Variation of terpene alkaloids in Daphniphyllum macropodum across plants and tissues.

Daphniphyllum macropodum produces alkaloids that are structurally complex with polycyclic, stereochemically rich carbon skeletons. Understanding how these compounds are formed by the plant may enable exploration of their biological function and bioactivities. We employed multiple metabolomics techniques, including a workflow to annotate compounds in the absence of standards, to compare alkaloid content across plants and tissues. Different alkaloid structural types were found to have distinct distributions between genotypes, between tissues and within tissues. Alkaloid structural types also showed different isotope labelling enrichments that matched their biosynthetic relationships. The work suggests that mevalonate derived 30-carbon alkaloids are formed in the phloem region before their conversion to 22-carbon alkaloids which accumulate in the epidermis. This sets the stage for further investigation into the biosynthetic pathway.

Antigenic epitope targets of rhesus macaques self-curing from Schistosoma mansoni infection

The self-cure of rhesus macaques from a schistosome infection and their subsequent strong immunity to a cercarial challenge should provide novel insights into the way these parasites can be eliminated by immunological attack. High-density arrays comprising overlapping 15-mer peptides from target proteins printed on glass slides can be used to screen sera from host species to determine antibody reactivity at the single epitope level. Careful selection of proteins, based on compositional studies, is crucial to encompass only those exposed on or secreted from the intra-mammalian stages and is intended to focus the analysis solely on targets mediating protection. We report the results of this approach using two pools of sera from hi- and lo-responder macaques undergoing self-cure, to screen arrays comprising tegument, esophageal gland, and gastrodermis proteins. We show that, overall, the target epitopes are the same in both groups, but the intensity of response is twice as strong in the high responders. In addition, apart from Sm25, tegument proteins elicit much weaker responses than those originating in the alimentary tract, as was apparent in IFNγR KO mice. We also highlight the most reactive epitopes in key proteins. Armed with this knowledge, we intend to use multi-epitope constructs in vaccination experiments, which seek to emulate the self-cure process in experimental animals and potentially in humans.

Whole genome structural predictions reveal hidden diversity in putative oxidative enzymes of the lignocellulose-degrading ascomycete Parascedosporium putredinis NO1.

IMPORTANCE: An annotated reference genome has revealed P. putredinis NO1 as a useful resource for the identification of new lignocellulose-degrading enzymes for biorefining of woody plant biomass. Utilizing a "structure-omics"-based searching strategy, we identified new potentially lignocellulose-active sequences that would have been missed by traditional sequence searching methods. These new identifications, alongside the discovery of novel enzymatic functions from this underexplored lineage with the recent discovery of a new phenol oxidase that cleaves the main structural ß-O-4 linkage in lignin from P. putredinis NO1, highlight the underexplored and poorly represented family Microascaceae as a particularly interesting candidate worthy of further exploration toward the valorization of high value biorenewable products.

Identification of a protein expression signature distinguishing early from organising diffuse alveolar damage in COVID-19 patients.

Diffuse alveolar damage (DAD) is the histological expression of acute respiratory distress syndrome and characterises lung pathology due to infection with SARS-CoV-2, and other respiratory pathogens of clinical significance. DAD reflects a time-dependent immunopathological process, progressing from an early/exudative stage through to an organising/fibrotic stage, yet within an individual these different stages of DAD may coexist. Understanding the progression of DAD is central to the development of new therapeutics to limit progressive lung damage. Here, we applied highly multiplexed spatial protein profiling to autopsy lung tissues derived from 27 patients who died from COVID-19 and identified a protein signature (ARG1, CD127, GZMB, IDO1, Ki67, phospho-PRAS40 (T246) and VISTA) that distinguishes early DAD from late DAD with good predictive accuracy. These proteins warrant further investigation as potential regulators of DAD progression.

Antigenic epitope targets of rhesus macaques self-curing from Schistosoma mansoni infection.

The self-cure of rhesus macaques from a schistosome infection and their subsequent strong immunity to a cercarial challenge should provide novel insights into the way these parasites can be eliminated by immunological attack. High-density arrays comprising overlapping 15-mer peptides from target proteins printed on glass slides can be used to screen sera from host species to determine antibody reactivity at the single epitope level. Careful selection of proteins, based on compositional studies, is crucial to encompass only those exposed on or secreted from the intra-mammalian stages and is intended to focus the analysis solely on targets mediating protection. We report the results of this approach using two pools of sera from hi- and lo-responder macaques undergoing self-cure, to screen arrays comprising tegument, esophageal gland, and gastrodermis proteins. We show that, overall, the target epitopes are the same in both groups, but the intensity of response is twice as strong in the high responders. In addition, apart from Sm25, tegument proteins elicit much weaker responses than those originating in the alimentary tract, as was apparent in IFNγR KO mice. We also highlight the most reactive epitopes in key proteins. Armed with this knowledge, we intend to use multi-epitope constructs in vaccination experiments, which seek to emulate the self-cure process in experimental animals and potentially in humans.

Corrigendum: CD317-Positive Immune Stromal Cells in Human "Mesenchymal Stem Cell" Populations.

[This corrects the article DOI: 10.3389/fimmu.2022.903796.].

Development and spread of health literacy eLearning: A partnership across Scotland and England.

With international health challenges, there are opportunities for collaboration between nations on health issues, including developing and sharing resources for teaching and learning. This article outlines collaboration across Scotland and England to develop a core resource for eLearning on health literacy. It describes the development of the resource with case studies of the implementation in Scotland and England, demonstrating the balance between shared development and tailored implementation. The eLearning was developed to increase awareness of NHS workforce and community partners, supplemented by training for NHS librarians and public health specialists to enable them to provide more tailored training on health literacy techniques.

CD317-Positive Immune Stromal Cells in Human "Mesenchymal Stem Cell" Populations.

Heterogeneity of bone marrow mesenchymal stromal cells (MSCs, frequently referred to as "mesenchymal stem cells") clouds biological understanding and hampers their clinical development. In MSC cultures most commonly used in research and therapy, we have identified an MSC subtype characterized by CD317 expression (CD317pos (29.77 ± 3.00% of the total MSC population), comprising CD317dim (28.10 ± 4.60%) and CD317bright (1.67 ± 0.58%) MSCs) and a constitutive interferon signature linked to human disease. We demonstrate that CD317pos MSCs induced cutaneous tissue damage when applied a skin explant model of inflammation, whereas CD317neg MSCs had no effect. Only CD317neg MSCs were able to suppress proliferative cycles of activated human T cells in vitro, whilst CD317pos MSCs increased polarization towards pro-inflammatory Th1 cells and CD317neg cell lines did not. Using an in vivo peritonitis model, we found that CD317neg and CD317pos MSCs suppressed leukocyte recruitment but only CD317neg MSCs suppressed macrophage numbers. Using MSC-loaded scaffolds implanted subcutaneously in immunocompromised mice we were able to observe tissue generation and blood vessel formation with CD317neg MSC lines, but not CD317pos MSC lines. Our evidence is consistent with the identification of an immune stromal cell, which is likely to contribute to specific physiological and pathological functions and influence clinical outcome of therapeutic MSCs.

Endosomal cargo recycling mediated by Gpa1 and phosphatidylinositol 3-kinase is inhibited by glucose starvation.

Cell surface protein trafficking is regulated in response to nutrient availability, with multiple pathways directing surface membrane proteins to the lysosome for degradation in response to suboptimal extracellular nutrients. Internalized protein and lipid cargoes recycle back to the surface efficiently in glucose-replete conditions, but this trafficking is attenuated following glucose starvation. We find that cells with either reduced or hyperactive phosphatidylinositol 3-kinase (PI3K) activity are defective for endosome to surface recycling. Furthermore, we find that the yeast Gα subunit Gpa1, an endosomal PI3K effector, is required for surface recycling of cargoes. Following glucose starvation, mRNA and protein levels of a distinct Gα subunit Gpa2 are elevated following nuclear translocation of Mig1, which inhibits recycling of various cargoes. As Gpa1 and Gpa2 interact at the surface where Gpa2 concentrates during glucose starvation, we propose that this disrupts PI3K activity required for recycling, potentially diverting Gpa1 to the surface and interfering with its endosomal role in recycling. In support of this model, glucose starvation and overexpression of Gpa2 alter PI3K endosomal phosphoinositide production. Glucose deprivation therefore triggers a survival mechanism to increase retention of surface cargoes in endosomes and promote their lysosomal degradation.

Integrated miRNA/cytokine/chemokine profiling reveals severity-associated step changes and principal correlates of fatality in COVID-19.

Inflammatory cytokines and chemokines (CC) drive COVID-19 pathology. Yet, patients with similar circulating CC levels present with different disease severity. Here, we determined 171 microRNAomes from 58 hospitalized COVID-19 patients (Cohort 1) and levels of 25 cytokines and chemokines (CC) in the same samples. Combining microRNA (miRNA) and CC measurements allowed for discrimination of severe cases with greater accuracy than using miRNA or CC levels alone. Severity group-specific associations between miRNAs and COVID-19-associated CC (e.g., IL6, CCL20) or clinical hallmarks of COVID-19 (e.g., neutrophilia, hypoalbuminemia) separated patients with similar CC levels but different disease severity. Analysis of an independent cohort of 108 patients from a different center (Cohort 2) demonstrated feasibility of CC/miRNA profiling in leftover hospital blood samples with similar severe disease CC and miRNA profiles, and revealed CCL20, IL6, IL10, and miR-451a as key correlates of fatal COVID-19. These findings highlight that systemic miRNA/CC networks underpin severe COVID-19.

Candidates for Balancing Selection in Leishmania donovani Complex Parasites.

The Leishmania donovani species complex is the causative agent of visceral leishmaniasis, which cause 20-40,000 fatalities a year. Here, we conduct a screen for balancing selection in this species complex. We used 384 publicly available L. donovani and L. infantum genomes, and sequence 93 isolates of L. infantum from Brazil to describe the global diversity of this species complex. We identify five genetically distinct populations that are sufficiently represented by genomic data to search for signatures of selection. We find that signals of balancing selection are generally not shared between populations, consistent with transient adaptive events, rather than long-term balancing selection. We then apply multiple diversity metrics to identify candidate genes with robust signatures of balancing selection, identifying a curated set of 24 genes with robust signatures. These include zeta toxin, nodulin-like, and flagellum attachment proteins. This study highlights the extent of genetic divergence between L. donovani complex parasites and provides genes for further study.

Superoxide is promoted by sucrose and affects amplitude of circadian rhythms in the evening.

Plants must coordinate photosynthetic metabolism with the daily environment and adapt rhythmic physiology and development to match carbon availability. Circadian clocks drive biological rhythms which adjust to environmental cues. Products of photosynthetic metabolism, including sugars and reactive oxygen species (ROS), are closely associated with the plant circadian clock, and sugars have been shown to provide metabolic feedback to the circadian oscillator. Here, we report a comprehensive sugar-regulated transcriptome of Arabidopsis and identify genes associated with redox and ROS processes as a prominent feature of the transcriptional response. We show that sucrose increases levels of superoxide (O2-), which is required for transcriptional and growth responses to sugar. We identify circadian rhythms of O2--regulated transcripts which are phased around dusk and find that O2- is required for sucrose to promote expression of TIMING OF CAB1 (TOC1) in the evening. Our data reveal a role for O2- as a metabolic signal affecting transcriptional control of the circadian oscillator in Arabidopsis.

Chromosomal assembly of the nuclear genome of the endosymbiont-bearing trypanosomatid Angomonas deanei.

Angomonas deanei is an endosymbiont-bearing trypanosomatid with several highly fragmented genome assemblies and unknown chromosome number. We present an assembly of the A. deanei nuclear genome based on Oxford Nanopore sequence that resolves into 29 complete or close-to-complete chromosomes. The assembly has several previously unknown special features; it has a supernumerary chromosome, a chromosome with a 340-kb inversion, and there is a translocation between two chromosomes. We also present an updated annotation of the chromosomal genome with 10,365 protein-coding genes, 59 transfer RNAs, 26 ribosomal RNAs, and 62 noncoding RNAs.

LIMPRINT in Australia.

Background and Study Objective: Australia was one of nine participating countries in the epidemiology Phase II Lymphoedema Impact and Prevalence - International (LIMPRINT) project to determine the number of people with chronic edema (CO) in local health services. Methods and Results: Data collection occurred through questionnaire-based interviews and clinical assessment with provided LIMPRINT tools. Four different types of services across three states in Australia participated. A total of 222 adults participated with an age range from 22 to 102 years, and 60% were female. Site 1 included three residential care facilities (54% of participants had swelling), site 2 was community-delivered aged care services (24% of participants had swelling), site 3 was a hospital setting (facility-based prevalence study; 28% of participants had swelling), and site 4 was a wound treatment center (specific patient population; 100% of participants had swelling). Of those with CO or secondary lymphedema, 93% were not related to cancer, the lower limbs were affected in 51% of cases, and 18% of participants with swelling reported one or more episodes of cellulitis in the previous year. Wounds were identified in 47% (n = 105) of all participants with more than half of those with wounds coming from the dedicated wound clinic. Leg/foot ulcer was the most common type of wound (65%, n = 68). Conclusions: Distances between services, lack of specialized services, and various state funding models contribute to inequities in CO treatment. Understanding the high number of noncancer-related CO presentations will assist health services to provide timely effective care and improve referral pathways.

Tissue-specific transcriptomic changes associated with AmBisome® treatment of BALB/c mice with experimental visceral leishmaniasis.

Background: Liposomal amphotericin B (AmBisome®) as a treatment modality for visceral leishmaniasis (VL) has had significant impact on patient care in some but not all regions where VL is endemic.  As the mode of action of AmBisome® in vivo is poorly understood, we compared the tissue-specific transcriptome in drug-treated vs untreated mice with experimental VL.    Methods:  BALB/c mice infected with L. donovani were treated with 8mg/kg AmBisome®, resulting in parasite elimination from liver and spleen over a 7-day period. At day 1 and day 7 post treatment (R x+1 and R x+7), transcriptomic profiling was performed on spleen and liver tissue from treated and untreated mice and uninfected mice.  BALB/c mice infected with M. bovis BCG (an organism resistant to amphotericin B) were analysed to distinguish between direct effects of AmBisome® and those secondary to parasite death.   Results: AmBisome® treatment lead to rapid parasitological clearance.  At R x+1, spleen and liver displayed only 46 and 88 differentially expressed (DE) genes (P<0.05; 2-fold change) respectively. In liver, significant enrichment was seen for pathways associated with TNF, fatty acids and sterol biosynthesis.  At R x+7, the number of DE genes was increased (spleen, 113; liver 400).  In spleen, these included many immune related genes known to be involved in anti-leishmanial immunity. In liver, changes in transcriptome were largely accounted for by loss of granulomas.   PCA analysis indicated that treatment only partially restored homeostasis.  Analysis of BCG-infected mice treated with AmBisome® revealed a pattern of immune modulation mainly targeting macrophage function.   Conclusions: Our data indicate that the tissue response to AmBisome® treatment varies between target organs and that full restoration of homeostasis is not achieved at parasitological cure.  The pathways required to restore homeostasis deserve fuller attention, to understand mechanisms associated with treatment failure and relapse and to promote more rapid restoration of immune competence.

Subtype-specific regulatory network rewiring in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a heterogeneous disease caused by a variety of alterations in transcription factors, epigenetic regulators and signaling molecules. To determine how different mutant regulators establish AML subtype-specific transcriptional networks, we performed a comprehensive global analysis of cis-regulatory element activity and interaction, transcription factor occupancy and gene expression patterns in purified leukemic blast cells. Here, we focused on specific subgroups of subjects carrying mutations in genes encoding transcription factors (RUNX1, CEBPα), signaling molecules (FTL3-ITD, RAS) and the nuclear protein NPM1). Integrated analysis of these data demonstrates that each mutant regulator establishes a specific transcriptional and signaling network unrelated to that seen in normal cells, sustaining the expression of unique sets of genes required for AML growth and maintenance.

Epidermal growth factor can signal via ß-catenin to control proliferation of mesenchymal stem cells independently of canonical Wnt signalling.

Bone marrow mesenchymal stem/stromal cells (MSCs) maintain bone homeostasis and repair through the ability to expand in response to mitotic stimuli and differentiate into skeletal lineages. Signalling mechanisms that enable precise control of MSC function remain unclear. Here we report that by initially examining differences in signalling pathway expression profiles of individual MSC clones, we identified a previously unrecognised signalling mechanism regulated by epidermal growth factor (EGF) in primary human MSCs. We demonstrate that EGF is able to activate ß-catenin, a key component of the canonical Wnt signalling pathway. EGF is able to induce nuclear translocation of ß-catenin in human MSCs but does not drive expression of Wnt target genes or T cell factor (TCF) activity in MSC reporter cell lines. Using an efficient Design of Experiments (DoE) statistical analysis, with different combinations and concentrations of EGF and Wnt ligands, we were able to confirm that EGF does not influence the Wnt/ß-catenin pathway in MSCs. We show that the effects of EGF on MSCs are temporally regulated to initiate early "classical" EGF signalling mechanisms (e.g via mitogen activated protein kinase) with delayed activation of ß-catenin. By RNA-sequencing, we identified gene sets that were exclusively regulated by the EGF/ß-catenin pathway, which were distinct from classical EGF-regulated genes. However, subsets of classical EGF gene targets were significantly influenced by EGF/ß-catenin activation. These signalling pathways cooperate to enable EGF-mediated proliferation of MSCs by alleviating the suppression of cell cycle pathways induced by classical EGF signalling.

Characterization of ulcerative colitis-associated constipation syndrome (proximal constipation).

BACKGROUND: The syndrome of constipation with other abdominal symptoms ("proximal constipation") in ulcerative colitis (UC) is commonly recognized by practitioners but is poorly described, with no recognized definition and little understanding with regard to prevalence and effect of therapies on disease outcomes. This study aimed to address these issues in a cross-sectional, consecutive series of patients with UC. METHODS: A working definition of proximal constipation was established. Consecutive patients were recruited, and their disease activity, recent medications, and investigations plus abdominal symptoms were assessed at a study visit. Relevant clinical data were also extracted from medical records. RESULTS: Of 125 patients with UC, (mean age 47, range 14-84 years, 61 male), 58 (46%) fulfilled the definition of proximal constipation. The main symptoms were reduced stool frequency (69%), hard stools (43%), abdominal pain (40%), excessive flatus (29%), straining (24%), and sensation of incomplete emptying (14%). Proximal constipation was associated with female gender (OR 3.45 [1.45-8.24]), left-sided (OR 2.84 [1.14-7.11]) and concurrently active disease (OR 5.56 [1.96-16.67]), but not age, disease duration or therapy. A total of 88% had an increase in anti-inflammatory therapy, with the use of laxatives or fiber supplements in 63% compared with 1.4% of those without proximal constipation. CONCLUSIONS: Proximal constipation is common, and its risk increases in active and distal disease, especially in women. Validation of its definition and evaluation of therapeutic strategies are needed. A new term "ulcerative colitis-associated constipation syndrome" is proposed to more accurately depict its nature.

Microexon gene transcriptional profiles and evolution provide insights into blood processing by the Schistosoma japonicum esophagus.

BACKGROUND: Adult schistosomes have a well-developed alimentary tract comprising an oral sucker around the mouth, a short esophagus and a blind ending gut. The esophagus is not simply a muscular tube for conducting blood from the mouth to gut but is divided into compartments, surrounded by anterior and posterior glands, where processing of ingested blood is initiated. Self-cure of rhesus macaques from a Schistosoma japonicum infection appears to operate by blocking the secretory functions of these glands so that the worms cease feeding and slowly starve to death. Here we use subtractive RNASeq to characterise the genes encoding the principal secretory products of S. japonicum esophageal glands, preparatory to evaluating their relevance as targets of the self-cure process. METHODOLOGY/PRINCIPAL FINDINGS: The heads and a small portion of the rear end of male and female S. japonicum worms were separately enriched by microdissection, for mRNA isolation and library construction. The sequence reads were then assembled de novo using Trinity and those genes enriched more than eightfold in the head preparation were subjected to detailed bioinformatics analysis. Of the 62 genes selected from the male heads, more than one third comprised MEGs encoding secreted or membrane-anchored proteins. Database searching using conserved motifs revealed that the MEG-4 and MEG-8/9 families had counterparts in the bird schistosome Trichobilharzia regenti, indicating an ancient association with blood processing. A second group of MEGs, including a MEG-26 family, encoded short peptides with amphipathic properties that most likely interact with ingested host cell membranes to destabilise them. A number of lysosomal hydrolases, two protease inhibitors, a secreted VAL and a putative natterin complete the line-up. There was surprisingly little difference between expression patterns in males and females despite the latter processing much more blood. SIGNIFICANCE/CONCLUSIONS: The mixture of approximately 40 proteins specifically secreted by the esophageal glands is responsible for initiating blood processing in the adult worm esophagus. They comprise the potential targets for the self-cure process in the rhesus macaque, and thus represent a completely new cohort of secreted proteins that can be investigated as vaccine candidates.

Tissue and host species-specific transcriptional changes in models of experimental visceral leishmaniasis.

Background: Human visceral leishmaniasis, caused by infection with Leishmania donovani or L. infantum, is a potentially fatal disease affecting 50,000-90,000 people yearly in 75 disease endemic countries, with more than 20,000 deaths reported. Experimental models of infection play a major role in understanding parasite biology, host-pathogen interaction, disease pathogenesis, and parasite transmission. In addition, they have an essential role in the identification and pre-clinical evaluation of new drugs and vaccines. However, our understanding of these models remains fragmentary. Although the immune response to Leishmania donovani infection in mice has been extensively characterized, transcriptomic analysis capturing the tissue-specific evolution of disease has yet to be reported. Methods: We provide an analysis of the transcriptome of spleen, liver and peripheral blood of BALB/c mice infected with L. donovani. Where possible, we compare our data in murine experimental visceral leishmaniasis with transcriptomic data in the public domain obtained from the study of L. donovani-infected hamsters and patients with human visceral leishmaniasis. Digitised whole slide images showing the histopathology in spleen and liver are made available via a dedicated website, www.leishpathnet.org. Results: Our analysis confirms marked tissue-specific alterations in the transcriptome of infected mice over time and identifies previously unrecognized parallels and differences between murine, hamster and human responses to infection. We show commonality of interferon-regulated genes whilst confirming a greater activation of type 2 immune pathways in infected hamsters compared to mice. Cytokine genes and genes encoding immune checkpoints were markedly tissue specific and dynamic in their expression, and pathways focused on non-immune cells reflected tissue specific immunopathology. Our data also addresses the value of measuring peripheral blood transcriptomics as a potential window into underlying systemic disease.  Conclusions: Our transcriptomic data, coupled with histopathologic analysis of the tissue response, provide an additional resource to underpin future mechanistic studies and to guide clinical research.