Δ44-45) were found within the United Dystrophinopathy Project database, and this mutation has only been reported twice before, which suggests an ascertainment bias attributable to a very mild phenotype., Interpretation: The observation that Δ45-46 patients have typical DMD suggests that the conformation of the resultant protein may result in protein instability or altered binding of critical partners. We conclude that in DMD patients with Δ45, skipping of exon 44 and multiexon skipping of exons 46 and 47 (or exons 46-48) are better potential therapies than skipping of exon 46 alone., (© 2015 American Neurological Association.)

Most mutations that truncate the reading frame of the DMD gene cause loss of dystrophin expression and lead to Duchenne muscular dystrophy. However, amelioration of disease severity has been shown to result from alternative translation initiation beginning in DMD exon 6 that leads to expression of a highly functional N-truncated dystrophin. Here we demonstrate that this isoform results from usage of an internal ribosome entry site (IRES) within exon 5 that is glucocorticoid inducible. We confirmed IRES activity by both peptide sequencing and ribosome profiling in muscle from individuals with minimal symptoms despite the presence of truncating mutations. We generated a truncated reading frame upstream of the IRES by exon skipping, which led to synthesis of a functional N-truncated isoform in both human subject-derived cell lines and in a new DMD mouse model, where expression of the truncated isoform protected muscle from contraction-induced injury and corrected muscle force to the same level as that observed in control mice. These results support a potential therapeutic approach for patients with mutations within the 5' exons of DMD.

There is a wealth of information on the genetic regulation and biochemical properties of bacterial C4-dicarboxylate transport systems. In sharp contrast, there are far fewer studies describing the transport and assimilation of C5-dicarboxylates among bacteria. In an effort to better our understanding on this subject, we identified the structural and regulatory genes necessary for the utilization of α-ketoglutarate (α-KG) in Pseudomonas aeruginosa PAO1. The PA5530 gene, encoding a putative dicarboxylate transporter, was found to be essential for the growth of P. aeruginosa PAO1 on both α-KG and glutarate (another C5-dicarboxylate). Metabolite analysis confirmed that the PA5530 gene was necessary for the uptake of extracellular α-KG. Like other substrate-inducible transporter genes, expression of the PA5530 gene was induced by extracellular C5-dicarboxylates. It was later found that the expression of the PA5530 gene was driven solely by a -24/-12 promoter recognized by the alternative sigma factor RpoN. Surprisingly, the enhancer binding protein MifR, which is known to have an essential role in biofilm development, was required for the expression of the PA5530 gene. The MifR protein is homologous to other transcriptional regulators involved in dicarboxylate assimilation, suggesting that MifR might interact with RpoN to activate the expression of the PA5530 gene in response to extracellular C5-dicarboxylates, especially α-KG. The results of this study provide a framework for exploring the assimilation of α-KG in other pseudomonads., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

The breastfeeding experience for the mother and infant is often complicated by a constellation of challenges that are difficult for lactation consultants alone to treat. To address this issue, a breastfeeding consultation clinic at Children's Hospital Colorado developed a multidisciplinary team: a pediatrician specializing in breastfeeding medicine, a lactation consultant, and a clinical psychologist specializing in infant mental health and child development. This Trifecta Breastfeeding Approach meets families' needs by addressing the infant's medical care, functional breastfeeding challenges, and the developing mother-infant relationship, and by screening for concurrent pregnancy-related mood disorders. The Approach also recognizes family dynamics and the transition to parenthood within the breastfeeding consultation. Issues of lost expectations, grief, infertility, high-risk infants, and fussiness often need to be addressed. Case examples here illustrate the benefits of this multidisciplinary, integrated health model. This type of integrated care will likely have an increased presence in health care systems as reimbursement for psychologists' fees and innovative models of care continue to emerge.

Symbiotic associations between animals and microbes are ubiquitous in nature, with an estimated 15% of all insect species harboring intracellular bacterial symbionts. Most bacterial symbionts share many genomic features including small genomes, nucleotide composition bias, high coding density, and a paucity of mobile DNA, consistent with long-term host association. In this study, we focus on the early stages of genome degeneration in a recently derived insect-bacterial mutualistic intracellular association. We present the complete genome sequence and annotation of Sitophilus oryzae primary endosymbiont (SOPE). We also present the finished genome sequence and annotation of strain HS, a close free-living relative of SOPE and other insect symbionts of the Sodalis-allied clade, whose gene inventory is expected to closely resemble the putative ancestor of this group. Structural, functional, and evolutionary analyses indicate that SOPE has undergone extensive adaptation toward an insect-associated lifestyle in a very short time period. The genome of SOPE is large in size when compared with many ancient bacterial symbionts; however, almost half of the protein-coding genes in SOPE are pseudogenes. There is also evidence for relaxed selection on the remaining intact protein-coding genes. Comparative analyses of the whole-genome sequence of strain HS and SOPE highlight numerous genomic rearrangements, duplications, and deletions facilitated by a recent expansion of insertions sequence elements, some of which appear to have catalyzed adaptive changes. Functional metabolic predictions suggest that SOPE has lost the ability to synthesize several essential amino acids and vitamins. Analyses of the bacterial cell envelope and genes encoding secretion systems suggest that these structures and elements have become simplified in the transition to a mutualistic association.