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The morphology of the deep pineal gland of the Sprague Dawley rat was investigated by serial block face scanning electron microscopy. Cells were three-dimensionally (3-D) reconstructed using the software Fiji TrackEM. The deep pineal gland consisted of 2-5 layers of electron-lucent pinealocytes, with a euchromatic nucleus, endowed with one or two processes. Laterally, the deep pineal merged with the habenula and the stria medullaris thalami, via an intermediate area containing cells with more electron-dense cytoplasm and an indented nucleus with heterochromatin. Neither nerve terminals nor capillaries were observed in the deep pineal itself but present in the intermediate parts of the gland. The deep pineal was in contact with the third ventricle via the pineal and suprahabenular recesses. The ependymal lining in these recesses was an epithelium connected by tight junctions between their lateral cell membranes. Several intraventricular nerve terminals were in contact with the ependyma. 3-D reconstructions showed the ependymal cells endowed with long slender process penetrating the underlying pineal parenchyma. Few "tanocyte-like" ependymal cells, endowed with a process, reaching the subarachnoid space on the inferior surface of the deep pineal were observed. In addition, pinealocyte and astrocyte processes, often connected by gap junctions, bordered the inferior surface. In summary, the rat deep pineal gland is a neuroendocrine structure connected to the habenula. We here report specialized ependymal cells that might transmit signals from the cerebrospinal fluid to the deep pineal parenchyma and a "trans-pineal tanocyte-like cell" that connects the ventricular system with the subarachnoid space.

Toxoplasma infection in intermediate host species closely associates with inflammation. This association has led to suggestions that the behavioural changes associated with infection may be indirectly driven by the resulting sustained inflammation rather than a direct behavioural manipulation by the parasite. If this is correct, sustained inflammation in chronically infected rodents should present as widespread differences in the gastrointestinal microbiota due to the dependency between the composition of these microbiota and sustained inflammation. We conducted a randomized controlled experiment in rats that were assigned to a Toxoplasma-treatment, placebo-treatment or negative control group. We euthanised rats during the chronic phase of infection, collected their caecal stool samples and sequenced the V3-V4 region of the 16S rRNA gene to characterize the bacterial community in these samples. Toxoplasma infection did not induce widespread differences in the bacterial community composition of the gastrointestinal tract of rats. Rather, we found sex differences in the bacterial community composition of rats. We conclude that it is unlikely that sustained inflammation is the mechanism driving the highly specific behavioural changes observed in Toxoplasma-positive rats.

The highly contagious SARS-CoV-2 is mainly transmitted by respiratory droplets and aerosols. Consequently, people are required to wear masks and maintain a social distance to avoid spreading of the virus. Despite the success of the commercially available vaccines, the virus is still uncontained globally. Given the tropism of SARS-CoV-2, a mucosal immune reaction would help to reduce viral shedding and transmission locally. Only seven out of hundreds of ongoing clinical trials are testing the intranasal delivery of a vaccine against COVID-19.In the current study, we evaluated the immunogenicity of a traditional vaccine platform based on virus-like particles (VLPs) displaying RBD of SARS-CoV-2 for intranasal administration in a murine model. The candidate vaccine platform, CuMVCuMVOur data demonstrate that intranasal administration of CuMV

Background SARS‐CoV‐2 caused one of the most devastating pandemics in the recent history of mankind. Due to various countermeasures, including lock‐downs, wearing masks, and increased hygiene, the virus has been controlled in some parts of the world. More recently, the availability of vaccines, based on RNA or adenoviruses, has greatly added to our ability to keep the virus at bay; again, however, in some parts of the world only. While available vaccines are effective, it would be desirable to also have more classical vaccines at hand for the future. Key feature of vaccines for long‐term control of SARS‐CoV‐2 would be inexpensive production at large scale, ability to make multiple booster injections, and long‐term stability at 4℃. Methods Here, we describe such a vaccine candidate, consisting of the SARS‐CoV‐2 receptor‐binding motif (RBM) grafted genetically onto the surface of the immunologically optimized cucumber mosaic virus, called CuMVTT‐RBM. Results Using bacterial fermentation and continuous flow centrifugation for purification, the yield of the production process is estimated to be >2.5 million doses per 1000‐litre fermenter run. We demonstrate that the candidate vaccine is highly immunogenic in mice and rabbits and induces more high avidity antibodies compared to convalescent human sera. The induced antibodies are more cross‐reactive to mutant RBDs of variants of concern (VoC). Furthermore, antibody responses are neutralizing and long‐lived. In addition, the vaccine candidate was stable for at least 14 months at 4℃. Conclusion Thus, the here presented VLP‐based vaccine may be a good candidate for use as conventional vaccine in the long term., In this study, we describe a novel conventional COVID‐19 vaccine that consists of the RBM of SARS‐CoV‐2 genetically grafted onto the surface of our optimized cucumber‐mosaic virus‐like particles. We demonstrate that the vaccine candidate (mCuMVTT‐RBM) is highly immunogenic in mice and rabbits, can efficiently neutralize SARS‐CoV‐2, and is stable and highly scalable. The induced antibodies show cross‐reactivity with VoC.

Pluripotent cells in the mammalian embryo undergo state transitions marked by changes in patterns of gene expression and developmental potential as they progress from pre-implantation through post-implantation stages of development. Recent studies of cultured mouse and human pluripotent stem cells (hPSCs) have identified cells representative of an intermediate stage (referred to as the formative state) between naive pluripotency (equivalent to pre-implantation epiblast) and primed pluripotency (equivalent to late post-implantation epiblast). We examine these recent findings in light of our knowledge of peri-implantation mouse and human development, and we consider the implications of this work for deriving human embryo models from pluripotent cells.

The poly-Immunoglobulin Receptor (pIgR) transcytoses J chain-containing antibodies through mucosal epithelia. In mammals, two cis-duplicates of PIGR, FCMR and FCAMR, flank the PIGR gene. A PIGR duplication is first found in amphibians, previously annotated as PIGR2 (herein xlFCAMR), and is expressed by antigen-presenting cells. We demonstrate that xlFcamR is the equivalent of mammalian FcamR. It has been assumed that pIgR is the oldest member of this family, yet our data could not distinguish whether PIGR or FCAMR emerged first; however, FCMR was the last family member to emerge. Interestingly, bony fish 'pIgR' is not an orthologue of tetrapod pIgR, and possibly acquired its function via convergent evolution. PIGR/FCAMR/FCMR are members of a larger superfamily including TREM, CD300, and NKp44, which we name the 'double-disulfide Ig superfamily' (ddIgSF). Domains related to each ddIgSF family were identified in cartilaginous fish (sharks, chimeras) and encoded in a single gene cluster syntenic to the human pIgR locus. Thus, the ddIgSF families date back to earliest antibody-based adaptive immunity, but apparently not before. Finally, our data strongly suggest that the J chain arose in evolution only for Ig multimerization. This study provides a framework for further studies of pIgR and the ddIgSF in vertebrates. This article is protected by copyright. All rights reserved.

Background Coronavirus disease 19 (COVID‐19)–associated coagulopathy is a hallmark of disease severity and poor prognosis. The key manifestations of this prothrombotic syndrome—microvascular thrombosis, stroke, and venous and pulmonary clots—are also observed in severe and catastrophic antiphospholipid syndrome. Antiphospholipid antibodies (aPL) are detectable in COVID‐19 patients, but their association with the clinical course of COVID‐19 remains unproven. Objectives To analyze the presence and relevance of lipid‐binding aPL in hospitalized COVID‐19 patients. Methods Two cohorts of 53 and 121 patients from a single center hospitalized for PCR‐proven severe acute respiratory syndrome–coronavirus 2 infection were analyzed for the presence of aPL and clinical severity of COVID‐19. Results We here demonstrate that lipid‐binding aPL are common in COVID‐19. COVID‐19 patients with lipid‐binding aPL have higher median concentrations of C‐reactive protein and D‐dimer, and are more likely to have a critical clinical course and fatal outcome. Lipid‐binding aPL isolated from COVID‐19 patients target the recently described cell surface complex of lysobisphosphatidic acid (LBPA) with the protein C receptor (EPCR) to induce prothrombotic and inflammatory responses in monocytes and endothelial cells. We show that B1a cells producing lipid‐reactive aPL of the IgG isotype circulate in the blood of COVID‐19 patients. In vivo, COVID‐19 aPL accelerate thrombus formation in an experimental mouse model dependent on the recently delineated signaling pathway involving EPCR‐LBPA. Conclusions COVID‐19 patients rapidly expand B1a cells secreting pathogenic lipid‐binding aPL with broad thrombotic and inflammatory effects. The association with markers of inflammation and coagulation, clinical severity, and mortality suggests a causal role of aPL in COVID‐19–associated coagulopathy.

The increased frequency of extreme weather events due to climate change has complicated the epidemiological pattern of mosquito-borne diseases, as the host and vector dynamics shift to adapt. However, little is known about the seroprevalence of common mosquito-borne virus infections in horses in Australia. In this study, serological surveys for multiple alphaviruses were performed on samples taken from 622 horses across two horse populations (racehorses and horses residing on The University of Queensland (UQ) campus) in Queensland using the gold standard virus neutralization test. As is the case in humans across Australia, Ross River virus (RRV) is the most common arbovirus infection in horses, followed by Barmah Forest virus, with an overall apparent seroprevalence of 48.6% (302/622) and 4.3% (26/607), respectively. Horses aged over 6 years old (OR 1.86, p = 0.01) and residing at UQ (OR 5.8, p < 0.001) were significantly associated with seroconversion to RRV. A significant medium correlation (r = 0.626, p < 0.001) between RRV and Getah virus (GETV) neutralizing antibody titers was identified. Collectively, these results advance the current epidemiological knowledge of arbovirus exposure in a susceptible host in Australia. The potential use of horses as sentinels for arbovirus monitoring should be considered. Furthermore, since GETV is currently exotic to Australia, antibodies cross-reactivity between RRV and GETV should be further investigated for cross-protection, which may also help to inform vaccine developments.

Dofetilide is a rapid delayed rectifier potassium current inhibitor widely used to prevent the recurrence of atrial fibrillation and flutter. The clinical use of this drug is associated with increases in QTc interval, which predispose patients to ventricular cardiac arrhythmias. The mechanisms involved in the disposition of dofetilide, including its movement in and out of cardiomyocytes, remain unknown. Using a xenobiotic transporter screen, we identified MATE1 (SLC47A1) as a transporter of dofetilide and found that genetic knockout or pharmacological inhibition of MATE1 in mice was associated with enhanced retention of dofetilide in cardiomyocytes and increased QTc prolongation. The urinary excretion of dofetilide was also dependent on the MATE1 genotype, and we found that this transport mechanism provides a mechanistic basis for previously recorded drug-drug interactions of dofetilide with various contraindicated drugs, including bictegravir, cimetidine, ketoconazole, and verapamil. The translational significance of these observations was examined with a physiologically-based pharmacokinetic model that adequately predicted the drug-drug interaction liabilities in humans. These findings support the thesis that MATE1 serves a conserved cardioprotective role by restricting excessive cellular accumulation and warrant caution against the concurrent administration of potent MATE1 inhibitors and cardiotoxic substrates with a narrow therapeutic window.

PurposeEwing sarcoma (EwS) is a highly aggressive bone- or soft tissue-associated malignancy mostly affecting children, adolescents, and young adults. Although multimodal therapies have strongly improved patients’ overall survival over the past decades, the development of prognostic biomarkers for risk-based patient stratification and more effective therapies with less adverse effects is stagnating. Thus, new personalized medicine approaches are urgently required.Experimental designGene expression data of EwS and normal tissues were crossed with survival data to identify highly overexpressed, prognostically relevant, and actionable potential targets. RNA-interference and dose-response assays as well as tissue-microarray analyses were carried out to explore the functional role and druggability of a prominent candidate gene in vitro and in vivo, and to validate its suitability as a prognostic biomarker.ResultsEmploying a multilayered screening approach, we discover ribonucleotide reductase regulatory subunit M2 (RRM2) as a promising therapeutic target and prognostic biomarker in EwS. Through analysis of two independent EwS patient cohorts, we show that RRM2 mRNA and protein overexpression is associated with an aggressive clinical phenotype and poor patients’ overall survival. In agreement, RRM2 silencing as well as pharmacological inhibition by the specific inhibitor triapine (3-AP) significantly reduces EwS growth in vitro and in vivo. Furthermore, we present evidence that pharmacological RRM2 inhibition by triapine can overcome chemoresistance against doxorubicin or gemcitabine, and synergize with cell cycle checkpoint inhibitors (CHEK1 or WEE1).ConclusionsBased on the aggressive phenotype mediated by and the druggability of RRM2 our results provide a translational rationale for exploiting RRM2 as a novel therapeutic target in EwS and prompt further clinical investigations.

Intellectual disability (ID) and autism spectrum disorder (ASD) are the most common neurodevelopmental disorders and are characterized by substantial impairment in intellectual and adaptive functioning, with their genetic and molecular basis remaining largely unknown. Here, we identify biallelic variants in the gene encoding one of the Elongator complex subunits, ELP2, in patients with ID and ASD. Modelling the variants in mice recapitulates the patient features, with brain imaging and tractography analysis revealing microcephaly, loss of white matter tract integrity and an aberrant functional connectome. We show that the Elp2 mutations negatively impact the activity of the complex and its function in translation via tRNA modification. Further, we elucidate that the mutations perturb protein homeostasis leading to impaired neurogenesis, myelin loss and neurodegeneration. Collectively, our data demonstrate an unexpected role for tRNA modification in the pathogenesis of monogenic ID and ASD and define Elp2 as a key regulator of brain development.

In recent years, a diverse array of in vitro cell-derived models of mammalian development have been described that hold immense potential for exploring fundamental questions in developmental biology, particularly in the case of the human embryo where ethical and technical limitations restrict research. These models open up new avenues toward biomedical advances in in vitro fertilization, clinical research, and drug screening with potential to impact wider society across many diverse fields. These technologies raise challenging questions with profound ethical, regulatory, and social implications that deserve due consideration. Here, we discuss the potential impacts of embryo-like models, and their biomedical potential and current limitations., In this article Moris, Alev and colleagues discuss the possibilities of pluripotent stem cell models of embryonic development in our understanding of early human development and their biomedical potential as well as ethical, regulatory, and social implications raised by their emergence.

Harnessing new technologies is vital to achieve global imperatives to restore degraded ecosystems. We explored the potential of genomics as one such tool. We aimed to understand barriers hindering the uptake of genomics, and how to overcome them, via exploratory interviews with leading scholars in both restoration and its sister discipline of conservation—a discipline that has successfully leveraged genomics. We also conducted an examination of research trends to explore some insights that emerged from the interviews, including publication trends that have used genomics to address restoration and conservation questions. Our qualitative findings revealed varied perspectives on harnessing genomics. For example, scholars in restoration without genomics experience felt genomics was over-hyped. Scholars with genomics experience emphatically emphasized the need to proceed cautiously in using genomics in restoration. Both genomics-experienced and less-experienced scholars called for case studies to demonstrate the benefits of genomics in restoration. These qualitative data contrasted with our examination of research trends, which revealed 70 restoration genomics studies, particularly studies using environmental DNA as a monitoring tool. We provide a roadmap to facilitate the uptake of genomics into restoration, to help the restoration sector meet the monumental task of restoring huge areas to biodiverse and functional ecosystems. This article is part of the theme issue ‘Ecological complexity and the biosphere: the next 30 years’.

Polyploidy has the potential to allow organisms to outcompete their diploid progenitor(s) and occupy new environments. Shark Bay, Western Australia, is a World Heritage Area dominated by temperate seagrass meadows including Poseidon's ribbon weed, Posidonia australis . This seagrass is at the northern extent of its natural geographic range and experiences extremes in temperature and salinity. Our genomic and cytogenetic assessments of 10 meadows identified geographically restricted, diploid clones (2 n = 20) in a single location, and a single widespread, high-heterozygosity, polyploid clone (2 n = 40) in all other locations. The polyploid clone spanned at least 180 km, making it the largest known example of a clone in any environment on earth. Whole-genome duplication through polyploidy, combined with clonality, may have provided the mechanism for P. australis to expand into new habitats and adapt to new environments that became increasingly stressful for its diploid progenitor(s). The new polyploid clone probably formed in shallow waters after the inundation of Shark Bay less than 8500 years ago and subsequently expanded via vegetative growth into newly submerged habitats.

Objective The aim of this study was to evaluate the influence of near-cortical over-drilling holes on the mechanical behaviour of locking plate constructs applied in maned wolf's femur by using mechanical testing and finite element method (FEM). Study Design Seven pairs of adult maned wolves (Chrysocyon brachyurus) femur bones were randomly distributed into four groups. In all groups, a 3.5 mm locking compression plate, designed with 12 combi-holes and one locked, was applied to the lateral surface of the femur. G1 (n = 4) received bicortical locking screws placed in holes 1, 3, 5, 8, 10 and 12. In G2 (n = 5), the plate was applied as used in G1, but the application of the locked screws involved the near-cortical over-drilling technique. In G3 (n = 4), the plate was applied as used in G2, but the size of the near-cortical over-drilling was larger. The combi-holes 6 and 7 were maintained over a 10 mm fracture gap without screws. All constructs were tested for failure in the axial load. The axial load was applied eccentrically to the femoral head. Results Statistical differences were observed in the maximum load with G3 > G1 and G3 > G2, and in the deflection with G2 > G1 and G2 > G3. The FEM showed the lowest total displacement of the bone-plate constructs as well as of the plate in G1 compared with G2 and G3. Conclusion The near-cortical over-drilling technique used in unstable fractures induced in the maned wolf's femur showed by static axial compression test that maximum load and deflection are dependent on drill hole size induced in the near-cortex. Based on FEM, the lowest total displacement of the bone-plate constructs was observed in Group 1.

Cancer metastasis requires the transient activation of cellular programs enabling dissemination and seeding in distant organs1. Genetic, transcriptional and translational heterogeneity contributes to this dynamic process2,3. Metabolic heterogeneity has also been observed4, yet its role in cancer progression is less explored. Here we find that the loss of phosphoglycerate dehydrogenase (PHGDH) potentiates metastatic dissemination. Specifically, we find that heterogeneous or low PHGDH expression in primary tumours of patients with breast cancer is associated with decreased metastasis-free survival time. In mice, circulating tumour cells and early metastatic lesions are enriched with Phgdhlow cancer cells, and silencing Phgdh in primary tumours increases metastasis formation. Mechanistically, Phgdh interacts with the glycolytic enzyme phosphofructokinase, and the loss of this interaction activates the hexosamine-sialic acid pathway, which provides precursors for protein glycosylation. As a consequence, aberrant protein glycosylation occurs, including increased sialylation of integrin αvβ3, which potentiates cell migration and invasion. Inhibition of sialylation counteracts the metastatic ability of Phgdhlow cancer cells. In conclusion, although the catalytic activity of PHGDH supports cancer cell proliferation, low PHGDH protein expression non-catalytically potentiates cancer dissemination and metastasis formation. Thus, the presence of PHDGH heterogeneity in primary tumours could be considered a sign of tumour aggressiveness. ispartof: NATURE vol:605 issue:7911 pages:747-+ ispartof: location:England status: published

Prostate cancer is a major global health concern with limited treatment options for advanced disease. Its heterogeneity challenges the identification of crucial driver genes implicated in disease progression. Activating protein-1 (AP-1) transcription factor is associated with cancer since the first identification of its subunits, the proto-oncogenes JUN and FOS. Whereas both JUN and FOS have been implicated in prostate cancer, this study provides the first functional evidence that FOS acts as a tumor suppressor during prostate cancer progression and invasion. Data mining revealed decreased FOS expression in prostate cancer and a further downregulation in metastatic disease, consistent with FOS expression in cell lines derived from different prostate cancer stages. FOS deficiency in prostate cancer cell lines increases cell proliferation and induces oncogenic pathway alterations. Importantly, in vivo CRISPR/Cas9-mediated Fos and Pten double mutation in murine prostate epithelium results in increased proliferation and invasiveness compared to the abrogation of Pten alone. Interestingly, enhanced Jun expression is observed in the murine prostatic intraepithelial neoplasia lacking Fos. CRISPR/Cas9-mediated knockout of Jun combined with Fos and Pten deficiency diminishes the increased proliferation rate in vivo but not the ability to form invasive disease. Overall, we demonstrate that loss of Fos promotes disease progression from clinical latent prostate cancer to advanced disease through accelerated proliferation and invasiveness, partly through Jun.

Immunoglobulins and T cell receptors (TCR) have obvious structural similarities as well as similar immunogenetic diversification and selection mechanisms. Nevertheless, the two receptor systems and the loci that encode them are distinct in humans and classical murine models, and the gene segments comprising each repertoire are mutually exclusive. Additionally, while both B and T cells employ recombination-activating genes (RAG) for primary diversification, immunoglobulins are afforded a supplementary set of activation-induced cytidine deaminase (AID)-mediated diversification tools. As the oldest-emerging vertebrates sharing the same adaptive B and T cell receptor systems as humans, extant cartilaginous fishes allow a potential view of the ancestral immune system. In this review, we discuss breakthroughs we have made in studies of nurse shark (Ginglymostoma cirratum) T cell receptors demonstrating substantial integration of loci and diversification mechanisms in primordial B and T cell repertoires. We survey these findings in this shark model where they were first described, while noting corroborating examples in other vertebrate groups. We also consider other examples where the gnathostome common ancestry of the B and T cell receptor systems have allowed dovetailing of genomic elements and AID-based diversification approaches for the TCR. The cartilaginous fish seem to have retained this T/B cell plasticity to a greater extent than more derived vertebrate groups, but representatives in all vertebrate taxa except bony fish and placental mammals show such plasticity.

This paper describes the automatic measurement of the spatial organization of the visual axes of insect compound eyes, which consist of several thousands of visual units called ommatidia. Each ommatidium samples the optical information from a small solid angle, with an approximate Gaussian-distributed sensitivity (half-width on the order of 1˚) centered around a visual axis. Together, the ommatidia gather the visual information from a nearly panoramic field of view. The spatial distribution of the visual axes thus determines the eye's spatial resolution. Knowledge of the optical organization of a compound eye and its visual acuity is crucial for quantitative studies of neural processing of the visual information. Here we present an automated procedure for mapping a compound eye's visual axes, using an intrinsic, in vivo optical phenomenon, the pseudopupil, and the pupil mechanism of the photoreceptor cells. We outline the optomechanical setup for scanning insect eyes and use experimental results obtained from a housefly, Musca domestica, to illustrate the steps in the measurement procedure.

STING is essential for control of infections and for tumor immunosurveillance, but it can also drive pathological inflammation. STING resides on the endoplasmic reticulum (ER) and traffics following stimulation to the ERGIC/Golgi, where signaling occurs. Although STING ER exit is the rate-limiting step in STING signaling, the mechanism that drives this process is not understood. Here we identify STEEP as a positive regulator of STING signaling. STEEP was associated with STING and promoted trafficking from the ER. This was mediated through stimulation of phosphatidylinositol-3-phosphate (PtdIns(3)P) production and ER membrane curvature formation, thus inducing COPII-mediated ER-to-Golgi trafficking of STING. Depletion of STEEP impaired STING-driven gene expression in response to virus infection in brain tissue and in cells from patients with STING-associated diseases. Interestingly, STING gain-of-function mutants from patients interacted strongly with STEEP, leading to increased ER PtdIns(3)P levels and membrane curvature. Thus, STEEP enables STING signaling by promoting ER exit. STING ER exit is the rate-limiting step in STING signaling, but the mechanism that drives this process is not understood. Paludan and colleagues identify CxORF56, called STEEP here, as a positive regulator of STING signaling.

In addition to canonical TCR and BCR, cartilaginous fish assemble noncanonical TCR that employ various B-cell components. For example, shark T cells associate alpha (TCR-α) or delta (TCR-δ) constant (C) regions with Ig heavy chain (H) variable (V) segments or TCR-associated Ig-like V (TAILV) segments to form chimeric IgV-TCR, and combine TCRδC with both Ig-like and TCR-like V segments to form the doubly rearranging NAR-TCR. Activation-induced (cytidine) deaminase-catalyzed somatic hypermutation (SHM), typically used for B-cell affinity maturation, also is used by TCR-α during selection in the shark thymus presumably to salvage failing receptors. Here, we found that the use of SHM by nurse shark TCR varies depending on the particular V segment or C region used. First, SHM significantly alters alpha/delta V (TCRαδV) segments using TCR αC but not δC. Second, mutation to IgHV segments associated with TCR δC was reduced compared to mutation to TCR αδV associated with TCR αC. Mutation was present but limited in V segments of all other TCR chains including NAR-TCR. Unexpectedly, we found preferential rearrangement of the noncanonical IgHV-TCRδC over canonical TCR αδV-TCRδC receptors. The differential use of SHM may reveal how activation-induced (cytidine) deaminase targets V regions.

In a decade when Industry 4.0 and quality by design are major technology drivers of biopharma, automated and adaptive process monitoring and control are inevitable requirements and model-based solutions are key enablers in fulfilling these goals. Despite strong advancement in process digitalization, in most cases, the generated datasets are not sufficient for relying on purely data-driven methods, whereas the underlying complex bioprocesses are still not completely understood. In this regard, hybrid models are emerging as a timely pragmatic solution to synergistically combine available process data and mechanistic understanding. In this study, we show a novel application of the hybrid-EKF framework, that is, hybrid models coupled with an extended Kalman filter for real-time monitoring, control, and automated decision-making in mammalian cell culture processing. We show that, in the considered application, the predictive monitoring accuracy of such a framework improves by at least 35% when developed with hybrid models with respect to industrial benchmark tools based on PLS models. In addition, we also highlight the advantages of this approach in industrial applications related to conditional process feeding and process monitoring. With regard to the latter, for an industrial use case, we demonstrate that the application of hybrid-EKF as a soft sensor for titer shows a 50% improvement in prediction accuracy compared with state-of-the-art soft sensor tools.

Mouse sex chromosomes are enriched for co-amplified gene families, present in tens to hundreds of copies. Co-amplification of Slx/Slxl1 on the X chromosome and Sly on the Y chromosome are involved in dose-dependent meiotic drive, however the role of other co-amplified genes remains poorly understood. Here we demonstrate that the co-amplified gene family on the X chromosome, Srsx, along with two additional partial gene annotations, is actually part of a larger transcription unit, which we name Laidx. Laidx is harbored in a 229 kb amplicon that represents the ancestral state as compared to a 525 kb Y-amplicon containing the rearranged Laidy. Laidx contains a 25,011 nucleotide open reading frame, predominantly expressed in round spermatids, predicted to encode an 871 kD protein. Laidx has orthologous copies with the rat and also the 825-MY diverged parasitic Chinese liver fluke, Clonorchis sinensis, the likely result of a horizontal gene transfer of rodent Laidx to an ancestor of the liver fluke. To assess the male reproductive functions of Laidx, we generated mice carrying a multi-megabase deletion of the Laidx-ampliconic region. Laidx-deficient male mice do not show detectable reproductive defects in fertility, fecundity, testis histology, and offspring sex ratio. We speculate that Laidx and Laidy represent a now inactive X vs. Y chromosome conflict that occurred in an ancestor of present day mice.

Vaccines need to be rationally designed in order be delivered to the immune system for maximizing induction of dynamic immune responses. Virus‐like particles (VLPs) are ideal platforms for such 3D vaccines, as they allow the display of complex and native antigens in a highly repetitive form on their surface and can easily reach lymphoid organs in intact form for optimal activation of B and T cells. Adjusting size and zeta potential may allow investigators to further fine‐tune delivery to lymphoid organs. An additional way to alter vaccine transfer to lymph nodes and spleen may be the formulation with micron‐sized adjuvants that creates a local depot and results in a slow release of antigen and adjuvant. Ideally, the adjuvant in addition stimulates the innate immune system. The dynamics of the immune response may be further enhanced by inclusion of Toll‐like receptor ligands, which many VLPs naturally package. Hence, considering the 3Ds in vaccine development may allow for enhancement of their attributes to tackle complex diseases, not usually amenable to conventional vaccine strategies.

The masses of particles in a bovine milk extracellular vesicle (EV) preparation enriched for exosomes were directly determined for the first time by charge detection mass spectrometry (CDMS). In CDMS, both the mass-to-charge ratio (m/z) and z are determined simultaneously for individual particles, enabling mass determinations for particles that are far beyond the mass limit (∼1.0 MDa) of conventional mass spectrometry (MS). Particle masses and charges span a wide range from m ∼ 2 to ∼90 MDa and z ∼ 50 to ∼1300 e (elementary charges) and are highly dependent upon the conditions used to extract and isolate the EVs. EV particles span a continuum of masses, reflecting the highly heterogeneous nature of these samples. However, evidence for unique populations of particles is obtained from correlation of the charges and masses. An analysis that uses a two-dimensional Gaussian model, provides evidence for six families of particles, four of which having masses in the range expected for exosomes. Complementary proteomics measurements and electron microscopy (EM) imaging are used to further characterize the EVs and confirm that these samples have been enriched in exosomes. The ability to characterize such extremely heterogeneous mixtures of large particles with rapid, sensitive, and high-resolution MS techniques is critical to ongoing analytical efforts to separate and purify exosomes and exosome subpopulations. Direct measurement of each particle's mass and charge is a new means of characterizing the physical and chemical properties of exosomes and other EVs.

Despite the recent clinical success of T cell checkpoint inhibition targeting the CTLA-4 and PD-1 pathways, many patients either fail to achieve objective responses or they develop resistance to therapy. In some cases, poor responses to checkpoint blockade have been linked to suboptimal CD28 costimulation and the inability to generate and maintain a productive adaptive anti-tumor immune response. To address this, here we utilize directed evolution to engineer a CD80 IgV domain with increased PD-L1 affinity and fuse this to an immunoglobulin Fc domain, creating a therapeutic (ALPN-202, davoceticept) capable of providing CD28 costimulation in a PD-L1-dependent fashion while also antagonizing PD-1 - PD-L1 and CTLA-4-CD80/CD86 interactions. We demonstrate that by combining CD28 costimulation and dual checkpoint inhibition, ALPN-202 enhances T cell activation and anti-tumor efficacy in cell-based assays and mouse tumor models more potently than checkpoint blockade alone and thus has the potential to generate potent, clinically meaningful anti-tumor immunity in humans.

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Elevated circulating levels of nutrient-derived trimethylamine N-oxide (TMAO) have been associated with the onset and progression of cardiovascular disease by promoting athero-thrombosis. However, in conditions like bariatric surgery (Roux-en-Y gastric bypass, RYGB), stable increases of plasma TMAO are associated with improved endothelial function and reduced cardiovascular morbidity and mortality, thus questioning whether a mechanistic relationship between TMAO and endothelial dysfunction exists. Herein, we translationally assessed the effects of acute TMAO exposure on endothelial dysfunction, thrombosis and stroke. After RYGB, fasting circulating levels of TMAO increased in patients and obese rats, in parallel with an improved gluco-lipid profile and higher circulating bile acids. The latter enhanced FXR-dependent signalling in rat livers, which may lead to higher TMAO synthesis post RYGB. In lean rats, acute TMAO injection (7 mg kg(-1)) 1.5-h before sacrifice and ex-vivo 30-min incubation of thoracic aortas with 10(-6) M TMAO did not impair vasodilation in response to acetylcholine (Ach), glucagon-like peptide 1, or insulin. Similarly, in lean WT mice (n = 5-6), TMAO injection prior to subjecting mice to ischemic stroke or arterial thrombosis did not increase its severity compared to vehicle treated mice. Endothelial nitric oxide synthase (eNOS) activity and intracellular stress-activated pathways remained unaltered in aorta of TMAO-injected rats, as assessed by Western Blot. Pre-incubation of human aortic endothelial cells with TMAO (10(-6) M) did not alter NO release in response to Ach. Our results indicate that increased plasmatic TMAO in the near-physiological range seems to be a neutral bystander to vascular function as translationally seen in patients after bariatric surgery or in healthy lean rodent models and in endothelial cells exposed acutely to TMAO., Scientific Reports, 12 (1), ISSN:2045-2322

Virus-like particles (VLPs) are used in different marketed vaccines and are able to induce potent antibody responses. The innate pattern recognition receptors TLR7/8 recognize single stranded (ss) RNA naturally packaged into some VLPs and have been shown to enhance the production of IgG antibodies upon immunization. Here we demonstrate that, upon immunization with RNA-loaded bacteriophage-derived VLP Qβ, TLR7 signaling accelerates germinal center formation, promotes affinity/avidity maturation of VLP-specific IgG and isotype switching to IgG2b/2c. These findings extrapolated to antigens displayed on Qβ; as Fel d 1, the major cat allergen, chemically attached to Qβ also induced higher affinity/avidity IgG2b/2c antibodies in a TLR7-dependent fashion. Chimeric mice lacking TLR7-expression exclusively in B cells demonstrated that the enhanced IgG responses were driven by a B cell intrinsic mechanism. Importantly, deep sequencing of the BCR repertoire of antigen-specific B cells demonstrated higher diversity in mice with TLR7 signaling in B cells, suggesting that TLR7-signaling drives BCR repertoire development and diversity. Furthermore, the current data demonstrate that high levels of clonal diversity are reached early in the response and maintained by TLR7 signaling. In conclusion, TLR7 signaling enhances levels and quality of IgG antibodies, and this finding has major implications for vaccine design., Frontiers in Immunology, 12, ISSN:1664-3224

European journal of pharmaceutical sciences 175, 106216 (2022). doi:10.1016/j.ejps.2022.106216, A strong inflammatory immune response drives the lung pathology in neonatal acute respiratory distress syndrome (nARDS). Anti-inflammatory therapy is therefore a promising strategy for improved treatment of nARDS. We demonstrate a new function of the anionic phospholipids POPG, DOPG, and PIP2 as inhibitors of IL-1β release by LPS and ATP-induced inflammasome activation in human monocyte-derived and lung macrophages. Curosurf® surfactant was enriched with POPG, DOPG, PIP2 and the head-group derivative IP3, biophysically characterized and applicability was evaluated in a piglet model of nARDS. The composition of pulmonary surfactant from piglets was determined by shotgun lipidomics screens. After 72 h of nARDS, levels of POPG, DOPG, and PIP2 were enhanced in the respective treatment groups. Otherwise, we did not observe changes of individual lipid species in any of the groups. Surfactant proteins were not affected, with the exception of the IP3 treated group. Our data show that POPG, DOPG, and PIP2 are potent inhibitors of inflammasome activation; their enrichment in a surfactant preparation did not induce any negative effects on lipid profile and reduced biophysical function in vitro was mainly observed for PIP2. These results encourage to rethink the current strategies of improving surfactant preparations by inclusion of anionic lipids as potent anti-inflammatory immune regulators., Published by Elsevier, New York, NY [u.a.]

International audience; The family Cactaceae comprises a diverse group of typically succulent plants that are native to the American continent but have been introduced to nearly all other continents, predominantly for ornamental purposes. Despite their economic, cultural, and ecological importance, very little research has been conducted on the viral community that infects them. We previously identified a highly divergent geminivirus that is the first known to infect cacti. Recent research efforts in non-cultivated and asymptomatic plants have shown that the diversity of this viral family has been under-sampled. As a consequence, little is known about the effects and interactions of geminiviruses in many plants, such as cacti. With the objective to expand knowledge on the diversity of geminiviruses infecting cacti, we used previously acquired high-throughput sequencing results to search for viral sequences using BLASTx against a viral RefSeq protein database. We identified two additional sequences with similarity to geminiviruses, for which we designed abutting primers and recovered full-length genomes. From 42 cacti and five scale insects, we derived 42 complete genome sequences of a novel geminivirus species that we have tentatively named Opuntia virus 2 (OpV2) and 32 genomes of an Opuntia-infecting becurtovirus (which is a new strain of the spinach curly top Arizona virus species). Interspecies recombination analysis of the OpV2 group revealed several recombinant regions, in some cases spanning half of the genome. Phylogenetic analysis demonstrated that OpV2 is a novel geminivirus more closely related to viruses of the genus Curtovirus, which was further supported by the detection of three recombination events between curtoviruses and OpV2. Both OpV2 and Opuntia becurtoviruses were identified in mixed infections, which also included the previously characterized Opuntia virus 1. Viral quantification of the co-infected cactus plants compared with single infections did not show any clear trend in viral dynamics that might be associated with the mixed infections. Using experimental Rhizobium-mediated inoculations, we found that the initial accumulation of OpV2 is facilitated by co-infection with OpV1. This study shows that the diversity of geminiviruses that infect cacti is under-sampled and that cacti harbor diverse geminiviruses. The detection of the Opuntia becurtoviruses suggests spill-over events between viruses of cultivated species and native vegetation. The threat this poses to cacti needs to be further investigated.

Toluene-diisocyanate (TDI) is mainly used in the manufacturing process of polyurethane foams, and is a potent inducer of occupational asthma characterized by airway inflammation and airway hyperreactivity. Thymic stromal lymphopoietin (TSLP) plays an important role in the development of asthma, and correlating with the differentiation of Th2 and Th17 cells. However, the role of TSLP in TDI-induced asthma remains unclear. In this study, 96 TDI-exposed workers as well as a mouse model of TDI-induced asthma were investigated. The air exposure assessment result of TDI in the workplace showed that workers were exposed to inhalation of a very high concentration of TDI, approximately 8 times the recommended level, leading to a decrease in pulmonary function and an increase in inflammatory cells, as well as TSLP and IgE levels in the supernatant of sputum obtained from exposed workers. In order to further investigate the role of TSLP in the pathogenesis of TDI-induced asthma, a mouse model of TDI-induced asthma was also employed. Histopathological analysis of mouse lung and bronchus showed an obvious infiltration of inflammatory cells around the bronchus. The levels of inflammatory cells, IFN-γ, IL-4 and IL-17 in bronchoalveolar lavage fluid (BALF), the expression levels of TSLP protein and ROR-γt and IL-17 mRNA in mouse lung tissues were also significantly increased. However, after treatment with TSLP neutralizing antibody (TSLP-Ab), the degree of pulmonary and bronchial inflammation in mice was significantly alleviated, and the levels of inflammatory cells, IFN-γ, IL-4 and IL-17 in BALF, and the expression levels of ROR-γt and IL-17 mRNA in lung tissue were significantly decreased. Our data shows that TSLP plays an important role in the pathogenesis of TDI-induced asthma, and that TSLP-Ab can effectively alleviate TDI-induced airway inflammation of asthma.

Fluid and solute transporters of the retinal pigment epithelium (RPE) are core components of the outer blood-retinal barrier. Characterizing these transporters and their role in retinal homeostasis may provide insights into ocular function and disease. Here, we describe RPE defects in

Intravenous (IV) iron nanoparticle preparations are widely used to treat iron deficiency. The mechanism of mononuclear phagocyte system-mediated clearance of IV iron nanoparticles is unknown. The early uptake and homeostasis of iron after injection of ferric carboxymaltose (FCM) in mice was studied. An increase in serum iron was observed at 2.5 h followed by a return to baseline by 24 h. An increase in circulating monocytes was observed, particularly Ly6Chi and Ly6Clow. FCM was also associated with a time-dependent decrease in liver Kupffer cells (KCs) and increase in liver monocytes. The increase in liver monocytes suggests an influx of iron-rich blood monocytes, while some KCs underwent apoptosis. Adoptive transfer experiments demonstrated that following liver infiltration, blood monocytes differentiated to KCs. KCs were also critical for IV iron uptake and biodegradation. Indeed, anti-Colony Stimulating Factor 1 Receptor (CSF1R)-mediated depletion of KCs resulted in elevated serum iron levels and impaired iron uptake by the liver. Gene expression profiling indicated that C-C chemokine receptor type 5 (CCR5) might be involved in monocyte recruitment to the liver, confirmed by pharmaceutical inhibition of CCR5. Liver KCs play a pivotal role in the clearance and storage of IV iron and KCs appear to be supported by the expanded blood monocyte population.

Inhibition of PIKfyve phosphoinositide kinase selectively kills autophagy-dependent cancer cells by disrupting lysosome homeostasis. Here, we show that PIKfyve inhibitors can also selectively eliminate pluripotent embryonal carcinoma cells (ECCs), embryonic stem cells, and induced pluripotent stem cells under conditions where differentiated cells remain viable. PIKfyve inhibitors prevented lysosome fission, induced autophagosome accumulation, and reduced cell proliferation in both pluripotent and differentiated cells, but they induced death only in pluripotent cells. The ability of PIKfyve inhibitors to distinguish between pluripotent and differentiated cells was confirmed with xenografts derived from ECCs. Pretreatment of ECCs with the PIKfyve specific inhibitor WX8 suppressed their ability to form teratocarcinomas in mice, and intraperitoneal injections of WX8 into mice harboring teratocarcinoma xenografts selectively eliminated pluripotent cells. Differentiated cells continued to proliferate, but at a reduced rate. These results provide a proof of principle that PIKfyve specific inhibitors can selectively eliminate pluripotent stem cells in vivo as well as in vitro.