Pathogenic bacteria modulate pheromone response to promote mating.

Pathogens generate ubiquitous selective pressures and host-pathogen interactions alter social behaviours in many animals1-4. However, very little is known about the neuronal mechanisms underlying pathogen-induced changes in social behaviour. Here we show that in adult Caenorhabditis elegans hermaphrodites, exposure to a bacterial pathogen (Pseudomonas aeruginosa) modulates sensory responses to pheromones by inducing the expression of the chemoreceptor STR-44 to promote mating. Under standard conditions, C. elegans hermaphrodites avoid a mixture of ascaroside pheromones to facilitate dispersal5-13. We find that exposure to the pathogenic Pseudomonas bacteria enables pheromone responses in AWA sensory neurons, which mediate attractive chemotaxis, to suppress the avoidance. Pathogen exposure induces str-44 expression in AWA neurons, a process regulated by a transcription factor zip-5 that also displays a pathogen-induced increase in expression in AWA. STR-44 acts as a pheromone receptor and its function in AWA neurons is required for pathogen-induced AWA pheromone response and suppression of pheromone avoidance. Furthermore, we show that C. elegans hermaphrodites, which reproduce mainly through self-fertilization, increase the rate of mating with males after pathogen exposure and that this increase requires str-44 in AWA neurons. Thus, our results uncover a causal mechanism for pathogen-induced social behaviour plasticity, which can promote genetic diversity and facilitate adaptation of the host animals.

CREB mediates the C. elegans dauer polyphenism through direct and cell-autonomous regulation of TGF-ß expression.

Animals can adapt to dynamic environmental conditions by modulating their developmental programs. Understanding the genetic architecture and molecular mechanisms underlying developmental plasticity in response to changing environments is an important and emerging area of research. Here, we show a novel role of cAMP response element binding protein (CREB)-encoding crh-1 gene in developmental polyphenism of C. elegans. Under conditions that promote normal development in wild-type animals, crh-1 mutants inappropriately form transient pre-dauer (L2d) larvae and express the L2d marker gene. L2d formation in crh-1 mutants is specifically induced by the ascaroside pheromone ascr#5 (asc-ωC3; C3), and crh-1 functions autonomously in the ascr#5-sensing ASI neurons to inhibit L2d formation. Moreover, we find that CRH-1 directly binds upstream of the daf-7 TGF-ß locus and promotes its expression in the ASI neurons. Taken together, these results provide new insight into how animals alter their developmental programs in response to environmental changes.

Chemical Signaling Regulates Axon Regeneration via the GPCR-Gqα Pathway in Caenorhabditis elegans.

Chemical communication controls a wide range of behaviors via conserved signaling networks. Axon regeneration in response to injury is determined by the interaction between the extracellular environment and intrinsic growth potential. In this study, we investigated the role of chemical signaling in axon regeneration in Caenorhabditis elegans We find that the enzymes involved in ascaroside pheromone biosynthesis, ACOX-1.1, ACOX-1.2, and DAF-22, participate in axon regeneration by producing a dauer-inducing ascaroside, ascr#5. We demonstrate that the chemoreceptor genes, srg-36 and srg-37, which encode G-protein-coupled receptors for ascr#5, are required for adult-specific axon regeneration. Furthermore, the activating mutation in egl-30 encoding Gqα suppresses axon regeneration defective phenotype in acox-1.1 and srg-36 srg-37 mutants. Therefore, the ascaroside signaling system provides a unique example of a signaling molecule that regulates the regenerative pathway in the nervous system.SIGNIFICANCE STATEMENT In Caenorhabditis elegans, axon regeneration is positively regulated by the EGL-30 Gqα-JNK MAP kinase cascade. However, it remains unclear what signals activate the EGL-30 pathway in axon regeneration. Here, we show that SRG-36 and SRG-37 act as upstream G-protein-coupled receptors (GPCRs) that activate EGL-30. C. elegans secretes a family of small-molecule pheromones called ascarosides, which serve various functions in chemical signaling. SRG-36 and SRG-37 are GPCRs for the dauer-inducing ascaroside ascr#5. Consistent with this, we found that ascr#5 activates the axon regeneration pathway via SRG-36/SRG-37 and EGL-30. Thus, ascaroside signaling promotes axon regeneration by activating the GPCR-Gqα pathway.

Decision making in metoidioplasty and phalloplasty gender-affirming surgery: a mixed methods study.

BACKGROUND: Gender-affirming surgical procedures, such as metoidioplasty and phalloplasty for those assigned female at birth, are complex and multistaged and involve risks. Individuals considering these procedures experience greater uncertainty or decisional conflict, compounded by difficulty finding trustworthy information. AIM: (1) To explore the factors contributing to decisional uncertainty and the needs of individuals considering metoidioplasty and phalloplasty gender-affirming surgery (MaPGAS) and (2) to inform development of a patient-centered decision aid. METHODS: This cross-sectional study was based on mixed methods. Adult transgender men and nonbinary individuals assigned female at birth at various stages of MaPGAS decision making were recruited from 2 study sites in the United States to participate in semistructured interviews and an online gender health survey, which included measures of gender congruence, decisional conflict, urinary health, and quality of life. Trained qualitative researchers conducted all interviews with questions to explore constructs from the Ottawa decision support framework. OUTCOMES: Outcomes included goals and priorities for MaPGAS, expectations, knowledge, and decisional needs, as well as variations in decisional conflict by surgical preference, surgical status, and sociodemographic variables. RESULTS: We interviewed 26 participants and collected survey data from 39 (24 interviewees, 92%) at various stages of MaPGAS decision making. In surveys and interviews, affirmation of gender identity, standing to urinate, sensation, and the ability to "pass" as male emerged as highly important factors for deciding to undergo MaPGAS. A third of survey respondents reported decisional conflict. Triangulation of data from all sources revealed that conflict emerged most when trying to balance the strong desire to resolve gender dysphoria through surgical transition against the risks and unknowns in urinary and sexual function, appearance, and preservation of sensation post-MaPGAS. Insurance coverage, age, access to surgeons, and health concerns further influenced surgery preferences and timing. CLINICAL IMPLICATIONS: The findings add to the understanding of decisional needs and priorities of those considering MaPGAS while revealing new complexities among knowledge, personal factors, and decisional uncertainty. STRENGTHS AND LIMITATIONS: This mixed methods study was codeveloped by members of the transgender and nonbinary community and yielded important guidance for providers and individuals considering MaPGAS. The results provide rich qualitative insights for MaPGAS decision making in US contexts. Limitations include low diversity and sample size; both are being addressed in work underway. CONCLUSIONS: This study increases understanding of the factors important to MaPGAS decision making, and results are being used to guide development of a patient-centered surgical decision aid and informed survey revision for national distribution.

IL-17 is a neuromodulator of Caenorhabditis elegans sensory responses.

Interleukin-17 (IL-17) is a major pro-inflammatory cytokine: it mediates responses to pathogens or tissue damage, and drives autoimmune diseases. Little is known about its role in the nervous system. Here we show that IL-17 has neuromodulator-like properties in Caenorhabditis elegans. IL-17 can act directly on neurons to alter their response properties and contribution to behaviour. Using unbiased genetic screens, we delineate an IL-17 signalling pathway and show that it acts in the RMG hub interneurons. Disrupting IL-17 signalling reduces RMG responsiveness to input from oxygen sensors, and renders sustained escape from 21% oxygen transient and contingent on additional stimuli. Over-activating IL-17 receptors abnormally heightens responses to 21% oxygen in RMG neurons and whole animals. IL-17 deficiency can be bypassed by optogenetic stimulation of RMG. Inducing IL-17 expression in adults can rescue mutant defects within 6 h. These findings reveal a non-immunological role of IL-17 modulating circuit function and behaviour.

Structure and integration of specialty palliative care in three NCI-designated cancer centers: a mixed methods case study.

INTRODUCTION: Early access to specialty palliative care is associated with better quality of life, less intensive end-of-life treatment and improved outcomes for patients with advanced cancer. However, significant variation exists in implementation and integration of palliative care. This study compares the organizational, sociocultural, and clinical factors that support or hinder palliative care integration across three U.S. cancer centers using an in-depth mixed methods case study design and proposes a middle range theory to further characterize specialty palliative care integration. METHODS: Mixed methods data collection included document review, semi-structured interviews, direct clinical observation, and context data related to site characteristics and patient demographics. A mixed inductive and deductive approach and triangulation was used to analyze and compare sites' palliative care delivery models, organizational structures, social norms, and clinician beliefs and practices. RESULTS: Sites included an urban center in the Midwest and two in the Southeast. Data included 62 clinician and 27 leader interviews, observations of 410 inpatient and outpatient encounters and seven non-encounter-based meetings, and multiple documents. Two sites had high levels of "favorable" organizational influences for specialty palliative care integration, including screening, policies, and other structures facilitating integration of specialty palliative care into advanced cancer care. The third site lacked formal organizational policies and structures for specialty palliative care, had a small specialty palliative care team, espoused an organizational identity linked to treatment innovation, and demonstrated strong social norms for oncologist primacy in decision making. This combination led to low levels of specialty palliative care integration and greater reliance on individual clinicians to initiate palliative care. CONCLUSION: Integration of specialty palliative care services in advanced cancer care was associated with a complex interaction of organization-level factors, social norms, and individual clinician orientation. The resulting middle range theory suggests that formal structures and policies for specialty palliative care combined with supportive social norms are associated with greater palliative care integration in advanced cancer care, and less influence of individual clinician preferences or tendencies to continue treatment. These results suggest multi-faceted efforts at different levels, including social norms, may be needed to improve specialty palliative care integration for advanced cancer patients.

Problematic Integration: Racial Discordance in End-of-Life Decision Making.

We describe racially discordant oncology encounters involving EOL decision-making. Fifty-eight provider interviews were content analyzed using the tenets of problematic integration theory. We found EOL discussions between non-Black providers and their Black patients were often complex and anxiety-inducing. That anxiety consisted of (1) ontological uncertainty in which providers characterized the nature of Black patients as distrustful, especially in the context of clinical trials; (2) ontological and epistemological uncertainty in which provider intercultural incompetency and perceived lack of patient health literacy were normalized and intertwined with provider assumptions about patients' religion and support systems; (3) epistemological uncertainty as ambivalence in which providers' feelings conflicted when deciding whether to speak with family members they perceived as lacking health literacy; (4) divergence in which the provider advised palliative care while the family desired surgery or cancer-directed medical treatment; and (5) impossibility when an ontological uncertainty stance of Black distrust was seen as natural by providers and therefore impossible to change. Some communication strategies used were indirect stereotyping, negotiating, asking a series of value questions, blame-guilt framing, and avoidance. We concluded that provider perceptions of Black distrust, religion, and social support influenced their ability to communicate effectively with patients.

A novel soluble complement receptor 1 fragment with enhanced therapeutic potential.

Human complement receptor 1 (HuCR1) is a pivotal regulator of complement activity, acting on all three complement pathways as a membrane-bound receptor of C3b/C4b, C3/C5 convertase decay accelerator, and cofactor for factor I-mediated cleavage of C3b and C4b. In this study, we sought to identify a minimal soluble fragment of HuCR1, which retains the complement regulatory activity of the wildtype protein. To this end, we generated recombinant, soluble, and truncated versions of HuCR1 and compared their ability to inhibit complement activation in vitro using multiple assays. A soluble form of HuCR1, truncated at amino acid 1392 and designated CSL040, was found to be a more potent inhibitor than all other truncation variants tested. CSL040 retained its affinity to both C3b and C4b as well as its cleavage and decay acceleration activity and was found to be stable under a range of buffer conditions. Pharmacokinetic studies in mice demonstrated that the level of sialylation is a major determinant of CSL040 clearance in vivo. CSL040 also showed an improved pharmacokinetic profile compared with the full extracellular domain of HuCR1. The in vivo effects of CSL040 on acute complement-mediated kidney damage were tested in an attenuated passive antiglomerular basement membrane antibody-induced glomerulonephritis model. In this model, CSL040 at 20 and 60 mg/kg significantly attenuated kidney damage at 24 h, with significant reductions in cellular infiltrates and urine albumin, consistent with protection from kidney damage. CSL040 thus represents a potential therapeutic candidate for the treatment of complement-mediated disorders.

Feeding state regulates pheromone-mediated avoidance behavior via the insulin signaling pathway in Caenorhabditis elegans.

Animals change sensory responses and their eventual behaviors, depending on their internal metabolic status and external food availability. However, the mechanisms underlying feeding state-dependent behavioral changes remain undefined. Previous studies have shown that Caenorhabditis elegans hermaphrodite exhibits avoidance behaviors to acute exposure of a pheromone, ascr#3 (asc-ΔC9, C9). Here, we show that the ascr#3 avoidance behavior is modulated by feeding state via the insulin signaling pathway. Starvation increases ascr#3 avoidance behavior, and loss-of-function mutations in daf-2 insulin-like receptor gene dampen this starvation-induced ascr#3 avoidance behavior. DAF-2 and its downstream signaling molecules, including the DAF-16 FOXO transcription factor, act in the ascr#3-sensing ADL neurons to regulate synaptic transmission to downstream target neurons, including the AVA command interneurons. Moreover, we found that starvation decreases the secretion of INS-18 insulin-like peptides from the intestine, which antagonizes DAF-2 function in the ADL neurons. Altogether, this study provides insights about the molecular communication between intestine and sensory neurons delivering hunger message to sensory neurons, which regulates avoidance behavior from pheromones to facilitate survival chance.

Balancing selection shapes density-dependent foraging behaviour.

The optimal foraging strategy in a given environment depends on the number of competing individuals and their behavioural strategies. Little is known about the genes and neural circuits that integrate social information into foraging decisions. Here we show that ascaroside pheromones, small glycolipids that signal population density, suppress exploratory foraging in Caenorhabditis elegans, and that heritable variation in this behaviour generates alternative foraging strategies. We find that natural C. elegans isolates differ in their sensitivity to the potent ascaroside icas#9 (IC-asc-C5). A quantitative trait locus (QTL) regulating icas#9 sensitivity includes srx-43, a G-protein-coupled icas#9 receptor that acts in the ASI class of sensory neurons to suppress exploration. Two ancient haplotypes associated with this QTL confer competitive growth advantages that depend on ascaroside secretion, its detection by srx-43 and the distribution of food. These results suggest that balancing selection at the srx-43 locus generates alternative density-dependent behaviours, fulfilling a prediction of foraging game theory.

Modular Platform for the Development of Recombinant Hemoglobin Scavenger Biotherapeutics.

Cell-free hemoglobin (Hb) is a driver of disease progression in conditions with intravascular or localized hemolysis. Genetic and acquired anemias or emergency medical conditions such as aneurysmal subarachnoid hemorrhage involve tissue Hb exposure. Haptoglobin (Hp) captures Hb in an irreversible protein complex and prevents its pathophysiological contributions to vascular nitric oxide depletion and tissue oxidation. Preclinical proof-of-concept studies suggest that human plasma-derived Hp is a promising therapeutic candidate for several Hb-driven diseases. Optimizing the efficacy and safety of Hb-targeting biotherapeutics may require structural and functional modifications for specific indications. Improved Hp variants could be designed to achieve the desired tissue distribution, metabolism, and elimination to target hemolytic disease states effectively. However, it is critical to ensure that these modifications maintain the function of Hp. Using transient mammalian gene expression of Hp combined with co-transfection of the pro-haptoglobin processing protease C1r-LP, we established a platform for generating recombinant Hp-variants. We designed an Hpß-scaffold, which was expressed in this system at high levels as a monomeric unit (mini-Hp) while maintaining the key protective functions of Hp. We then used this Hpß-scaffold as the basis to develop an initial proof-of-concept Hp fusion protein using human serum albumin as the fusion partner. Next, a hemopexin-Hp fusion protein with bispecific heme and Hb detoxification capacity was generated. Further, we developed a Hb scavenger devoid of CD163 scavenger receptor binding. The functions of these proteins were then characterized for Hb and heme-binding, binding of the Hp-Hb complexes with the clearance receptor CD163, antioxidant properties, and vascular nitric oxide sparing capacity. Our platform is designed to support the generation of innovative Hb scavenger biotherapeutics with novel modes of action and potentially improved formulation characteristics, function, and pharmacokinetics.

Psychometric characteristics of the health care empowerment questionnaire in a sample of patients with arthritis and rheumatic conditions.

BACKGROUND: Patient empowerment can improve health-related outcomes and is important in chronic conditions, such as arthritis. This study aimed to validate the Health Care Empowerment Questionnaire (HCEQ), a patient-reported experience measure of empowerment, for use with patients with arthritis and other rheumatic diseases. METHODS: The HCEQ measures Patient Information Seeking (or Involvement in Decisions) and Healthcare Interaction Results (or Involvement in Interactions) and asks respondents to answer questions in two ways: whether they feel something happened and its importance to them. Face validity was assessed through qualitative data (n = 8, nominal group technique; n = 55, focus groups). Measure structure was assessed through confirmatory factor analysis (CFA); internal consistency was also assessed (n = 9226). Test-retest reliability was assessed with sub-sample of participants (n = 182). RESULTS: We found adequate face validity of the HCEQ for patients with arthritis. The CFA indicated good fit to the data for the two-factor structure of the HCEQ (RMSEA = 0.075; CFI = 0.987; TLI = 0.978; SRMR = 0.026). Internal consistency was strong (α=0.94 for both subscales). Test-retest reliability was moderate for Patient Information Seeking (ICC=0.67) and good for Healthcare Interaction Results (ICC=0.77). CONCLUSIONS: The HCEQ, with modifications, demonstrated promising psychometric properties within this sample, laying the foundation for further assessment. This work supports the HCEQ as an appropriate instrument for examining experiences with and perceived importance of empowerment in individuals with arthritis and other rheumatic conditions. PATIENT CONTRIBUTION: Patients contributed to the assessment of face validity. As a measure of patient empowerment, the HCEQ's use can enable further participation of patients in health care.

Toward Improved Identification of Parental Substance Misuse: An Examination of Current Practices and Gaps in One US State.

INTRODUCTION: The use of illicit substances, including opioids, is a serious public health issue in the United States. While there are reports of the impact of the ongoing opioid crisis on adults, a new focus has emerged on how parental substance misuse (PSM) affects children. This study explored existing screening and assessment practices and services for children and families affected by PSM across different service sectors in one state. The purpose of the study was to identify opportunities for training, policy development, and practice improvement related to identifying PSM and linking children and parents to services. METHODS: Interviews (n = 15) with professionals from five service sectors (mental health, primary care, schools, community programs, and law enforcement) were used to inform development of a state-wide survey of the same groups (n = 498) to assess current practices, attitudes, knowledge, and training needs related to child screening of PSM. The survey was piloted using cognitive interviewing (n = 9) before it was distributed. RESULTS: Fewer than 20% of survey respondents reported using standardized tools specific to screening PSM. Informal assessment practices predominate, though 60% of respondents saw value in adopting more standardized PSM screening. Attitudes about PSM and screening varied among sectors but interest in training was high. DISCUSSION: Results indicate a need for more systematic PSM screening, cross-sector training and practice discussions, and policies to support early identification of children affected by PSM. Ramifications of these findings and recommendations are discussed.

A Large Family of Enzymes Responsible for the Modular Architecture of Nematode Pheromones.

The nematode Caenorhabditis elegans produces a broad family of pheromones, known as the ascarosides, that are modified with a variety of groups derived from primary metabolism. These modifications are essential for the diverse activities of the ascarosides in development and various behaviors, including attraction, aggregation, avoidance, and foraging. The mechanism by which these different groups are added to the ascarosides is poorly understood. Here, we identify a family of over 30 enzymes, which are homologous to mammalian carboxylesterase (CES) enzymes, and show that a number of these enzymes are responsible for the selective addition of specific modifications to the ascarosides. Through stable isotope feeding experiments, we demonstrate the in vivo activity of the CES-like enzymes and provide direct evidence that the acyl-CoA synthetase ACS-7, which was previously implicated in the attachment of certain modifications to the ascarosides in C. elegans, instead activates the side chains of certain ascarosides for shortening through ß-oxidation. Our data provide a key to the combinatorial logic that gives rise to different modified ascarosides, which should greatly facilitate the exploration of the specific biological functions of these pheromones in the worm.

rIgG1 Fc Hexamer Inhibits Antibody-Mediated Autoimmune Disease via Effects on Complement and FcγRs.

Activation of Fc receptors and complement by immune complexes is a common important pathogenic trigger in many autoimmune diseases and so blockade of these innate immune pathways may be an attractive target for treatment of immune complex-mediated pathomechanisms. High-dose IVIG is used to treat autoimmune and inflammatory diseases, and several studies demonstrate that the therapeutic effects of IVIG can be recapitulated with the Fc portion. Further, recent data indicate that recombinant multimerized Fc molecules exhibit potent anti-inflammatory properties. In this study, we investigated the biochemical and biological properties of an rFc hexamer (termed Fc-µTP-L309C) generated by fusion of the IgM µ-tailpiece to the C terminus of human IgG1 Fc. Fc-µTP-L309C bound FcγRs with high avidity and inhibited FcγR-mediated effector functions (Ab-dependent cell-mediated cytotoxicity, phagocytosis, respiratory burst) in vitro. In addition, Fc-µTP-L309C prevented full activation of the classical complement pathway by blocking C2 cleavage, avoiding generation of inflammatory downstream products (C5a or sC5b-9). In vivo, Fc-µTP-L309C suppressed inflammatory arthritis in mice when given therapeutically at approximately a 10-fold lower dose than IVIG, which was associated with reduced inflammatory cytokine production and complement activation. Likewise, administration of Fc-µTP-L309C restored platelet counts in a mouse model of immune thrombocytopenia. Our data demonstrate a potent anti-inflammatory effect of Fc-µTP-L309C in vitro and in vivo, likely mediated by blockade of FcγRs and its unique inhibition of complement activation.

Role of norms in variation in cancer centers' end-of-life quality: qualitative case study protocol.

BACKGROUND: A critical barrier to improving the quality of end-of-life (EOL) cancer care is our lack of understanding of the mechanisms underlying variation in EOL treatment intensity. This study aims to fill this gap by identifying 1) organizational and provider practice norms at major US cancer centers, and 2) how these norms influence provider decision making heuristics and patient expectations for EOL care, particularly for minority patients with advanced cancer. METHODS: This is a multi-center, qualitative case study at six National Comprehensive Cancer Network (NCCN) and National Cancer Institute (NCI) Comprehensive Cancer Centers. We will theoretically sample centers based upon National Quality Forum (NQF) endorsed EOL quality metrics and demographics to ensure heterogeneity in EOL intensity and region. A multidisciplinary team of clinician and non-clinician researchers will conduct direct observations, semi-structured interviews, and artifact collection. Participants will include: 1) cancer center and clinical service line administrators; 2) providers from medical, surgical, and radiation oncology; palliative or supportive care; intensive care; hospital medicine; and emergency medicine who see patients with cancer and have high clinical practice volume or high local influence (provider interviews and observations); and 3) adult patients with metastatic solid tumors and whom the provider would not be surprised if they died in the next 12 months and their caregivers (patient and caregiver interviews). Leadership interviews will probe about EOL institutional norms and organization. We will observe inpatient and outpatient care for two weeks. Provider interviews will use vignettes to probe explicit and implicit motivations for treatment choices. Semi-structured interviews with patients near EOL, or their family members and caregivers will explore past, current, and future decisions related to their cancer care. We will import transcribed field notes and interviews into Dedoose software for qualitative data management and analysis, and we will develop and apply a deductive and inductive codebook to the data. DISCUSSION: This study aims to improve our understanding of organizational and provider practice norms pertinent to EOL care in U.S. cancer centers. This research will ultimately be used to inform a provider-oriented intervention to improve EOL care for racial and ethnic minority patients with advanced cancer. TRIAL REGISTRATION: Clinicaltrials.gov ; NCT03780816 ; December 19, 2018.

Small-molecule pheromones and hormones controlling nematode development.

The existence of small-molecule signals that influence development in Caenorhabditis elegans has been known for several decades, but only in recent years have the chemical structures of several of these signals been established. The identification of these signals has enabled connections to be made between these small molecules and fundamental signaling pathways in C. elegans that influence not only development but also metabolism, fertility, and lifespan. Spurred by these important discoveries and aided by recent advances in comparative metabolomics and NMR spectroscopy, the field of nematode chemistry has the potential to expand dramatically in the coming years. This Perspective will focus on small-molecule pheromones and hormones that influence developmental events in the nematode life cycle (ascarosides, dafachronic acids, and nemamides), will cover more recent work regarding the biosynthesis of these signals, and will explore how the discovery of these signals is transforming our understanding of nematode development and physiology.

Optimizing high throughput antibody purification by using continuous chromatography media.

The ability to engineer monoclonal antibodies (mAbs) with high specificity made mAbs the fastest growing segment in the drug market. mAbs represent 8 of the top 20 selling drugs with combined sales of more than 57 billion US$ per year. The ability to purify large numbers of mAbs with sufficient yields for initial screening campaigns has direct impact on the timelines of a project. Automated liquid handling (ALH)-based mAb purification platforms have been used to facilitate the production of large numbers of mAbs. However, the ongoing pressure to de-risk potential lead molecules at an early development stage by including bio-physical characterization of mAbs has further increased the demand to produce sufficient quantities from limited sample volumes. A bottleneck so far has been the limited dynamic binding capacity of these systems, which is partly due to the binding properties of commonly used Protein A affinity matrices. The present publication suggests that by using a Protein A matrix optimized for continuous chromatography applications the yields of ALH-based but also standard lab-scale mAb purifications can be significantly increased without the need to change established protocols.

Influence of symbiotic and non-symbiotic bacteria on pheromone production in Steinernema nematodes (Nematoda, Steinernematidae).

In this study, we assessed the effect of symbiotic (cognate and non-cognate) and non-symbiotic bacteria on ascaroside production of first-generation adults in two Steinernema spp.: S. carpocapsae All strain and S. feltiae SN strain. Each nematode species was reared under three bacterial scenarios: (1) cognate symbiotic, (2) non-cognate symbiotic strain and (3) non-cognate symbiotic species. Our results showed S. carpocapsae produced four quantifiable ascaroside molecules: asc-C5, asc-C6, asc-C7 and asc-C11, whereas in S. feltiae only three molecules were detected: asc-C5, asc-C7 and asc-C11. Bacterial conditions did not significantly affect the quantity of the secreted ascarosides in first-generation adults of S. carpocapsae However, in S. feltiae, Xenorhabdus nematophila All strain influenced the production of two ascaroside molecules: asc-C5 and asc-C11.

Structural characterization of acyl-CoA oxidases reveals a direct link between pheromone biosynthesis and metabolic state in Caenorhabditis elegans.

Caenorhabditis elegans secretes ascarosides as pheromones to communicate with other worms and to coordinate the development and behavior of the population. Peroxisomal ß-oxidation cycles shorten the side chains of ascaroside precursors to produce the short-chain ascaroside pheromones. Acyl-CoA oxidases, which catalyze the first step in these ß-oxidation cycles, have different side chain-length specificities and enable C. elegans to regulate the production of specific ascaroside pheromones. Here, we determine the crystal structure of the acyl-CoA oxidase 1 (ACOX-1) homodimer and the ACOX-2 homodimer bound to its substrate. Our results provide a molecular basis for the substrate specificities of the acyl-CoA oxidases and reveal why some of these enzymes have a very broad substrate range, whereas others are quite specific. Our results also enable predictions to be made for the roles of uncharacterized acyl-CoA oxidases in C. elegans and in other nematode species. Remarkably, we show that most of the C. elegans acyl-CoA oxidases that participate in ascaroside biosynthesis contain a conserved ATP-binding pocket that lies at the dimer interface, and we identify key residues in this binding pocket. ATP binding induces a structural change that is associated with tighter binding of the FAD cofactor. Mutations that disrupt ATP binding reduce FAD binding and reduce enzyme activity. Thus, ATP may serve as a regulator of acyl-CoA oxidase activity, thereby directly linking ascaroside biosynthesis to ATP concentration and metabolic state.