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Introduction: Prolyl-4-hydroxylases (P4H) catalyse the irreversible conversion of proline to hydroxyproline, constituting a common posttranslational modification of proteins found in humans, plants, and microbes. Hydroxyproline residues can be further modified in plants to yield glycoproteins containing characteristic O-glycans. It is currently unknown how these plant endogenous modifications impact protein functionality and they cause considerable concerns for the recombinant production of therapeutic proteins in plants. In this study, we carried out host engineering to generate a therapeutic glycoprotein largely devoid of plant-endogenous O-glycans for functional characterization.Methods: Genome editing was used to inactivate two genes coding for enzymes of the P4H10 subfamily in the widely used expression host Nicotiana benthamiana. Using glycoengineering in plants and expression in human HEK293 cells we generated four variants of a potent, SARS-CoV-2 neutralizing antibody, COVA2-15 IgA1. The variants that differed in the number of modified proline residues and O-glycan compositions of their hinge region were assessed regarding their physicochemical properties and functionality.Results: We found that plant endogenous O-glycan formation was strongly reduced on IgA1 when transiently expressed in the P4H10 double mutant N. benthamiana plant line. The IgA1 glycoforms displayed differences in proteolytic stability and minor differences in receptor binding thus highlighting the importance of O-glycosylation in the hinge region of human IgA1.Discussion: This work reports the successful protein O-glycan engineering of an important plant host for recombinant protein expression. While the complete removal of endogenous hydroxyproline residues from the hinge region of plant-produced IgA1 is yet to be achieved, our engineered line is suitable for structure-function studies of O-glycosylated recombinant glycoproteins produced in plants.

Secretory IgA (SIgA) presents a promising avenue for mucosal immunotherapy yet faces challenges in expression, purification, and stability. IgA exists in two primary isotypes, IgA1 and IgA2, with IgA2 further subdivided into two common allotypes: IgA2m(1) and IgA2m(2). The major differences between IgA1 and IgA2 are located in the hinge region, with IgA1 featuring a 13-amino acid elongation that includes up to six O-glycosylation sites. Furthermore, the IgA2m(1) allotype lacks a covalent disulfide bond between heavy and light chains, which is present in IgA1 and IgA2m(2). While IgA1 demonstrates superior epitope binding and pathogen neutralization, IgA2 exhibits enhanced effector functions and stability against mucosal bacterial degradation. However, the noncovalent linkage in the IgA2m(1) allotype raises production and stability challenges. The introduction of distinct single mutations aims to facilitate an alternate disulfide bond formation to mitigate these challenges. We compare four different IgA2 versions with IgA1 to further develop secretory IgA antibodies against SARS-CoV-2 for topical delivery to mucosal surfaces. Our results indicate significantly improved expression levels and assembly efficacy of SIgA2 (P221R) in Nicotiana benthamiana. Moreover, engineered SIgA2 displays heightened thermal stability under physiological as well as acidic conditions and can be aerosolized using a mesh nebulizer. In summary, our study elucidates the benefits of stability-enhancing mutations in overcoming hurdles associated with SIgA expression and stability.

Abstract Background Premature birth and/or low birthweight have long-lasting effects on cognition. The purpose of the present systematic review is to examine whether the effects of prematurity and/or low birth weight on neurodevelopmental outcomes differ between males and females. Methods Web of Science, Scopus, and Ovid MEDLINE were searched for studies of humans born premature and/or of low birthweight, where neurodevelopmental phenotypes were measured at 1 year of age or older. Studies must have reported outcomes in such a way that it was possible to assess whether effects were greater in one sex than the other. Risk of bias was assessed using both the Newcastle–Ottawa scale and the National Institutes of Health Quality assessment tool for observational cohort and cross-sectional studies. Results Seventy-five studies were included for descriptive synthesis, although only 24 presented data in a way that could be extracted for meta-analyses. Meta-analyses found that severe and moderate prematurity/low birthweight impaired cognitive function, and severe prematurity/low birthweight also increased internalizing problem scores. Moderate, but not severe, prematurity/low birthweight significantly increased externalizing problem scores. In no case did effects of prematurity/low birthweight differ between males and females. Heterogeneity among studies was generally high and significant, although age at assessment was not a significant moderator of effect. Descriptive synthesis did not identify an obvious excess or deficiency of male-biased or female-biased effects for any trait category. Individual study quality was generally good, and we found no evidence of publication bias. Conclusions We found no evidence that the sexes differ in their susceptibility to the effects of severe or moderate prematurity/low birthweight on cognitive function, internalizing traits or externalizing traits. Result heterogeneity tended to be high, but this reflects that one sex is not consistently more affected than the other. Frequently stated generalizations that one sex is more susceptible to prenatal adversity should be re-evaluated.

Abstract Catheter ablation is the most effective treatment for atrial fibrillation (AF). Electrical pulmonary veins isolation (PVI) forms the cornerstone ablation strategy. Radiofrequency (RF) in combination with 3D mapping system is the traditional ablation approach to treat AF. As a single-shot ablation system, cryoballoon (CB) has been an established ablation modality to facilitate PVI procedure. As a novel non-thermal single-shot ablation technology, pulsed field ablation (PFA) has its unique biophysical profile. Recent studies have demonstrated that PFA allows for efficient and durable PVI. However, the manipulation of single-shot ablation catheter may be different from the conventional point-by-point RF ablation catheter; ablation catheter with fixed size may have practical difficulties in variable anatomy and different clinical scenarios. The present article focuses on the technical aspects, describes the procedural approaches and illustrates the practical techniques of using CB and PFA for PVI, ultimately with the purpose to facilitate the ablation procedure and improve the patients’ clinical outcome.

Early-life conditions such as prenatal nutrition can have long-term effects on metabolic health, and these effects may differ between males and females. Understanding the biological mechanisms underlying sex differences in the response to early-life environment will improve interventions, but few such mechanisms have been identified, and there is no overall framework for understanding sex differences. Biological sex differences may be due to chromosomal sex, gonadal sex, or interactions between the two. This review describes approaches to distinguish between the roles of chromosomal and gonadal sex, and summarizes findings regarding sex differences in metabolism. The Four Core Genotypes (FCG) mouse model allows dissociation of the sex chromosome genotype from gonadal type, whereas the XY* mouse model can be used to distinguish effects of X chromosome dosage vs the presence of the Y chromosome. Gonadectomy can be used to distinguish between organizational (permanent) and activational (reversible) effects of sex hormones. Baseline sex differences in a variety of metabolic traits are influenced by both activational and organizational effects of gonadal hormones, as well as sex chromosome complement. Thus far, these approaches have not been widely applied to examine sex-dependent effects of prenatal conditions, although a number of studies have found activational effects of estradiol to be protective against the development of hypertension following early-life adversity. Genes that escape X chromosome inactivation (XCI), such as Kdm5c, contribute to baseline sex-differences in metabolism, while Ogt, another XCI escapee, leads to sex-dependent responses to prenatal maternal stress. Genome-wide approaches to the study of sex differences include mapping genetic loci influencing metabolic traits in a sex-dependent manner. Seeking enrichment for binding sites of hormone receptors among genes showing sexually-dimorphic expression can elucidate the relative roles of hormones. Using the approaches described herein to identify mechanisms underlying sex-dependent effects of early nutrition on metabolic health may enable the identification of fundamental mechanisms and potential interventions.

BackgroundAdministration of recombinant erythropoietin (EPO), a kidney-produced hormone with erythropoietic functions, has been shown to have multiple immunoregulatory effects in mice and humans, but whether physiological levels of EPO regulate immune function in vivo has not been previously evaluated.MethodsWe generated mice in which we could downregulate EPO production using a doxycycline (DOX)-inducible, EPO-specific silencing RNA (shEPOrtTAPOS), and we crossed them with B6.MRL-Faslpr/J mice that develop spontaneous lupus. We treated these B6.MRL/lpr shEPOrtTAPOS with DOX and serially measured anti-dsDNA antibodies, analyzed immune subsets by flow cytometry, and evaluated clinical signs of disease activity over 6 months of age in B6.MRL/lpr shEPOrtTAPOS and in congenic shEPOrtTANEG controls.ResultsIn B6.MRL/lpr mice, Epo downregulation augmented anti-dsDNA autoantibody levels and increased disease severity and percentages of germinal center B cells compared with controls. It also increased intracellular levels of IL-6 and MCP-1 in macrophages.DiscussionOur data in a murine model of lupus document that endogenous EPO reduces T- and B-cell activation and autoantibody production, supporting the conclusion that EPO physiologically acts as a counterregulatory mechanism to control immune homeostasis.

Biomimetic adhesives with a stiff fibre-reinforced base layer generate strong attachment, even without bioinspired micropatterning of the contact surface. However, current fibre-reinforced adhesive designs are still less versatile with respect to substrate variability than their biological counterparts. In this study, we enhance the comformability of a fibre-reinforced adhesive on curved substrates by adding bioinspired soft backings. We designed and fabricated soft backing variations (polyurethane foams and silicone hydroskeletons) with varying compressive stiffnesses that mimic the soft viscoelastic structures in the adhesive appendages of tree frogs, geckos and other animals. The backings were mounted on a smooth silicone layer enforced with a polyester mesh, and we experimentally investigated the contact area and friction performance of these adhesives on a curved substrate. The results show that the contact area and friction created by a fibre-reinforced adhesive with a soft backing in contact with a non-flat substrate scale inversely with backing stiffness. The integration of stiff fibre-reinforcement with a compressible backing represents an important step in bringing bioinspired adhesives out of the laboratory and into the real world, for example in soft robotic grippers. Moreover, our findings stimulate further research into the role of soft tissues in biological adhesive systems.

IntroductionBroadly neutralising antibodies are promising candidates for preventing and treating Human Immunodeficiency Virus/Acquired Immunodeficiency Syndrome (HIV/AIDS), as an alternative to or in combination with antiretroviral therapy (ART). These mAbs bind to sites on the virus essential for virus attachment and entry, thereby inhibiting entry into the host cell. However, the cost and availability of monoclonal antibodies, especially combinations of antibodies, hampers implementation of anti-HIV bNAb therapies in low- to middle- income countries (LMICs) where HIV-1 prevalence is highest.MethodsWe have produced three HIV broadly neutralizing antibodies (bNAbs), 10-1074, VRC01 and 3BNC117 in the Nicotiana benthamiana transient expression system. The impact of specific modifications to enhance potency and efficacy were assessed. To prolong half-life and increase bioavailability, a M252Y/S254T/T256E (YTE) or M428L/N434S (LS) mutation was introduced. To increase antibody dependent cellular cytotoxicity (ADCC), we expressed an afucosylated version of each antibody using a glycoengineered plant line.ResultsThe majority of bNAbs and their variants could be expressed at yields of up to 47 mg/kg. Neither the expression system nor the modifications impacted the neutralization potential of the bNAbs. Afucosylated bNAbs exhibit enhanced ability to bind to FcγRIIIa and trigger ADCC, regardless of the presence of Fc amino acid mutations. Lastly, we demonstrated that Fc-modified variants expressed in plants show enhanced binding to FcRn, which results in a favourable in vivo pharmacokinetic profile compared to their unmodified counterparts. ConclusionTobacco plants are suitable expression hosts for anti-HIV bNAbs with increased efficacy and an improved pharmacokinetic profile.

Background Survival after heart transplantation (HT) has improved considerably since the first HT was performed in 1967 in Cape Town, South Africa. Understanding immunology behind organ rejection has paved way for advances in the assessment of pre-transplant compatibility, development of newer and more specific immunosuppressive drugs, and management of rejection. Objectives Unlike medical therapy for heart failure, transplant protocols vary considerably between different centers. These variations in protocols generally reflect unique population characteristics and the availability of resources. This review article aims to provide a consolidated update on contemporary cardiac transplant medicine. We also aim to highlight local practice and its difference from our international counterparts. Methods A literature search was performed on Pubmed and Cochrane Central Register of Controlled Trials to identify trials and review articles that discussed heart transplant immunology and protocols. The International Society for Heart and Lung Transplant ( ISHLT ) guidelines were also reviewed. We focused on risk factors, prevention strategies, and treatment of cardiac rejection. Results A total of 48 articles were selected to provide a comprehensive overview of the contemporary practice of cardiac transplant immunosuppressive therapy. Comparisons were made with local data and practice protocols to highlight key differences. Conclusion Heart transplant covers a small subset of cardiac patients and much of the evidence is derived from empirical observations and retrospective analysis. This accounts for the heterogeneity in care and treatment protocols. More studies are needed to select best practices from around the world to further improve outcomes.

Molecular pharming in plants offers exciting possibilities to address global access to modern biologics. However, differences in the N-glycosylation pathway including the presence of β(1,2)-xylose and core α(1,3)-fucose can affect activity, potency and immunogenicity of plant-derived proteins. Successful glycoengineering approaches toward human-like structures with no changes in plant phenotype, growth, or recombinant protein expression levels have been reported for Arabidopsis thaliana and Nicotiana benthamiana. Such engineering of N-glycosylation would also be desirable for Nicotiana tabacum, which remains the crop of choice for recombinant protein pharmaceuticals required at massive scale and for manufacturing technology transfer to less developed countries. Here, we generated N. tabacum cv. SR-1 β(1,2)-xylosyltransferase (XylT) and α(1,3)-fucosyltransferase (FucT) knockout lines using CRISPR/Cas9 multiplex genome editing, targeting three conserved regions of the four FucT and two XylT genes. These two enzymes are responsible for generating non-human N-glycan structures. We confirmed full functional knockout of transformants by immunoblotting of total soluble protein by antibodies recognizing β(1,2)-xylose and core α(1,3)-fucose, mass spectrometry analysis of recombinantly produced VRC01, a broadly neutralizing anti-HIV-1 hIgG1 antibody, and Sanger sequencing of targeted regions of the putative transformants. These data represent an important step toward establishing Nicotiana tabacum as a biologics platform for Global Health.

Antibody-dependent enhancement of infection (ADE) is clinically relevant to Dengue virus (DENV) infection and poses a major risk to the application of monoclonal antibody (mAb)-based therapeutics against related flaviviruses such as the Zika virus (ZIKV). Here, we tested a two-tier approach for selecting non-cross-reactive mAbs combined with modulating Fc glycosylation as a strategy to doubly secure the elimination of ADE while preserving Fc effector functions. To this end, we selected a ZIKV-specific mAb (ZV54) and generated three ZV54 variants using Chinese hamster ovary cells and wild-type (WT) and glycoengineered ΔXF Nicotiana benthamiana plants as production hosts (ZV54CHO, ZV54WT, and ZV54ΔXF). The three ZV54 variants shared an identical polypeptide backbone, but each exhibited a distinct Fc N-glycosylation profile. All three ZV54 variants showed similar neutralization potency against ZIKV but no ADE activity for DENV infection, validating the importance of selecting the virus/serotype-specific mAbs for avoiding ADE by related flaviviruses. For ZIKV infection, however, ZV54CHO and ZV54ΔXF showed significant ADE activity while ZV54WT completely forwent ADE, suggesting that Fc glycan modulation may yield mAb glycoforms that abrogate ADE even for homologous viruses. In contrast to the current strategies for Fc mutations that abrogate all effector functions along with ADE, our approach allowed the preservation of effector functions as all ZV54 glycovariants retained antibody-dependent cellular cytotoxicity (ADCC) against the ZIKV-infected cells. Furthermore, the ADE-free ZV54WT demonstrated in vivo efficacy in a ZIKV-infection mouse model. Collectively, our study provides further support for the hypothesis that antibody–viral surface antigen and Fc-mediated host cell interactions are both prerequisites for ADE, and that a dual-approach strategy, as shown herein, contributes to the development of highly safe and efficacious anti-ZIKV mAb therapeutics. Our findings may be impactful to other ADE-prone viruses, including SARS-CoV-2.

Abstract Background HIV remains one of the most important health issues worldwide, with almost 40 million people living with HIV. Although patients develop antibodies against the virus, its high mutation rate allows evasion of immune responses. Some patients, however, produce antibodies that are able to bind to, and neutralise different strains of HIV. One such ‘broadly neutralising’ antibody is ‘N6’. Identified in 2016, N6 can neutralise 98% of HIV-1 isolates with a median IC50 of 0.066 µg/mL. This neutralisation breadth makes N6 a very promising therapeutic candidate. Results N6 was expressed in a glycoengineered line of N. benthamiana plants (pN6) and compared to the mammalian cell-expressed equivalent (mN6). Expression at 49 mg/kg (fresh leaf tissue) was achieved in plants, although extraction and purification are more challenging than for most plant-expressed antibodies. N-glycoanalysis demonstrated the absence of xylosylation and a reduction in α(1,3)-fucosylation that are typically found in plant glycoproteins. The N6 light chain contains a potential N-glycosylation site, which was modified and displayed more α(1,3)-fucose than the heavy chain. The binding kinetics of pN6 and mN6, measured by surface plasmon resonance, were similar for HIV gp120. pN6 had a tenfold higher affinity for FcγRIIIa, which was reflected in an antibody-dependent cellular cytotoxicity assay, where pN6 induced a more potent response from effector cells than that of mN6. pN6 demonstrated the same potency and breadth of neutralisation as mN6, against a panel of HIV strains. Conclusions The successful expression of N6 in tobacco supports the prospect of developing a low-cost, low-tech production platform for a monoclonal antibody cocktail to control HIV in low-to middle income countries. Graphic abstract

Erythropoietin (EPO) has multiple nonerythropoietic functions, including immune modulation, but EPO’s effects in transplantation remain incompletely understood. We tested the mechanisms linking EPO administration to prolongation of murine heterotopic heart transplantation using WT and conditional EPO receptor–knockout (EPOR-knockout) mice as recipients. In WT controls, peritransplant administration of EPO synergized with CTLA4-Ig to prolong allograft survival (P < 0.001), reduce frequencies of donor-reactive effector CD8+ T cells in the spleen (P < 0.001) and in the graft (P < 0.05), and increase frequencies and total numbers of donor-reactive Tregs (P < 0.01 for each) versus CTLA4-Ig alone. Studies performed in conditional EPOR-knockout recipients showed that each of these differences required EPOR expression in myeloid cells but not in T cells. Analysis of mRNA isolated from spleen monocytes showed that EPO/EPOR ligation upregulated macrophage-expressed, antiinflammatory, regulatory, and pro-efferocytosis genes and downregulated selected proinflammatory genes. Taken together, the data support the conclusion that EPO promotes Treg-dependent murine cardiac allograft survival by crucially altering the phenotype and function of macrophages. Coupled with our previous documentation that EPO promotes Treg expansion in humans, the data support the need for testing the addition of EPO to costimulatory blockade-containing immunosuppression regimens in an effort to prolong human transplant survival.

Abstract Background Males and females may experience different effects of early-life adversity on life-long health. One hypothesis is that male foetuses invest more in foetal growth and relatively less in placental growth, and that this makes them susceptible to poor nutrition in utero, particularly if nutrition is reduced part-way through gestation. Objectives Our objectives were to examine whether (1) food and/ or protein restriction in rats and mice has consistent sex-dependent effects, (2) sex-dependency differs between types of outcomes, and (3) males are more severely affected when restriction starts part-way through gestation. Data sources PubMed and Web of Science were searched to identify eligible studies. Study eligibility criteria Eligible studies described controlled experiments that restricted protein or food during gestation in rats or mice, examined physiological traits in offspring from manipulated pregnancies, and tested whether effects differed between males and females. Results Our search identified 292 articles, of which the full texts of 72 were assessed, and 65 were included for further synthesis. A majority (50) used Wistar or Sprague-Dawley rats and so these were the primary focus. Among studies in which maternal diet was restricted for the duration of gestation, no type of trait was consistently more severely affected in one particular sex, although blood pressure was generally increased in both sexes. Meta-analysis found no difference between sexes in the effect of protein restriction throughout gestation on blood pressure. Among studies restricting food in the latter half of gestation only, there were again few consistent sex-dependent effects, although three studies found blood pressure was increased in males only. Meta-analysis found that food restriction in the second half of gestation increased adult blood pressure in both sexes, with a significantly greater effect in males. Birthweight was consistently reduced in both sexes, a result confirmed by meta-analysis. Conclusions We found little support for the hypotheses that males are more affected by food and protein restriction, or that effects are particularly severe if nutrition is reduced part-way through gestation. However, less than half of the studies tested for sex by maternal diet interactions to identify sex-dependent effects. As a result, many reported sex-specific effects may be false positives.

Due to their ability to self-assemble into complex structures, block copolymers are of great interest for use in a wide range of future applications, such as self-healing materials. Therefore, it is important to understand the mechanisms of their structure formation. In particular, the process engineering of the formation and transition of the polymer structures is required for ensuring reproducibility and scalability, but this has received little attention in the literature. In this article, the influence of the addition rate of the selective solvent on the homogeneity of self-assembled vesicles of polystyrene-block-polyacrylic acid is demonstrated, as well as the influence of the reaction time and the mixing intensity on the morphology of the polymer structures. For example, it was demonstrated that the higher the mixing intensity, the faster the transition from micelle to vesicle. The experimental results are further supported by CFD simulations, which visually and graphically show an increase in shear rate and narrower shear rate distributions at higher stirring rates. Furthermore, it was demonstrated that the vesicle size is not only kinetically determined, since flow forces above a critical size lead to the deformation and fission of the vesicles.

Abstract Maze‐like linear substrate modification in atrial fibrillation patients nonresponders to pulmonary vein isolation represents a feasible technique to gain left atrial appendage electrical isolation even in the presence of a Watchman occluder device.

TNF ligation of TNF receptor 1 (TNFR1) promotes either inflammation and cell survival by (a) inhibiting RIPK1’s death-signaling function and activating NF-κB or (b) causing RIPK1 to associate with the death-inducing signaling complex to initiate apoptosis or necroptosis. The cellular source of TNF that results in RIPK1-dependent cell death remains unclear. To address this, we employed in vitro systems and murine models of T cell–dependent transplant or tumor rejection in which target cell susceptibility to RIPK1-dependent cell death could be genetically altered. We show that TNF released by T cells is necessary and sufficient to activate RIPK1-dependent cell death in target cells and thereby mediate target cell cytolysis independently of T cell frequency. Activation of the RIPK1-dependent cell death program in target cells by T cell–derived TNF accelerates murine cardiac allograft rejection and synergizes with anti-PD1 administration to destroy checkpoint blockade–resistant murine melanoma. Together, the findings uncover a distinct immunological role for TNF released by cytotoxic effector T cells following cognate interactions with their antigenic targets. Manipulating T cell TNF and/or target cell susceptibility to RIPK1-dependent cell death can be exploited to either mitigate or augment T cell–dependent destruction of allografts and malignancies to improve outcomes.

Controlled, reversible attachment is widely spread throughout the animal kingdom: from ticks to tree frogs, whose weights span from 2 mg to 200 g, and from geckos to mosquitoes, who stick under vastly different situations, such as quickly climbing trees and stealthily landing on human hosts. A fascinating and complex interplay of adhesive and frictional forces forms the foundation of attachment of these highly diverse systems to various substrates. In this review, we present an overview of the techniques used to quantify the adhesion and friction of terrestrial animals, with the aim of informing future studies on the fundamentals of bioadhesion, and motivating the development and adoption of new or alternative measurement techniques. We classify existing methods with respect to the forces they measure, including magnitude and source, i.e., generated by the whole body, single limbs, or by sub-structures. Additionally, we compare their versatility, specifically what parameters can be measured, controlled, and varied. This approach reveals critical trade-offs of bioadhesion measurement techniques. Beyond stimulating future studies on evolutionary and physicochemical aspects of bioadhesion, understanding the fundamentals of biological attachment is key to the development of biomimetic technologies, from soft robotic grippers to gentle surgical tools.

Abstract Background Maternal nutrition during pregnancy has life-long consequences for offspring. However, the effects of maternal overnutrition and/ or obesity on fetal growth remain poorly understood, e.g., it is not clear why birthweight is increased in some obese pregnancies but not in others. Maternal obesity is frequently studied using rodents on high-fat diets, but effects on fetal growth are inconsistent. The purpose of this review is to identify factors that contribute to reduced or increased fetal growth in rodent models of maternal overnutrition. Methods We searched Web of Science and screened 2173 abstracts and 328 full texts for studies that fed mice or rats diets providing ~ 45% or ~ 60% calories from fat for 3 weeks or more prior to pregnancy. We identified 36 papers matching the search criteria that reported birthweight or fetal weight. Results Studies that fed 45% fat diets to mice or 60% fat diets to rats generally did not show effects on fetal growth. Feeding a 45% fat diet to rats generally reduced birth and fetal weight. Feeding mice a 60% fat diet for 4–9 weeks prior to pregnancy tended to increase in fetal growth, whereas feeding this diet for a longer period tended to reduce fetal growth. Conclusions The high-fat diets used most often with rodents do not closely match Western diets and frequently reduce fetal growth, which is not a typical feature of obese human pregnancies. Adoption of standard protocols that more accurately mimic effects on fetal growth observed in obese human pregnancies will improve translational impact in this field.

BackgroundLactation results in substantial maternal bone loss that is recovered following weaning. However, the mechanisms underlying this recovery, and in particular the role of insulin-like growth factor 1 (IGF-I), is not clear. Furthermore, there is little data regarding whether recovery is affected by advanced maternal age.MethodsUsing micro-computed tomography, we studied bone recovery following lactation in mice at 2, 5 and 7 months of age. We also investigated the effects of reduced IGF-I availability using mice lacking PAPP-A2, a protease of insulin-like growth factor binding protein 5 (IGFBP-5).ResultsIn 2 month old mice, lactation affected femoral trabecular and cortical bone, but only cortical bone showed recovery 3 weeks after weaning. This recovery was not affected by deletion of the Pappa2 gene. The amount of trabecular bone was reduced in 5 and 7 month old mice, and was not further reduced by lactation. However, the recovery of cortical bone was impaired at 5 and 7 months compared with at 2 months.ConclusionsRecovery of the maternal skeleton after lactation is impaired in moderately-aged mice compared with younger mice. Our results may be relevant to the long-term effects of breastfeeding on the maternal skeleton in humans, particularly given the increasing median maternal age at childbearing.

Passive immunization with antibodies is a promising approach against enterotoxigenic Escherichia coli diarrhea, a prevalent disease in LMICs. The objective of this study was to investigate expression of a monoclonal anti-ETEC CfaE secretory IgA antibody in N. benthamiana plants, with a view to facilitating access to ETEC passive immunotherapy. SIgA1 and SIgA2 forms of mAb 68–81 were produced by co-expressing the light and engineered heavy chains with J chain and secretory component in N. benthamiana. Antibody expression and assembly were compared with CHO-derived antibodies by SDS-PAGE, western blotting, size-exclusion chromatography and LC-MS peptide mapping. N-linked glycosylation was assessed by rapid fluorescence/mass spectrometry and LC-ESI-MS. Susceptibility to gastric digestion was assessed in an in vitro model. Antibody function was compared for antigen binding, a Caco-2 cell-based ETEC adhesion assay, an ETEC hemagglutination inhibition assay and a murine in vivo challenge study. SIgA1 assembly appeared superior to SIgA2 in plants. Both sub-classes exhibited resistance to degradation by simulated gastric fluid, comparable to CHO-produced 68–61 SIgA1. The plant expressed SIgAs had more homogeneous N-glycosylation than CHO-derived SIgAs, but no alteration of in vitro functional activity was observed, including antibodies expressed in a plant line engineered for mammalian-like N glycosylation. The plant-derived SIgA2 mAb demonstrated protection against diarrhea in a murine infection model. Although antibody yield and purification need to be optimized, anti-ETEC SIgA antibodies produced in a low-cost plant platform are functionally equivalent to CHO antibodies, and provide promise for passive immunotherapy in LMICs.

BACKGROUND:Spontaneous abortions, intrauterine growth restriction, and preeclampsia are thought to be caused by defective placentation and are associated with increased risk of adverse outcomes in subsequent pregnancies. However, it is not known whether the recurrence of adverse outcomes is associated with the recurrence of placental pathology. We hypothesized that recurrent maternal vascular malperfusion (MVM) underlies the recurrence of adverse outcomes. METHODS:Using data from the National Collaborative Perinatal Project, we assessed the recurrence of pregnancy complications and MVM lesions (N = 3865), associations between a history of spontaneous abortions and MVM lesions or adverse outcomes in subsequent pregnancies (N = 8312), and whether the recurrence of pregnancy complications occurred independently of the presence of MVM lesions. RESULTS:The odds of an MVM lesion were higher for a woman who had had an MVM lesion in a previous pregnancy (aOR = 1.6; 95% CI 1.3-1.9), although this was marginally non-significant after adjusting for covariates such as gestational age, race and BMI. The odds of preeclampsia, a small-for-gestational-age infant, premature delivery and early pregnancy loss were 2.7-5.0 times higher if there had been that same adverse outcome in a previous pregnancy. A history of spontaneous abortions was associated with higher risk of a small-for-gestational-age baby (aOR = 2.4; 95% CI 1.7-3.4) and prematurity (aOR = 5.1; 95% CI 2.3-11.5 for extremely preterm), but not preeclampsia. The recurrence of adverse outcomes was significant when restricting analyses to women without MVM lesions. Similarly, associations between adverse outcomes and previous spontaneous abortions were significant when statistically controlling for the presence of MVM lesions, or excluding pregnancies with MVM lesions. CONCLUSIONS:Women with adverse outcomes in one pregnancy are at higher risk of complications in subsequent pregnancies. However, there is significant recurrence of adverse outcomes even in the absence of MVM.

Plant molecular farming (PMF) is a convenient and cost-effective way to produce high-value recombinant proteins that can be used in the production of a range of health products, from pharmaceutical therapeutics to cosmetic products. New plant breeding techniques (NPBTs) provide a means to enhance PMF systems more quickly and with greater precision than ever before. However, the feasibility, regulatory standing and social acceptability of both PMF and NPBTs are in question. This paper explores the perceptions of key stakeholders on two European Union (EU) Horizon 2020 programmes-Pharma-Factory and Newcotiana-towards the barriers and facilitators of PMF and NPBTs in Europe. One-on-one qualitative interviews were undertaken with N = 20 individuals involved in one or both of the two projects at 16 institutions in seven countries (Belgium, France, Germany, Italy, Israel, Spain and the UK). The findings indicate that the current EU regulatory environment and the perception of the public towards biotechnology are seen as the main barriers to scaling-up PMF and NPBTs. Competition from existing systems and the lack of plant-specific regulations likewise present challenges for PMF developing beyond its current niche. However, respondents felt that the communication of the benefits and purpose of NPBT PMF could provide a platform for improving the social acceptance of genetic modification. The importance of the media in this process was highlighted. This article also uses the multi-level perspective to explore the ways in which NPBTs are being legitimated by interested parties and the systemic factors that have shaped and are continuing to shape the development of PMF in Europe.

Rhabdoviruses are enveloped negative-sense RNA viruses that have numerous biotechnological applications. However, recovering plant rhabdoviruses from cDNA remains difficult due to technical difficulties such as the need for concurrent in planta expression of the viral genome together with the viral nucleoprotein (N), phosphoprotein (P) and RNA-dependent RNA polymerase (L) and viral genome instability in E. coli. Here, we developed a negative-sense minigenome cassette for Lettuce necrotic yellows virus (LNYV). We introduced introns into the unstable viral ORF and employed Agrobacterium tumefaciens to co-infiltrate Nicotiana with the genes for the N, P, and L proteins together with the minigenome cassette. The minigenome cassette included the Discosoma sp. red fluorescent protein gene (DsRed) cloned in the negative-sense between the viral trailer and leader sequences which were placed between hammerhead and hepatitis delta ribozymes. In planta DsRed expression was demonstrated by western blotting while the appropriate splicing of introduced introns was confirmed by sequencing of RT-PCR product.

Abstract Tree frogs have the remarkable ability to attach to smooth, rough, dry, and wet surfaces using their versatile toe pads. Tree frog attachment involves the secretion of mucus into the pad-substrate gap, requiring adaptations towards mucus drainage and pad lubrication. Here, we present an overview of tree frog attachment, with focus on (i) the morphology and material of the toe pad; (ii) the functional demands on the toe pad arising from ecology, lifestyle, and phylogenetics; (iii) experimental data of attachment performance such as adhesion and friction forces; and (iv) potential perspectives on future developments in the field. By revisiting reported data and observations, we discuss the involved mechanisms of attachment and propose new hypotheses for further research. Among others, we address the following questions: Do capillary and hydrodynamic forces explain the strong friction of the toe pads directly, or indirectly by promoting dry attachment mechanisms? If friction primarily relies on van der Waals (vdW) forces instead, how much do these forces contribute to adhesion in the wet environment tree frogs live in and what role does the mucus play? We show that both pad morphology and measured attachment performance suggest the coaction of several attachment mechanisms (e.g. capillary and hydrodynamic adhesion, mechanical interlocking, and vdW forces) with situation-dependent relative importance. Current analytical models of capillary and hydrodynamic adhesion, caused by the secreted mucus and by environmental liquids, do not capture the contributions of these mechanisms in a comprehensive and accurate way. We argue that the soft pad material and a hierarchical surface pattern on the ventral pad surface enhance the effective contact area and facilitate gap-closure by macro- to nanoscopic drainage of interstitial liquids, which may give rise to a significant contribution of vdW interactions to tree frog attachment. Increasing the comprehension of the complex mechanism of tree frog attachment contributes to a better understanding of other biological attachment systems (e.g. in geckos and insects) and is expected to stimulate the development of a wide array of bioinspired adhesive applications.

Abstract Background Pregnancy-associated plasma protein-A2 (PAPP-A2) is consistently upregulated in the placentae of pregnancies complicated by preeclampsia and fetal growth restriction. The causes and significance of this upregulation remain unknown, but it has been hypothesized that it is a compensatory response to improve placental growth and development. We predicted that, if the upregulation of PAPP-A2 in pregnancy complications reflects a compensatory response, then deletion of Pappa2 in mice would exacerbate the effects of a gene deletion previously reported to impair placental development: deficiency of matrix metalloproteinase-9 (MMP9). Methods We crossed mice carrying deletions in Pappa2 and Mmp9 to produce pregnancies deficient in one, both, or neither of these genes. We measured pregnancy rates, number of conceptuses, fetal and placental growth, and the histological structure of the placenta. Results We found no evidence of reduced fertility, increased pregnancy loss, or increased fetal demise in Mmp9 −/− females. In pregnancies segregating for Mmp9, Mmp9 −/− fetuses were lighter than their siblings with a functional Mmp9 allele. However, deletion of Pappa2 did not exacerbate or reveal any effects of Mmp9 deficiency. We observed some effects of Pappa2 deletion on placental structure that were independent of Mmp9 deficiency, but no effects on fetal growth. At G16, male fetuses were heavier than female fetuses and had heavier placentae with larger junctional zones and smaller labyrinths. Conclusions Effects of Mmp9 deficiency were not exacerbated by the deletion of Pappa2. Our results do not provide evidence that upregulation of placental PAPP-A2 represents a mechanism to compensate for impaired fetal growth.

Abstract The southern blue‐ringed octopus, Hapalochlaena maculosa (Hoyle, 1883) lacks a planktonic dispersal phase, yet ranges across Australia's southern coastline. This species’ brief and holobenthic life history suggests gene flow might be limited, leaving distant populations prone to strong genetic divergence. This study used 17,523 genome‐wide SNP loci to investigate genetic structuring and local adaptation patterns of H. maculosa among eight sampling sites along its reported range. Within sites, interrelatedness was very high, consistent with the limited dispersal of this taxon. However, inbreeding coefficients were proportionally lower among sites where substructuring was not detected, suggesting H. maculosa might possess a mechanism for inbreeding avoidance. Genetic divergence was extremely high among all sites, with the greatest divergence observed between both ends of the distribution, Fremantle, WA, and Stanley, TAS. Genetic distances closely followed an isolation by geographic distance pattern. Outlier analyses revealed distinct selection signatures at all sites, with the strongest divergence reported between Fremantle and the other Western Australian sites. Phylogenetic reconstructions using the described sister taxon H. fasciata (Hoyle, 1886) further supported that the genetic divergence between distal H. maculosa sites in this study was equivalent to that of between established heterospecifics within this genus. However, it is advocated that taxonomic delineations within this species should be made with caution. These data indicate that H. maculosa forms a clinal species pattern across its geographic range, with gene flow present through allele sharing between adjacent populations. Morphological investigations are recommended for a robust resolution of the taxonomic identity and ecotype boundaries of this species.

Reference-free genome phasing is vital for understanding allele inheritance and the impact of single-molecule DNA variation on phenotypes. To achieve thorough phasing across homozygous or repetitive regions of the genome, long-read sequencing technologies are often used to perform phased de novo assembly. As a step toward reducing the cost and complexity of this type of analysis, we describe new methods for accurately phasing Oxford Nanopore Technologies (ONT) sequence data with the Shasta genome assembler and a modular tool for extending phasing to the chromosome scale called GFAse. We test using new variants of ONT PromethION sequencing, including those using proximity ligation, and show that newer, higher accuracy ONT reads substantially improve assembly quality.

The pandemic disease chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis (Bd), is a major threat to amphibian biodiversity. For most species, the exact mechanisms of chytridiomycosis that lead to negative population dynamics remain uncertain, though mounting evidence suggests that sublethal effects could be an important driver. In this review, we propose that tree frog attachment is a promising case to study the sublethal effects of a Bd infection on amphibians. A synthesis of the current knowledge on the functional morphology of the adhesive toe pads of tree frogs, on the underlying mechanisms of tree frog attachment, and on the epidermal pathology of chytridiomycosis substantiates the hypothesis that Bd-induced epidermal alterations have the potential to disrupt tree frog attachment. We highlight a series of (biomechanical) experiments to test this hypothesis and to shed some light on the sublethal disease mechanisms of chytridiomycosis. The knowledge generated from such an approach could contribute to future research on Bd epidemiology and ultimately to the conservation of the biodiversity of arboreal anurans.

Abstract Background Maternal and neonatal outcomes are influenced by the nature of antenatal care. Standard pregnancy care is provided on an individual basis, with one-on-one appointments between a client and family doctor, midwife or obstetrician. A novel, group-based antenatal care delivery model was developed in the United States in the 1990s and is growing in popularity beyond the borders of the USA. The purpose of this study was to evaluate outcomes in clients receiving interprofessional group perinatal care versus interprofessional individual care in a Canadian setting. Methods Clients attending the South Community Birth Program (SCBP), an interprofessional, collaborative, primary care maternity program, offering both individual and group care, were invited to participate in the study. Pregnancy knowledge and satisfaction scores, and perinatal outcomes were compared between those receiving group versus individual care. Chi-square tests, general linear models and logistic regression were used to compare the questionnaire scores and perinatal outcomes between cohorts. Results Three hundred three clients participated in the study. Group care was comparable to individual care in terms of mode of birth, gestational age at birth, infant birth weight, breastfeeding rates, pregnancy knowledge, preparedness for labour and baby care, and client satisfaction. The rates of adverse perinatal outcomes were extremely low amongst SCBP clients, regardless of the type of care received (preterm birth rates ~5%). Breastfeeding rates were very high amongst all study participants (> 78% exclusive breastfeeding), as were measures of pregnancy knowledge and satisfaction. Conclusions This is the first Canadian study to compare outcomes in clients receiving interprofessional group care versus individual care. Our observation that interprofessional group care outcomes and satisfaction were as good as interprofessional individual care has important implications for the antenatal care of clients and for addressing the projected maternity provider crisis facing Canada, particularly in small and rural communities. Further study of group-based care including not only client satisfaction, but also provider satisfaction, is needed. In addition, research into the role of interprofessional care in meeting the needs and improving perinatal outcomes of different populations is necessary.

Background. Gastric cancer is the fourth most common cancer worldwide and the second most common cause of cancer-related death. The majority of newly diagnosed gastric cancer cases present either as locally advanced tumor growth or with distant metastases. Case Report. Here, we describe a case of isolated brain metastases in a male patient with gastric cancer. Initially, our patient presented with dysphagia and was diagnosed with gastric cancer after a thorough evaluation. One year after chemotherapy and surgical resection of his gastric cancer, he presented with headaches, nausea, dizziness, and photophobia. Further evaluation of these symptoms led to the discovery of three metastatic brain lesions without evidence of extracranial metastases. Conclusions. Our review of the literature has found that such cases are rare. Additionally, our review of the literature demonstrates the poor outcomes associated with metastatic brain lesions from gastric cancer and highlights the importance of surgical resection in increasing overall survival time.

Deficiency of pregnancy-associated plasma protein-A2 (PAPP-A2), an IGF-1 availability regulator, causes postnatal growth failure and dysregulation of bone size and density. The present study aimed to determine the effects of recombinant murine IGF-1 (rmIGF-1) on bone composition and remodeling in constitutive Pappa2 knock-out (ko/ko) mice. To address this challenge, X-ray diffraction (XRD), attenuated total reflection-fourier transform infra-red (ATR-FTIR) spectroscopy and gene expression analysis of members of the IGF-1 system and bone resorption/formation were performed. Pappa2ko/ko mice (both sexes) had reduced body and bone length. Male Pappa2ko/ko mice had specific alterations in bone composition (mineral-to-matrix ratio, carbonate substitution and mineral crystallinity), but not in bone remodeling. In contrast, decreases in collagen maturity and increases in Igfbp3, osteopontin (resorption) and osteocalcin (formation) characterized the bone of Pappa2ko/ko females. A single rmIGF-1 administration (0.3 mg/kg) induced short-term changes in bone composition in Pappa2ko/ko mice (both sexes). rmIGF-1 treatment in Pappa2ko/ko females also increased collagen maturity, and Igfbp3, Igfbp5, Col1a1 and osteopontin expression. In summary, acute IGF-1 treatment modifies bone composition and local IGF-1 response to bone remodeling in mice with Pappa2 deficiency. These effects depend on sex and provide important insights into potential IGF-1 therapy for growth failure and bone loss and repair.

The global use of lithium-ion batteries of all types has been increasing at a rapid pace for many years. In order to achieve the goal of an economical and sustainable battery industry, the recycling and recirculation of materials is a central element on this path. As the achievement of high 95% recovery rates demanded by the European Union for some metals from today’s lithium ion batteries is already very challenging, the question arises of how the process chains and safety of battery recycling as well as the achievement of closed material cycles are affected by the new lithium battery generations, which are supposed to enter the market in the next 5 to 10 years. Based on a survey of the potential development of battery technology in the next years, where a diversification between high-performance and cost-efficient batteries is expected, and today’s knowledge on recycling, the challenges and chances of the new battery generations regarding the development of recycling processes, hazards in battery dismantling and recycling, as well as establishing a circular economy are discussed. It becomes clear that the diversification and new developments demand a proper separation of battery types before recycling, for example by a transnational network of dismantling and sorting locations, and flexible and high sophisticated recycling processes with case-wise higher safety standards than today. Moreover, for the low-cost batteries, recycling of the batteries becomes economically unattractive, so legal stipulations become important. However, in general, it must be still secured that closing the material cycle for all battery types with suitable processes is achieved to secure the supply of raw materials and also to further advance new developments.

Obesity increases the risk of a number of pregnancy complications, potentially due to chronic inflammation. We predicted that an obesogenic high-fat diet (HFD) in mice would create an inflammatory environment that would exacerbate the effects of lipopolysaccharide (LPS), an inflammatory insult, administered during pregnancy. Females were placed on a HFD or a low-fat diet (LFD) prior to mating, injected with 2 µg LPS or control on gestational day 7 and collected on day 14. Treatment with LPS increased the odds that a female thought to be pregnant at injection had no conceptuses at day 14 (p = 0.024), suggesting that injection with LPS was more likely to induce complete abortion. However, there was no effect of diet on the odds of having no conceptuses at day 14 and no interaction between diet and LPS injection. Diet and LPS injection had no effect on the number of viable fetuses in females still pregnant at day 14. For fetal weight, there was a significant interaction between diet and treatment (p = 0.017), whereby LPS reduced fetal weight in HFD females but not in LFD females. However, LPS treatment of HFD females reduced fetal weight to that observed in control-injected LFD females. Although LPS increased the odds of abortion, there was little evidence that a HFD exacerbated the effects of LPS.

Maternal overnutrition and obesity during pregnancy can have long-term effects on offspring physiology and behaviour. These developmental programming effects may be mediated by fetal exposure to glucocorticoids, which is regulated in part by placental 11β-hydroxysteroid dehydrogenase (11β-HSD) type 1 and 2. We tested whether a maternal high-fat, high-sucrose diet would alter expression of placental 11β-HSD1 and 2, thereby increasing fetal exposure to maternal glucocorticoids, with downstream effects on offspring physiology and behaviour. C57BL/6J mice were fed a high-fat, high-sucrose (HFHS) diet or a nutrient-matched low-fat, no-sucrose control diet prior to and during pregnancy and lactation. At day 17 of gestation, HFHS dams had ~20% lower circulating corticosterone levels than controls. Furthermore, there was a significant interaction between maternal diet and fetal sex for circulating corticosterone levels in the fetuses, whereby HFHS males tended to have higher corticosterone than control males, with no effect in female fetuses. However, placental 11β-HSD1 or 11β-HSD2 expression did not differ between diets or show an interaction between diet and sex. To assess potential long-term consequences of this sex-specific effect on fetal corticosterone, we studied locomotor activity and metabolic traits in adult offspring. Despite a sex-specific effect of maternal diet on fetal glucocorticoids, there was little evidence of sex-specific effects on offspring physiology or behaviour, although HFHS offspring of both sexes had higher circulating corticosterone at 9 weeks of age. Our results suggest the existence of as yet unknown mechanisms that mitigate the effects of altered glucocorticoid exposure early in development, making offspring resilient to the potentially negative effects of a HFHS maternal diet.

Here the Human Pangenome Reference Consortium presents a first draft of the human pangenome reference. The pangenome contains 47 phased, diploid assemblies from a cohort of genetically diverse individuals1. These assemblies cover more than 99% of the expected sequence in each genome and are more than 99% accurate at the structural and base pair levels. Based on alignments of the assemblies, we generate a draft pangenome that captures known variants and haplotypes and reveals new alleles at structurally complex loci. We also add 119 million base pairs of euchromatic polymorphic sequences and 1,115 gene duplications relative to the existing reference GRCh38. Roughly 90 million of the additional base pairs are derived from structural variation. Using our draft pangenome to analyse short-read data reduced small variant discovery errors by 34% and increased the number of structural variants detected per haplotype by 104% compared with GRCh38-based workflows, which enabled the typing of the vast majority of structural variant alleles per sample.

Research on associative learning suggests that marketers can enhance consumer attitudes by repeatedly pairing their brands with pleasant or "positively-valenced" stimuli (e.g. attractive models, babies, cute animals) rather than unpleasant or "negatively-valenced" stimuli (e.g. garbage cans and disgusting insects)—an evaluative conditioning effect also known as affect transfer. In this research, we combine the associative learning and the goal pursuit literatures to show that the influence of affect transfer on brands depends on the mindset that is active at the time of judgment. Four experiments and one field study uniquely demonstrate that negatively-valenced brand pairings may become desirable when consumers have an instrumentality mindset, which increases attention to the instrumentality, or effectiveness, of a given consumption behavior. This pattern of results occurs due to a bidirectional association between unpleasantness and instrumentality, making a brand with negative associations seem more effective. Results are robust across contexts (health, entertainment, news) and persist regardless of whether the (un)pleasant images are within or adjacent to the advertisement. The effect attenuates when consumers have a weaker association between unpleasantness and instrumentality, and reverses when consumers are cued to focus on favorability (vs. instrumentality). Contributions and implications for associative learning and brand management are discussed. [ABSTRACT FROM AUTHOR]

There has been tremendous progress in phased genome assembly production by combining long-read data with parental information or linked-read data. Nevertheless, a typical phased genome assembly generated by trio-hifiasm still generates more than 140 gaps. We perform a detailed analysis of gaps, assembly breaks, and misorientations from 182 haploid assemblies obtained from a diversity panel of 77 unique human samples. Although trio-based approaches using HiFi are the current gold standard, chromosome-wide phasing accuracy is comparable when using Strand-seq instead of parental data. Importantly, the majority of assembly gaps cluster near the largest and most identical repeats (including segmental duplications [35.4%], satellite DNA [22.3%], or regions enriched in GA/AT-rich DNA [27.4%]). Consequently, 1513 protein-coding genes overlap assembly gaps in at least one haplotype, and 231 are recurrently disrupted or missing from five or more haplotypes. Furthermore, we estimate that 6-7 Mbp of DNA are misorientated per haplotype irrespective of whether trio-free or trio-based approaches are used. Of these misorientations, 81% correspond to bona fide large inversion polymorphisms in the human species, most of which are flanked by large segmental duplications. We also identify large-scale alignment discontinuities consistent with 11.9 Mbp of deletions and 161.4 Mbp of insertions per haploid genome. Although 99% of this variation corresponds to satellite DNA, we identify 230 regions of euchromatic DNA with frequent expansions and contractions, nearly half of which overlap with 197 protein-coding genes. Such variable and incompletely assembled regions are important targets for future algorithmic development and pangenome representation.