Optimization of the Synthesis and Conjugation of the Methyl Rhamnan Tip of Pseudomonas aeruginosa A-Band Polysaccharide and Immunogenicity Evaluation for the Continued Development of a Potential Glycoconjugate Vaccine.

Pseudomonas aeruginosa is an antimicrobial-resistant bacterium that has no vaccine approved for human use. Additionally, it has been identified by the World Health Organization as a priority pathogen for novel vaccines and therapeutic development. We previously developed a synthetic mimic of the A-band polysaccharide tip that showed promise in terms of immunogenicity for use as a glycoconjugate vaccine. In this current manuscript, we improve upon the previous work to continue the development of this glycoconjugate vaccine. Herein, we report a higher-yielding synthesis of mimics containing a handle and a spacer that improved conjugation efficiency, resulting in better carbohydrate-to-protein ratios and also good immunogenicity of these conjugates in mice and rabbits. The data suggested that perhaps only a tetrasaccharide was required to induce an immune response capable of recognizing whole cells of P. aeruginosa.

Formation and immunological evaluation of Moraxella catarrhalis glycoconjugates based on synthetic oligosaccharides.

Published work has shown that glycoconjugate vaccines, based on truncated detoxified lipopolysaccharides from Moraxella catarrhalis attached through their reducing end to a carrier protein, gave good protection for all three serotypes A, B, and C in mice immunisation experiments. The (from the non-reducing end) truncated LPS structures were obtained from bacterial glycosyl transferase knock-out mutants and contained the de-esterified Lipid A, two Kdo residues and five glucose moieties. This work describes the chemical synthesis of the same outer Moraxella LPS structures, spacer-equipped and further truncated from the reducing end, i.e., without the Lipid A part and containing four or five glucose moieties or four glucose moieties and one Kdo residue, and their subsequent conjugation to a carrier protein via a five­carbon bifunctional spacer to form glycoconjugates. Immunisation experiments both in mice and rabbits of these gave a good antibody response, being 2-7 times that of pre-immune sera. However, the sera produced only recognized the immunizing glycan immunogens and failed to bind to native LPS or whole bacterial cells. Comparative molecular modelling of three alternative antigens shows that an additional (2 â†’ 4)-linked Kdo residue, not present in the synthetic structures, has a significant impact on the shape and volume of the molecule, with implications for antigen binding and cross-reactivity.

SLAM-ITseq identifies that Nrf2 induces liver regeneration through the pentose phosphate pathway.

The liver exhibits a remarkable capacity to regenerate following injury. Despite this unique attribute, toxic injury is a leading cause of liver failure. The temporal processes by which the liver senses injury and initiates regeneration remain unclear. Here, we developed a transgenic zebrafish model wherein hepatocyte-specific expression of uracil phosphoribosyltransferase (UPRT) enabled the implementation of SLAM-ITseq to investigate the nascent transcriptome during initiation of liver injury and regeneration. Using this approach, we identified a rapid metabolic transition from the fed to the fasted state that was followed by induction of the nuclear erythroid 2-related factor (Nrf2) antioxidant program. We find that activation of Nrf2 in hepatocytes is required to induce the pentose phosphate pathway (PPP) and improve survival following liver injury. Mechanistically, we demonstrate that inhibition of the PPP disrupts nucleotide biosynthesis to prevent liver regeneration. Together, these studies provide fundamental insights into the mechanism by which early metabolic adaptation to injury facilitates tissue regeneration.

The African killifish: A short-lived vertebrate model to study the biology of sarcopenia and longevity.

Sarcopenia, the age-related decline in muscle function, places a considerable burden on health-care systems. While the stereotypic hallmarks of sarcopenia are well characterized, their contribution to muscle wasting remains elusive, which is partly due to the limited availability of animal models. Here, we have performed cellular and molecular characterization of skeletal muscle from the African killifish-an extremely short-lived vertebrate-revealing that while many characteristics deteriorate with increasing age, supporting the use of killifish as a model for sarcopenia research, some features surprisingly reverse to an "early-life" state in the extremely old stages. This suggests that in extremely old animals, there may be mechanisms that prevent further deterioration of skeletal muscle, contributing to an extension of life span. In line with this, we report a reduction in mortality rates in extremely old killifish. To identify mechanisms for this phenomenon, we used a systems metabolomics approach, which revealed that during aging there is a striking depletion of triglycerides, mimicking a state of calorie restriction. This results in the activation of mitohormesis, increasing Sirt1 levels, which improves lipid metabolism and maintains nutrient homeostasis in extremely old animals. Pharmacological induction of Sirt1 in aged animals was sufficient to induce a late life-like metabolic profile, supporting its role in life span extension in vertebrate populations that are naturally long-lived. Collectively, our results demonstrate that killifish are not only a novel model to study the biological processes that govern sarcopenia, but they also provide a unique vertebrate system to dissect the regulation of longevity.

Variable thermal plasticity of leaf functional traits in Andean tropical montane forests.

Tropical montane forests (TMFs) are biodiversity hotspots and provide vital ecosystem services, but they are disproportionately vulnerable to climate warming. In the Andes, cold-affiliated species from high elevations are being displaced at the hot end of their thermal distributions by warm-affiliated species migrating upwards from lower elevations, leading to compositional shifts. Leaf functional traits are strong indicators of plant performance and at the community level have been shown to vary along elevation gradients, reflecting plant adaptations to different environmental niches. However, the plastic response of such traits to relatively rapid temperature change in Andean TMF species remains unknown. We used three common garden plantations within a thermosequence in the Colombian Andes to investigate the warming and cooling responses of key leaf functional traits in eight cold- and warm-affiliated species with variable thermal niches. Cold-affiliated species shifted their foliar nutrient concentrations when exposed to warming, while all other traits did not significantly change; contrastingly, warm-affiliated species were able to adjust structural, nutrient and water-use efficiency traits from acquisitive to conservative strategies in response to cooling. Our findings suggest that cold-affiliated species will struggle to acclimate functional traits to warming, conferring warm-affiliated species a competitive advantage under climate change.

Automated prediction of acute promyelocytic leukemia from flow cytometry data using a graph neural network pipeline.

OBJECTIVES: Our study aimed to develop a machine learning (ML) model to accurately classify acute promyelocytic leukemia (APL) from other types of acute myeloid leukemia (other AML) using multicolor flow cytometry (MFC) data. Multicolor flow cytometry is used to determine immunophenotypes that serve as disease signatures for diagnosis. METHODS: We used a data set of MFC files from 27 patients with APL and 41 patients with other AML, including those with uncommon immunophenotypes. Our ML pipeline involved training a graph neural network (GNN) to output graph-level labels and identifying the most crucial MFC parameters and cells for predictions using an input perturbation method. RESULTS: The top-performing GNN achieved 100% accuracy on the training/validation and test sets on classifying APL from other AML and used MFC parameters similarly to expert pathologists. Pipeline performance is amenable to use in a clinical decision support system, and our deep learning architecture readily enables prediction explanations. CONCLUSIONS: Our ML pipeline shows robust performance on predicting APL and could be used to screen for APL using MFC data. It also allowed for intuitive interrogation of the model's predictions by clinicians.

Vasculature is getting Hip(po): Hippo signaling in vascular development and disease.

The Hippo signaling pathway regulates developmental organ growth, regeneration, and cell fate decisions. Although the role of the Hippo pathway, and its transcriptional effectors YAP and TAZ, has been well documented in many cell types and species, only recently have the roles for this pathway come to light in vascular development and disease. Experiments in mice, zebrafish, and in vitro have uncovered roles for the Hippo pathway, YAP, and TAZ in vasculogenesis, angiogenesis, and lymphangiogenesis. In addition, the Hippo pathway has been implicated in vascular cancers and cardiovascular diseases, thus identifying it as a potential therapeutic target for the treatment of these conditions. However, despite recent advances, Hippo's role in the vasculature is still underappreciated compared with its role in epithelial tissues. In this review, we appraise our current understanding of the Hippo pathway in blood and lymphatic vessel development and highlight the current knowledge gaps and opportunities for further research.

Behavioral Adaptations of Nursing Brangus Cows to Virtual Fencing: Insights from a Training Deployment Phase.

Virtual fencing systems have emerged as a promising technology for managing the distribution of livestock in extensive grazing environments. This study provides comprehensive documentation of the learning process involving two conditional behavioral mechanisms and the documentation of efficient, effective, and safe animal training for virtual fence applications on nursing Brangus cows. Two hypotheses were examined: (1) animals would learn to avoid restricted zones by increasing their use of containment zones within a virtual fence polygon, and (2) animals would progressively receive fewer audio-electric cues over time and increasingly rely on auditory cues for behavioral modification. Data from GPS coordinates, behavioral metrics derived from the collar data, and cueing events were analyzed to evaluate these hypotheses. The results supported hypothesis 1, revealing that virtual fence activation significantly increased the time spent in containment zones and reduced time in restricted zones compared to when the virtual fence was deactivated. Concurrently, behavioral metrics mirrored these findings, with cows adjusting their daily travel distances, exploration area, and cumulative activity counts in response to the allocation of areas with different virtual fence configurations. Hypothesis 2 was also supported by the results, with a decrease in cueing events over time and increased reliance with animals on audio cueing to avert receiving the mild electric pulse. These outcomes underscore the rapid learning capabilities of groups of nursing cows in responding to virtual fence boundaries.

Real-Time Monitoring of Grazing Cattle Using LORA-WAN Sensors to Improve Precision in Detecting Animal Welfare Implications via Daily Distance Walked Metrics.

Animal welfare monitoring relies on sensor accuracy for detecting changes in animal well-being. We compared the distance calculations based on global positioning system (GPS) data alone or combined with motion data from triaxial accelerometers. The assessment involved static trackers placed outdoors or indoors vs. trackers mounted on cows grazing on pasture. Trackers communicated motion data at 1 min intervals and GPS positions at 15 min intervals for seven days. Daily distance walked was determined using the following: (1) raw GPS data (RawDist), (2) data with erroneous GPS locations removed (CorrectedDist), or (3) data with erroneous GPS locations removed, combined with the exclusion of GPS data associated with no motion reading (CorrectedDist_Act). Distances were analyzed via one-way ANOVA to compare the effects of tracker placement (Indoor, Outdoor, or Animal). No difference was detected between the tracker placement for RawDist. The computation of CorrectedDist differed between the tracker placements. However, due to the random error of GPS measurements, CorrectedDist for Indoor static trackers differed from zero. The walking distance calculated by CorrectedDist_Act differed between the tracker placements, with distances for static trackers not differing from zero. The fusion of GPS and accelerometer data better detected animal welfare implications related to immobility in grazing cattle.

The KEAP1-NRF2 pathway regulates TFEB/TFE3-dependent lysosomal biogenesis.

The maintenance of redox and metabolic homeostasis is integral to embryonic development. Nuclear factor erythroid 2-related factor 2 (NRF2) is a stress-induced transcription factor that plays a central role in the regulation of redox balance and cellular metabolism. Under homeostatic conditions, NRF2 is repressed by Kelch-like ECH-associated protein 1 (KEAP1). Here, we demonstrate that Keap1 deficiency induces Nrf2 activation and postdevelopmental lethality. Loss of viability is preceded by severe liver abnormalities characterized by an accumulation of lysosomes. Mechanistically, we demonstrate that loss of Keap1 promotes aberrant activation of transcription factor EB (TFEB)/transcription factor binding to IGHM Enhancer 3 (TFE3)-dependent lysosomal biogenesis. Importantly, we find that NRF2-dependent regulation of lysosomal biogenesis is cell autonomous and evolutionarily conserved. These studies identify a role for the KEAP1-NRF2 pathway in the regulation of lysosomal biogenesis and suggest that maintenance of lysosomal homeostasis is required during embryonic development.

Acclimation of photosynthetic capacity and foliar respiration in Andean tree species to temperature change.

Climate warming is causing compositional changes in Andean tropical montane forests (TMFs). These shifts are hypothesised to result from differential responses to warming of cold- and warm-affiliated species, with the former experiencing mortality and the latter migrating upslope. The thermal acclimation potential of Andean TMFs remains unknown. Along a 2000 m Andean altitudinal gradient, we planted individuals of cold- and warm-affiliated species (under common soil and irrigation), exposing them to the hot and cold extremes of their thermal niches, respectively. We measured the response of net photosynthesis (Anet ), photosynthetic capacity and leaf dark respiration (Rdark ) to warming/cooling, 5 months after planting. In all species, Anet and photosynthetic capacity at 25°C were highest when growing at growth temperatures (Tg ) closest to their thermal means, declining with warming and cooling in cold-affiliated and warm-affiliated species, respectively. When expressed at Tg , photosynthetic capacity and Rdark remained unchanged in cold-affiliated species, but the latter decreased in warm-affiliated counterparts. Rdark at 25°C increased with temperature in all species, but remained unchanged when expressed at Tg . Both species groups acclimated to temperature, but only warm-affiliated species decreased Rdark to photosynthetic capacity ratio at Tg as temperature increased. This could confer them a competitive advantage under future warming.

The vicious cycle of BRASH syndrome: A case report.

First described in 2016, BRASH syndrome is an underreported clinical entity characterized by bradycardia, renal dysfunction, atrioventricular nodal blockade (AVNB), shock, and hyperkalemia. The recognition of BRASH syndrome as a clinical entity is crucial for early and effective management. Patients with BRASH syndrome present with symptomatic bradycardia that is resistant to treatment with standard agents such as atropine. In this report, we present the case of a 67-year-old male patient who presented with symptomatic bradycardia with an ultimate diagnosis of BRASH syndrome. We also shed light on predisposing factors and challenges encountered during the management of affected patients.

Artificial intelligence in clinical multiparameter flow cytometry and mass cytometry-key tools and progress.

There are many research studies and emerging tools using artificial intelligence (AI) and machine learning to augment flow and mass cytometry workflows. Emerging AI tools can quickly identify common cell populations with continuous improvement of accuracy, uncover patterns in high-dimensional cytometric data that are undetectable by human analysis, facilitate the discovery of cell subpopulations, perform semi-automated immune cell profiling, and demonstrate potential to automate aspects of clinical multiparameter flow cytometric (MFC) diagnostic workflow. Utilizing AI in the analysis of cytometry samples can reduce subjective variability and assist in breakthroughs in understanding diseases. Here we review the diverse types of AI that are being applied to clinical cytometry data and how AI is driving advances in data analysis to improve diagnostic sensitivity and accuracy. We review supervised and unsupervised clustering algorithms for cell population identification, various dimensionality reduction techniques, and their utilities in visualization and machine learning pipelines, and supervised learning approaches for classifying entire cytometry samples.Understanding the AI landscape will enable pathologists to better utilize open source and commercially available tools, plan exploratory research projects to characterize diseases, and work with machine learning and data scientists to implement clinical data analysis pipelines.

Digital pathology and artificial intelligence as the next chapter in diagnostic hematopathology.

Digital pathology has a crucial role in diagnostic pathology and is increasingly a technological requirement in the field. Integration of digital slides into the pathology workflow, advanced algorithms, and computer-aided diagnostic techniques extend the frontiers of the pathologist's view beyond the microscopic slide and enable true integration of knowledge and expertise. There is clear potential for artificial intelligence (AI) breakthroughs in pathology and hematopathology. In this review article, we discuss the approach of using machine learning in the diagnosis, classification, and treatment guidelines of hematolymphoid disease, as well as recent progress of artificial intelligence in flow cytometric analysis of hematolymphoid diseases. We review these topics specifically through the potential clinical applications of CellaVision, an automated digital image analyzer of peripheral blood, and Morphogo, a novel artificial intelligence-based bone marrow analyzing system. Adoption of these new technologies will allow pathologists to streamline workflow and achieve faster turnaround time in diagnosing hematological disease.

Maternal Undernutrition Induces Cell Signalling and Metabolic Dysfunction in Undifferentiated Mouse Embryonic Stem Cells.

Peri-conceptional environment can induce permanent changes in embryo phenotype which alter development and associate with later disease susceptibility. Thus, mouse maternal low protein diet (LPD) fed exclusively during preimplantation is sufficient to lead to cardiovascular, metabolic and neurological dysfunction in adult offspring. Embryonic stem cell (ESC) lines were generated from LPD and control NPD C57BL/6 blastocysts and characterised by transcriptomics, metabolomics, bioinformatics and molecular/cellular studies to assess early potential mechanisms in dietary environmental programming. Previously, we showed these lines retain cellular and epigenetic characteristics of LPD and NPD embryos after several passages. Here, three main changes were identified in LPD ESC lines. First, their derivation capacity was reduced but pluripotency marker expression was similar to controls. Second, LPD lines had impaired Mitogen-activated protein kinase (MAPK) pathway with altered gene expression of several regulators (e.g., Maff, Rassf1, JunD), reduced ERK1/2 signalling capacity and poorer cell survival characteristics which may contribute to reduced derivation. Third, LPD lines had impaired glucose metabolism comprising reduced upstream enzyme expression (e.g., Gpi, Mpi) and accumulation of metabolites (e.g., glucose-6-P, fructose-6-P) above the phosphofructokinase (PFK) gateway with PFK enzyme activity reduced. ESC lines may therefore permit investigation of peri-conceptional programming mechanisms with reduced need for animal experimentation.

Structure of the lipopolysaccharide O-antigens from Fusobacterium nucleatum strains HM-994, HM-995, HM-997.

Fusobacterium nucleatum is an anaerobic bacterium found in the human mouth where it causes periodontitis. It was also found in colorectal cancer tissues and is linked with pregnancy complications, including pre-term and stillbirths. Cell surface structures of the bacterium could be implicated in pathogenesis. Here we report the structure of the lipopolysaccharide O-chain (OPS) of three strains of F. nucleatum HM-994, HM-995, and HM-997, isolated from cancerous tissues: -3-ß-D-ManNAc4Lac-4-ß-D-Glc6OAc-3-ß-D-FucNAc4N- HM-994. -4-α-L-GalNHBuA-3-α-D-QuiNAc4NHBu-3-α-L-Rha-6-α-D-GalN- HM-995. -3-[α-L-GulNAcA-4-]-ß-D-Glc-4-ß-D-ManNAcAN-3-ß-D-FucNAc4N-3- HM-997. where HBu is 3-hydroxybutyryl, ManNAc4Lac is 4-O-(1-carboxyethyl)-2-acetamido-2-deoxy-mannose. All monosaccharides are in the pyranose form. The structures were determined using standard NMR (2D homo- and hetero-nuclear techniques), MS and chemical methods following gtypical LPS isolation and purification methods. In some cases polymeric material was further degraded in order to produce compounds that gave improved NMR spectra that were easier to be fully interpreted. Structure of the OPS from strain HM-994 was identical to the OPS from F. nucleatum strain MJR 7757 B. Structures of the OPS from HM-995 and HM-997 are novel and to our knowledge have not been previously reported and include the often observed 6-deoxy- sugars found in several F. nucleatum strains and butyrate rather than acetate modifications in the HM-995 strain. This structural knowledge adds to the ever increasing variation found in LPS O-antigen structures from F. nucleatum strain from both oral and cancerous origin and suggests that there may be a multitude of different LPS O-antigen structures elaborated by this organism that may present challenges to any serotyping efforts.

The structure of the LPS O-chain from five Fusobacterium nucleatum strains CTX47T, CC2_6JVN3, CC2_3FMU1, CC2_1JVN3, HM-996, containing alditol and phosphate in the main chain and development of mouse monoclonal antibodies specific to the O-antigens.

Fusobacterium nucleatum is an anaerobic bacterium found in the human mouth where it causes periodontitis. It was also found in colorectal cancer tissues and is linked with pregnancy complications, including pre-term and stillbirths. Cell surface structures of the bacterium could be implicated in pathogenesis. Here we report four new structures of the lipopolysaccharide O-chain (OPS) from five strains of F. nucleatum CTX47T, CC2_6JVN3, CC2_3FMU1, CC2_1JVN3, HM-996, isolated from cancerous tissues. Three of the four structures have a common sequence of hexose-diaminofucose-hexitol-phosphate in the main chain.

Disruption of dopamine receptor 1 localization to primary cilia impairs signaling in striatal neurons.

A rod-shaped appendage called a primary cilium projects from the soma of most central neurons in the mammalian brain. The importance of cilia within the nervous system is highlighted by the fact that human syndromes linked to primary cilia dysfunction, collectively termed ciliopathies, are associated with numerous neuropathologies, including hyperphagia-induced obesity, neuropsychiatric disorders, and learning and memory deficits. Neuronal cilia are enriched with signaling molecules, including specific G protein-coupled receptors (GPCRs) and their downstream effectors, suggesting they act as sensory organelles that respond to neuromodulators in the extracellular space. We previously showed that GPCR ciliary localization is disrupted in neurons from mouse models of the ciliopathy Bardet-Biedl syndrome (BBS). Based on this finding we hypothesized that mislocalization of ciliary GPCRs may impact receptor signaling and contribute to the BBS phenotypes. Here, we show that disrupting localization of the ciliary GPCR dopamine receptor 1 (D1) in male and female mice, either by loss of a BBS protein or loss of the cilium itself, specifically in D1-expressing neurons, results in obesity. Interestingly, the weight gain is associated with reduced locomotor activity, rather than increased food intake. Moreover, loss of a BBS protein or cilia on D1-expressing neurons leads to a reduction in D1-mediated signaling. Together, these results indicate that cilia impact D1 activity in the nervous system and underscore the importance of neuronal cilia for proper GPCR signaling.SIGNIFICANCE STATEMENT:Most mammalian neurons possess solitary appendages called primary cilia. These rod-shaped structures are enriched with signaling proteins, such as G protein-coupled receptors (GPCRs), suggesting they respond to neuromodulators. This study examines the consequences of disrupting ciliary localization of the GPCR dopamine receptor 1 (D1) in D1-expressing neurons. Remarkably, mice that have either abnormal accumulation of D1 in cilia or loss of D1 ciliary localization become obese. In both cases the obesity is associated with lower locomotor activity rather than overeating. As D1 activation increases locomotor activity, these results are consistent with a reduction in D1 signaling. Indeed, we found that D1-mediated signaling is reduced in brain slices from both mouse models. Thus, cilia impact D1 signaling in the brain.

Synthesis and Immunogenicity of a Methyl Rhamnan Pentasaccharide Conjugate from Pseudomonas aeruginosa A-Band Polysaccharide.

Pseudomonas aeruginosa was added to the World Health Organization's priority pathogen list for research and development of new antibiotics in 2017. Alongside the development of new antibiotics to fight antimicrobial-resistant P. aeruginosa, vaccines would be an appealing addition to the toolbox health professionals have against this bacteria, which causes life-threatening respiratory infections. Recently, the structure of a novel immunogenic terminal carbohydrate moiety on the cell surface of P. aeruginosa was elucidated, consisting of a 3-O-methyl (1→4)-α-d-rhamnan pentasaccharide. As isolating this oligosaccharide from P. aeruginosa in sufficient amounts for producing a conjugate vaccine is challenging, herein we describe the synthesis of 3-O-methyl d-rhamnose oligosaccharide. We also report the conjugation of the synthetic pentasaccharide to human serum albumin and its resulting immunogenicity.