The Name Is Barrel, ß-Barrel.

ß-barrels are a class of membrane proteins made up of a cylindrical, anti-parallel ß-sheet with a hydrophobic exterior and a hydrophilic interior. The majority of proteins found in the outer membranes (OMs) of Gram-negative bacteria, mitochondria, and chloroplasts are ß-barrel outer membrane proteins (OMPs). ß-barrel OMPs have a diverse repertoire of functions, including nutrient transport, secretion, bacterial virulence, and enzymatic activity. Here, we discuss the broad functional classes of ß-barrel OMPs, how they are folded into the membrane, and the future of ß-barrel OMP research and its applications.

Energization of Outer Membrane Transport by the ExbB ExbD Molecular Motor.

The outer membranes (OM) of Gram-negative bacteria contain a class of proteins (TBDTs) that require energy for the import of nutrients and to serve as receptors for phages and protein toxins. Energy is derived from the proton motif force (pmf) of the cytoplasmic membrane (CM) through the action of three proteins, namely, TonB, ExbB, and ExbD, which are located in the CM and extend into the periplasm. The leaky phenotype of exbB exbD mutants is caused by partial complementation by homologous tolQ tolR. TonB, ExbB, and ExbD are genuine components of an energy transmission system from the CM into the OM. Mutant analyses, cross-linking experiments, and most recently X-ray and cryo-EM determinations were undertaken to arrive at a model that describes the energy transfer from the CM into the OM. These results are discussed in this paper. ExbB forms a pentamer with a pore inside, in which an ExbD dimer resides. This complex harvests the energy of the pmf and transmits it to TonB. TonB interacts with the TBDT at the TonB box, which triggers a conformational change in the TBDT that releases bound nutrients and opens the pore, through which nutrients pass into the periplasm. The structurally altered TBDT also changes the interactions of its periplasmic signaling domain with anti-sigma factors, with the consequence being that the sigma factors initiate transcription.

"The simple life experiences that every other human gets": Desire for normalcy among adolescents and young adults with advanced cancer.

BACKGROUND: Adolescents and young adults (AYAs) with advanced cancer identify normalcy as an important component of quality end-of-life care. We sought to define domains of normalcy and identify ways in which clinicians facilitate or hinder normalcy during advanced cancer care. PROCEDURE: This was a secondary analysis of a qualitative study that aimed to identify priority domains for end-of-life care. Content analysis of semi-structured interviews among AYAs aged 12-39 years with advanced cancer, caregivers, and clinicians was used to evaluate transcripts. Coded excerpts were reviewed to identify themes related to normalcy. RESULTS: Participants included 23 AYAs with advanced cancer, 28 caregivers, and 29 clinicians. Participants identified five domains of normalcy including relationships, activities, career/school, milestones, and appearance. AYAs and caregivers identified that clinicians facilitate normalcy through exploration of these domains with AYAs, allowing flexibility in care plans, identification of short-term and long-term goals across normalcy domains, and recognizing losses of normalcy that occur during cancer care. CONCLUSIONS: AYAs with cancer experience multiple threats to normalcy during advanced cancer care. Clinicians can attend to normalcy and improve AYA quality of life by acknowledging these losses through ongoing discussions on how best to support domains of normalcy and by reinforcing AYA identities beyond a cancer diagnosis.

Patient, Family, and Clinician Perspectives on Location of Death for Adolescents and Young Adults With Cancer.

PURPOSE: Adolescents and young adults (AYAs) with cancer have high rates of hospital deaths. It is not clear if this reflects their preferences or barriers to dying at home. METHODS: Between December 2018 and January 2021, we conducted in-depth interviews with AYAs (age 12-39 years) with stage IV or recurrent cancer, family caregivers including bereaved caregivers, and clinicians of AYAs with cancer. Patients were asked about their priorities for care including location of death, caregivers were asked what was most important in the care of their AYA family member, and clinicians were asked to reflect on priorities identified through caring for AYAs. Directed content analysis was applied to interview data, and themes regarding location of death were developed. RESULTS: Eighty individuals (23 AYAs, 28 caregivers, and 29 clinicians) participated in interviews. Most AYAs and caregivers preferred a home death. However, some AYAs and caregivers opted for a hospital death to alleviate caregiver burden or protect siblings from the perceived trauma of witnessing a home death. Lack of adequate services to manage intractable symptoms at home and insufficient caregiver support led some AYAs/caregivers to opt for hospital death despite a preference for home death. Participants acknowledged the value of hospice while also pointing out its limitations in attaining a home death. CONCLUSION: Although most AYAs prefer to die at home, this preference is not always achieved. Robust home-based services for effective symptom management and caregiver support are needed to close the gap between preferred and actual location of death for AYAs.

Mechanistic insights into fungal mitochondrial outer membrane protein biogenesis.

The majority of mitochondrial proteins are nuclear-encoded and need to be transported into the mitochondria, including the proteins in the outer mitochondrial membrane. For ß-barrel proteins, the preproteins are initially recognized and imported by the TOM complex, then shuttled to the SAM complex via small Tim proteins. For ⍺-helical proteins, some preproteins are recognized by the TOM complex and imported into the membrane by the MIM complex. In recent years multiple structures of the TOM complex and the SAM complex have been reported, increasing our understanding of the mechanism of protein biogenesis in the outer mitochondrial membrane.

The Ton Motor.

The Ton complex is a molecular motor at the inner membrane of Gram-negative bacteria that uses a proton gradient to apply forces on outer membrane (OM) proteins to permit active transport of nutrients into the periplasmic space. Recently, the structure of the ExbB-ExbD subcomplex was determined in several bacterial species, but the complete structure and stoichiometry of TonB have yet to be determined. The C-terminal end of TonB is known to cross the periplasm and interact with TonB-dependent outer membrane transport proteins with high affinity. Yet despite having significant knowledge of these transport proteins, it is not clear how the Ton motor opens a pathway across the outer membrane for nutrient import. Additionally, the mechanism by which energy is harnessed from the inner membrane subcomplex and transduced to the outer membrane via TonB is not well understood. In this review, we will discuss the gaps in the knowledge about the complete structure of the Ton motor complex and the relationship between ion flow used to generate mechanical work at the outer membrane and the nutrient transport process.

Persistent and toxic chemical pollutants in fish consumed by Asians in Chicago, United States.

Consumption of seafood brings health benefits but may increase the ingestion of contaminants. Compared with other ethnic groups in the U.S., Asians consume seafood more frequently. However, there is little information about how culturally specific fish consumption contributes to exposure to toxicants. In this work, we surveyed fish consumption among Chinese, Korean and Vietnamese communities and purchased 103 seafood samples from local markets in Chicago. Each sample was analyzed for mercury (Hg) and 92 organic chemicals including polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs), polybrominated diphenyl ethers (PBDEs), and Dechlorane Plus and related compounds (DPs). The rank order of pollutant concentration in all samples was Hg â‰« Σ66PCBs > Σ17OCPs > Σ8PBDEs > Σ8DPs. Positive correlations were noted among most contaminant groups. Bluefish, pike and tuna steak had the highest mean Hg (>1 mg/kg). The mean Σ66PCBs was highest in pike and bluefish (>100 ng/g) followed by pollock and mackerel (>40 ng/g). Overall, octopus, shrimp and tilapia were the least contaminated; while pike, bluefish, and pollock were the most contaminated. Omega-3 fatty acids were more strongly affiliated with the organic contaminants than mercury. A risk assessment identified seven types of fish that should have consumption limitations and six that should not be consumed. For these seafoods, consumption advice based on Hg levels would adequately protect health. In the survey participants, 17% of seafood mass consumed is from types of fish that should be limited to 1 or 2 meals/week, while 7% of the seafood mass consumed comes from types that should not be consumed at all. This work adds additional contaminants to the profile of health risks resulting from fish consumption among Asian Americans, which can be used in interventions aimed at conserving consumption of healthy fish while avoiding contaminants.

The Role of Voltage-Dependent Anion Channel in Mitochondrial Dysfunction and Human Disease.

The voltage-dependent anion channel (VDAC) is a ß-barrel membrane protein located in the outer mitochondrial membrane (OMM). VDAC has two conductance states: an open anion selective state, and a closed and slightly cation-selective state. VDAC conductance states play major roles in regulating permeability of ATP/ADP, regulation of calcium homeostasis, calcium flux within ER-mitochondria contact sites, and apoptotic signaling events. Three reported structures of VDAC provide information on the VDAC open state via X-ray crystallography and nuclear magnetic resonance (NMR). Together, these structures provide insight on how VDAC aids metabolite transport. The interaction partners of VDAC, together with the permeability of the pore, affect the molecular pathology of diseases including Parkinson's disease (PD), Friedreich's ataxia (FA), lupus, and cancer. To fully address the molecular role of VDAC in disease pathology, major questions must be answered on the structural conformers of VDAC. For example, further information is needed on the structure of the closed state, how binding partners or membrane potential could lead to the open/closed states, the function and mobility of the N-terminal α-helical domain of VDAC, and the physiological role of VDAC oligomers. This review covers our current understanding of the various states of VDAC, VDAC interaction partners, and the roles they play in mitochondrial regulation pertaining to human diseases.

Patient, Family, and Clinician Perspectives on End-of-Life Care Quality Domains and Candidate Indicators for Adolescents and Young Adults With Cancer.

Importance: End-of-life care quality indicators specific to adolescents and young adults (AYAs) aged 12 to 39 years with cancer have not been developed. Objective: To identify priority domains for end-of-life care from the perspectives of AYAs, family caregivers, and clinicians, and to propose candidate quality indicators reflecting priorities. Design, Setting, and Participants: This qualitative study was conducted from December 6, 2018, to January 5, 2021, with no additional follow-up. In-depth interviews were conducted with patients, family caregivers, and clinicians and included a content analysis of resulting transcripts. A multidisciplinary advisory group translated priorities into proposed quality indicators. Interviews were conducted at the Dana-Farber Cancer Institute, Kaiser Permanente Northern California, Kaiser Permanente Southern California, and an AYA cancer support community (lacunaloft.org). Participants included 23 AYAs, 28 caregivers, and 29 clinicians. Exposure: Stage IV or recurrent cancer. Main Outcomes and Measures: Care priorities. Results: Interviews were conducted with 23 patients (mean [SD] age, 29.3 [7.3] years; 12 men [52%]; 18 White participants [78%]), 28 family caregivers (23 women [82%]; 14 White participants [50%]), and 29 clinicians (20 women [69%]; 13 White participants [45%]). Caregivers included 22 parents (79%), 5 spouses or partners (18%), and 1 other family member (4%); the 29 clinicians included 15 physicians (52%), 6 nurses or nurse practitioners (21%), and 8 social workers or psychologists (28%). Interviews identified 7 end-of-life priority domains: attention to physical symptoms, attention to quality of life, psychosocial and spiritual care, communication and decision-making, relationships with clinicians, care and treatment, and independence. Themes were consistent across the AYA age range and participant type. Although some domains were represented in quality indicators developed for adults, unique domains were identified, as well as AYA-specific manifestations of existing domains. For example, quality of life included global quality of life; attainment of life goals, legacy, and meaning; support of personal relationships; and normalcy. Within communication and decision-making, domains included communication early in the disease course, addressing prognosis and what to expect at the end of life, and opportunity for AYAs to hold desired roles in decision-making. Care and treatment domains relevant to cancer therapy, use of life-prolonging measures, and location of death emphasized the need for preference sensitivity rather than a standard path. This finding differs from existing adult indicators that propose that late-life chemotherapy, intensive measures, and hospital death should be rare. Conclusions and Relevance: The findings of this qualitative study suggest that AYAs with cancer have priorities for care at the end of life that are not fully encompassed in existing indicators for adults. Use of new indicators for this young population may better reflect patient- and family-centered experiences of quality care.

A Biochemical and Structural Understanding of TOM Complex Interactions and Implications for Human Health and Disease.

The central role mitochondria play in cellular homeostasis has made its study critical to our understanding of various aspects of human health and disease. Mitochondria rely on the translocase of the outer membrane (TOM) complex for the bulk of mitochondrial protein import. In addition to its role as the major entry point for mitochondrial proteins, the TOM complex serves as an entry pathway for viral proteins. TOM complex subunits also participate in a host of interactions that have been studied extensively for their function in neurodegenerative diseases, cardiovascular diseases, innate immunity, cancer, metabolism, mitophagy and autophagy. Recent advances in our structural understanding of the TOM complex and the protein import machinery of the outer mitochondrial membrane have made structure-based therapeutics targeting outer mitochondrial membrane proteins during mitochondrial dysfunction an exciting prospect. Here, we describe advances in understanding the TOM complex, the interactome of the TOM complex subunits, the implications for the development of therapeutics, and our understanding of the structure/function relationship between components of the TOM complex and mitochondrial homeostasis.

Protein import and export across the bacterial outer membrane.

The bacterial outer membrane forms an impermeable barrier to the environment, but a wide variety of substances must cross it without compromising the membrane. Perhaps, the most fascinating transport phenomenon is the import and export of very large protein toxins using relatively small ß-barrel proteins residing in the outer membrane. Progress has been made on three systems in recent years that shed light on this process. In this review, we summarize bacteriocin (toxin) import using TonB-dependent transporters and protein secretion by autotransporters and two partner secretion systems.

Building Better Barrels - ß-barrel Biogenesis and Insertion in Bacteria and Mitochondria.

ß-barrel proteins are folded and inserted into outer membranes by multi-subunit protein complexes that are conserved across different types of outer membranes. In Gram-negative bacteria this complex is the barrel-assembly machinery (BAM), in mitochondria it is the sorting and assembly machinery (SAM) complex, and in chloroplasts it is the outer envelope protein Oep80. Mitochondrial ß-barrel precursor proteins are translocated from the cytoplasm to the intermembrane space by the translocase of the outer membrane (TOM) complex, and stabilized by molecular chaperones before interaction with the assembly machinery. Outer membrane bacterial BamA interacts with four periplasmic accessory proteins, whereas mitochondrial Sam50 interacts with two cytoplasmic accessory proteins. Despite these major architectural differences between BAM and SAM complexes, their core proteins, BamA and Sam50, seem to function the same way. Based on the new SAM complex structures, we propose that the mitochondrial ß-barrel folding mechanism follows the budding model with barrel-switching aiding in the release of new barrels. We also built a new molecular model for Tom22 interacting with Sam37 to identify regions that could mediate TOM-SAM supercomplex formation.

Ton motor complexes.

The Ton complex is a molecular motor that uses the proton gradient at the inner membrane of Gram-negative bacteria to apply forces on outer membrane proteins, allowing active transport of nutrients into the periplasmic space. For decades, contradictory data has been reported on the structure and stoichiometry of the Ton complex. However, recent reports strongly support a subunit stoichiometry of 5:2 for the ExbB-ExbD subcomplex. In this review, we summarize the recent discoveries of the structures and proposed mechanisms of the Ton system, as well as similar protein motor complexes in Gram-negative bacteria.

In-depth interrogation of protein thermal unfolding data with MoltenProt.

Protein stability is a key factor in successful structural and biochemical research. However, the approaches for systematic comparison of protein stability are limited by sample consumption or compatibility with sample buffer components. Here we describe how miniaturized measurement of intrinsic tryptophan fluorescence (NanoDSF assay) in combination with a simplified description of protein unfolding can be used to interrogate the stability of a protein sample. We demonstrate that improved protein stability measures, such as apparent Gibbs free energy of unfolding, rather than melting temperature Tm , should be used to rank the results of thermostability screens. The assay is compatible with protein samples of any composition, including protein complexes and membrane proteins. Our data analysis software, MoltenProt, provides an easy and robust way to perform characterization of multiple samples. Potential applications of MoltenProt and NanoDSF include buffer and construct optimization for X-ray crystallography and cryo-electron microscopy, screening for small-molecule binding partners and comparison of effects of point mutations.

Author Correction: Cryo-EM structure of the bacterial Ton motor subcomplex ExbB-ExbD provides information on structure and stoichiometry.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Levels of Ethylene Oxide Biomarker in an Exposed Residential Community.

The purpose of this study was to examine whether there is a difference in ethylene oxide (EtO) biomarker levels based on residential proximity to facilities emitting EtO, a carcinogen. We recruited residents living near two EtO-emitting facilities and administered a questionnaire on items such as address and length of residency, smoking habits, occupational exposures to EtO, and demographics. We also collected venous blood samples to measure an EtO biomarker, hemoglobin adduct N-2-hydroxyethyl-valine (HbEO), and cotinine, a metabolite of nicotine. Questionnaires and blood samples were collected from 93 participants. The overall geometric HbEO adduct level was 35.0 pmol/gmHb and for nonsmokers it was 29.7 pmol/gmHb. Mean HbEO adduct levels were not significantly associated with sex, age, race, BMI, or education level. HbEO adduct levels for nonsmoking participants who lived in a neighborhood approximately 0.8 km from one of the facilities were significantly higher compared to persons living farther away (p < 0.001). These results suggest that facilities that emit EtO may put nearby communities at risk of cancer and other associated health outcomes.

Structural insight into toxin secretion by contact-dependent growth inhibition transporters.

Bacterial contact-dependent growth inhibition (CDI) systems use a type Vb secretion mechanism to export large CdiA toxins across the outer membrane by dedicated outer membrane transporters called CdiB. Here, we report the first crystal structures of two CdiB transporters from Acinetobacter baumannii and Escherichia coli. CdiB transporters adopt a TpsB fold, containing a 16-stranded transmembrane ß-barrel connected to two periplasmic domains. The lumen of the CdiB pore is occluded by an N-terminal α-helix and the conserved extracellular loop 6; these two elements adopt different conformations in the structures. We identified a conserved DxxG motif located on strand ß1 that connects loop 6 through different networks of interactions. Structural modifications of DxxG induce rearrangement of extracellular loops and alter interactions with the N-terminal α-helix, preparing the system for α-helix ejection. Using structural biology, functional assays, and molecular dynamics simulations, we show how the barrel pore is primed for CdiA toxin secretion.

Exposure characteristics for congeners, isomers, and enantiomers of perfluoroalkyl substances in mothers and infants.

Per- and polyfluoroalkyl substances (PFASs) are ubiquitous in the environment, making it inevitable for humans to be exposed to these pollutants. The exposure begins while in utero and continues in infancy, during the potentially most sensitive early stages of life. This review summarizes the current knowledge on pre- and neo-natal exposures based on more than 200 articles published from 2000 to date. All relevant biological matrices used in the cited studies were included, such as maternal blood, umbilical cord blood, breast milk, placenta, amniotic fluid, fetal organs, newborns' dried blood spots, and infant serum. We show that such exposures are geographically global with significant discrepancies among countries and continents, and that while the levels of major legacy PFASs (PFOS and PFOA) have declined since 2000, those of others may have not. We also show that levels of PFOS and PFOA exceed those of some major environmental toxins, such as p,p'-DDE, BDE-47, PCB-153, PBB-153, and OH-PBDEs in maternal blood. Given that the behavior and potential effects have an origin in molecular structure, biomonitoring and research at the levels of isomers and enantiomers are critically important. Through critical analysis of these works, we summarize the major achievements, consensus, and the deficiencies of existing research. To our knowledge, this is the first review on the overall internal exposure status of mothers and infants to PFASs during pregnancy and lactation.

Structural insight into mitochondrial ß-barrel outer membrane protein biogenesis.

In mitochondria, ß-barrel outer membrane proteins mediate protein import, metabolite transport, lipid transport, and biogenesis. The Sorting and Assembly Machinery (SAM) complex consists of three proteins that assemble as a 1:1:1 complex to fold ß-barrel proteins and insert them into the mitochondrial outer membrane. We report cryoEM structures of the SAM complex from Myceliophthora thermophila, which show that Sam50 forms a 16-stranded transmembrane ß-barrel with a single polypeptide-transport-associated (POTRA) domain extending into the intermembrane space. Sam35 and Sam37 are located on the cytosolic side of the outer membrane, with Sam35 capping Sam50, and Sam37 interacting extensively with Sam35. Sam35 and Sam37 each adopt a GST-like fold, with no functional, structural, or sequence similarity to their bacterial counterparts. Structural analysis shows how the Sam50 ß-barrel opens a lateral gate to accommodate its substrates.