An allele-selective inter-chromosomal protein bridge supports monogenic antigen expression in the African trypanosome.

UPF1-like helicases play roles in telomeric heterochromatin formation and X-chromosome inactivation, and also in monogenic variant surface glycoprotein (VSG) expression via VSG exclusion-factor-2 (VEX2), a UPF1-related protein in the African trypanosome. We show that VEX2 associates with chromatin specifically at the single active VSG expression site on chromosome 6, forming an allele-selective connection, via VEX1, to the trans-splicing locus on chromosome 9, physically bridging two chromosomes and the VSG transcription and splicing compartments. We further show that the VEX-complex is multimeric and self-regulates turnover to tightly control its abundance. Using single cell transcriptomics following VEX2-depletion, we observed simultaneous derepression of many other telomeric VSGs and multi-allelic VSG expression in individual cells. Thus, an allele-selective, inter-chromosomal, and self-limiting VEX1-2 bridge supports monogenic VSG expression and multi-allelic VSG exclusion.

Competition among variants is predictable and contributes to the antigenic variation dynamics of African trypanosomes.

Several persistent pathogens employ antigenic variation to continually evade mammalian host adaptive immune responses. African trypanosomes use variant surface glycoproteins (VSGs) for this purpose, transcribing one telomeric VSG expression-site at a time, and exploiting a reservoir of (sub)telomeric VSG templates to switch the active VSG. It has been known for over fifty years that new VSGs emerge in a predictable order in Trypanosoma brucei, and differential activation frequencies are now known to contribute to the hierarchy. Switching of approximately 0.01% of dividing cells to many new VSGs, in the absence of post-switching competition, suggests that VSGs are deployed in a highly profligate manner, however. Here, we report that switched trypanosomes do indeed compete, in a highly predictable manner that is dependent upon the activated VSG. We induced VSG gene recombination and switching in in vitro culture using CRISPR-Cas9 nuclease to target the active VSG. VSG dynamics, that were independent of host immune selection, were subsequently assessed using RNA-seq. Although trypanosomes activated VSGs from repressed expression-sites at relatively higher frequencies, the population of cells that activated minichromosomal VSGs subsequently displayed a competitive advantage and came to dominate. Furthermore, the advantage appeared to be more pronounced for longer VSGs. Differential growth of switched clones was also associated with wider differences, affecting transcripts involved in nucleolar function, translation, and energy metabolism. We conclude that antigenic variants compete, and that the population of cells that activates minichromosome derived VSGs displays a competitive advantage. Thus, competition among variants impacts antigenic variation dynamics in African trypanosomes and likely prolongs immune evasion with a limited set of antigens.

Pediatric Organ and Tissue Donation-A 10-Year Retrospective Study in Portugal.

Organ and tissue donation can transform lives. One donor can ensure the survival of up to 8 people through their organs and improve the quality of life for dozens more through tissue donation. Portugal has an excellent transplantation rate, but deaths still occur while waiting for an organ. The study aimed to analyze pediatric organ and tissue donors nationally and evaluate brain deaths in a pediatric intensive care unit (PICU) over the past 10 years to identify any potential lost donors. We conducted a retrospective descriptive study of pediatric organ and tissue donors and diagnosed brain deaths from January 2011 to December 2021. Demographic and clinical data were analyzed, including those provided by the National Transplant Coordination. Over the past 10 years in Portugal, 121 pediatric donors (11.7 per million population) were collected, and 569 organs and tissues were collected. During the same period in the PICU, there were 125 deaths, including 20 brain deaths. Of this group, 4 were organ and tissue donors. In the non-donor group (n = 16), a potential lost donor case stands out. Pediatric specialists need to be more familiar with the donation process, which would enable the identification and optimization of all potential donors, thus minimizing the number of potentially lost organs.

Las láminas zootómicas del profesor Paul Pfurtscheller de Viena: un material científico renovador en las aulas españolas en el primer tercio de siglo XX / [The zootomic slides of Professor Paul Pfurtscheller from Vienna: innovative scientific material in Spanish classrooms in the first third of the 20th century]

Paul Pfurtscheller fue uno de los creadores de material didáctico más universales y re-conocidos dentro de la enseñanza de la Historia Natural, sus láminas de pared de zoología tuvieron presencia a nivel internacional en escuelas normales, institutos de segunda enseñanza e incluso en gabinetes y laboratorios universitarios. Doctor por la Universidad de Viena con una tesis de Botá-nica desarrolló una carrera prominente como profesor de secundaria de la asignatura de Historia Natural en el Gymnasium (Instituto) Franz Josef de Viena, desarrollando un gabinete museo para su asignatura y creando 39 láminas zootómicas como apoyo a su docencia personal. La edición de sus láminas a través de prestigiosa casa editorial vienesa A. Pichlers Witwe & Sohn y posteriormente por la holandesa Martinus Nijhoff permitió la difusión de su trabajo a través de Europa y América. Tras una aproximación biográfica del personaje basada en los documentos encontrados en la Biblioteca Nacional de Austria y en los de la Universidad de Viena, se ha indagado en su papel como docente y en la génesis de su colección, en su éxito pedagógico y el papel que tuvieron en las aulas espa-ñolas de principios de siglo XX. Dentro de las fuentes a considerar destacan las propias láminas, los catálogos comerciales, las leyendas explicativas, los manuales de clase, memorias anuales de los centros estudiados y las imágenes de las aulas, no como meras ilustraciones, sino como objeto de estudio para analizar el espacio escolar y el papel didáctico de las láminas. (AU)

Emergence and adaptation of the cellular machinery directing antigenic variation in the African trypanosome.

Survival of the African trypanosome within its mammalian hosts, and hence transmission between hosts, relies upon antigenic variation, where stochastic changes in the composition of their protective variant-surface glycoprotein (VSG) coat thwart effective removal of the pathogen by adaptive immunity. Antigenic variation has evolved remarkable mechanistic complexity in Trypanosoma brucei, with switching of the VSG coat executed by either transcriptional or recombination reactions. In the former, a single T. brucei cell selectively transcribes one telomeric VSG transcription site, termed the expression site (ES), from a pool of around 15. Silencing of the active ES and activation of one previously silent ES can lead to a co-ordinated VSG coat switch. Outside the ESs, the T. brucei genome contains an enormous archive of silent VSG genes and pseudogenes, which can be recombined into the ES to execute a coat switch. Most such recombination involves gene conversion, including copying of a complete VSG and more complex reactions where novel 'mosaic' VSGs are formed as patchworks of sequences from several silent (pseudo)genes. Understanding of the cellular machinery that directs transcriptional and recombination VSG switching is growing rapidly and the emerging picture is of the use of proteins, complexes and pathways that are not limited to trypanosomes, but are shared across the wider grouping of kinetoplastids and beyond, suggesting co-option of widely used, core cellular reactions. We will review what is known about the machinery of antigenic variation and discuss if there remains the possibility of trypanosome adaptations, or even trypanosome-specific machineries, that might offer opportunities to impair this crucial parasite-survival process.

Pinch2 regulates myelination in the mouse central nervous system.

The extensive morphological changes of oligodendrocytes during axon ensheathment and myelination involve assembly of the Ilk-Parvin-Pinch (IPP) heterotrimeric complex of proteins to relay essential mechanical and biochemical signals between integrins and the actin cytoskeleton. Binding of Pinch1 and Pinch2 isoforms to Ilk is mutually exclusive and allows the formation of distinct IPP complexes with specific signaling properties. Using tissue-specific conditional gene ablation in mice, we reveal an essential role for Pinch2 during central nervous system myelination. Unlike Pinch1 gene ablation, loss of Pinch2 in oligodendrocytes results in hypermyelination and in the formation of pathological myelin outfoldings in white matter regions. These structural changes concur with inhibition of Rho GTPase RhoA and Cdc42 activities and phenocopy aspects of myelin pathology observed in corresponding mouse mutants. We propose a dual role for Pinch2 in preventing an excess of myelin wraps through RhoA-dependent control of membrane growth and in fostering myelin stability via Cdc42-dependent organization of cytoskeletal septins. Together, these findings indicate that IPP complexes containing Pinch2 act as a crucial cell-autonomous molecular hub ensuring synchronous control of key signaling networks during developmental myelination.

CRISPR/Cas9-based precision tagging of essential genes in bloodstream form African trypanosomes.

Proteins of interest are frequently expressed with a fusion-tag to facilitate experimental analysis. In trypanosomatids, which are typically diploid, a tag-encoding DNA fragment is typically fused to one native allele. However, since recombinant cells represent ≪0.1% of the population following transfection, these DNA fragments also incorporate a marker cassette for positive selection. Consequently, native mRNA untranslated regions (UTRs) are replaced, potentially perturbing gene expression; in trypanosomatids, UTRs often impact gene expression in the context of widespread and constitutive polycistronic transcription. We sought to develop a tagging strategy that preserves native UTRs in bloodstream-form African trypanosomes, and here we describe a CRISPR/Cas9-based knock-in approach to drive precise and marker-free tagging of essential genes. Using simple tag-encoding amplicons, we tagged four proteins: a histone acetyltransferase, HAT2; a histone deacetylase, HDAC3; a cleavage and polyadenylation specificity factor, CPSF3; and a variant surface glycoprotein exclusion factor, VEX2. The approach maintained the native UTRs and yielded clonal strains expressing functional recombinant proteins, typically with both alleles tagged. We demonstrate utility for both immunofluorescence-based localisation and for enriching protein complexes; GFPHAT2 or GFPHDAC3 complexes in this case. This precision tagging approach facilitates the assembly of strains expressing essential recombinant genes with their native UTRs preserved.

Hematopoietic stem cell transplantation reverses white matter injury measured by diffusion-tensor imaging (DTI) in sickle cell disease patients.

Brain injury in sickle cell disease (SCD) comprises a wide spectrum of neurological damage. Neurocognitive deficits have been described even without established neurological lesions. DTI is a rapid, noninvasive, and non-contrast method that enables detection of normal-appearing white matter lesions not detected by conventional magnetic resonance imaging (MRI). The aim of the study was to evaluate if stem cell transplantation can revert white matter lesions in patients with SCD. Twenty-eight SCD patients were evaluated with MRI and DTI before and after allogeneic hematopoietic stem cell transplantation (HSCT), compared with 26 healthy controls (HC). DTI metrics included fractional anisotropy (FA), mean diffusivity (MD), radial (RD), and axial (AD) diffusivity maps, global efficiency, path length, and clustering coefficients. Compared to HC, SCD patients had a lower FA (p = 0.0086) before HSCT. After HSCT, FA increased and was not different from healthy controls (p = 0.1769). Mean MD, RD, and AD decreased after HSCT (p = 0.0049; p = 0.0029; p = 0.0408, respectively). We confirm previous data of white matter lesions in SCD and present evidence that HSCT promotes recovery of brain injury with potential improvement of brain structural connectivity.

Spatial integration of transcription and splicing in a dedicated compartment sustains monogenic antigen expression in African trypanosomes.

Highly selective gene expression is a key requirement for antigenic variation in several pathogens, allowing evasion of host immune responses and maintenance of persistent infections1. African trypanosomes-parasites that cause lethal diseases in humans and livestock-employ an antigenic variation mechanism that involves monogenic antigen expression from a pool of >2,600 antigen-coding genes2. In other eukaryotes, the expression of individual genes can be enhanced by mechanisms involving the juxtaposition of otherwise distal chromosomal loci in the three-dimensional nuclear space3-5. However, trypanosomes lack classical enhancer sequences or regulated transcription initiation6,7. In this context, it has remained unclear how genome architecture contributes to monogenic transcription elongation and transcript processing. Here, we show that the single expressed antigen-coding gene displays a specific inter-chromosomal interaction with a major messenger RNA splicing locus. Chromosome conformation capture (Hi-C) revealed a dynamic reconfiguration of this inter-chromosomal interaction upon activation of another antigen. Super-resolution microscopy showed the interaction to be heritable and splicing dependent. We found a specific association of the two genomic loci with the antigen exclusion complex, whereby VSG exclusion 1 (VEX1) occupied the splicing locus and VEX2 occupied the antigen-coding locus. Following VEX2 depletion, loss of monogenic antigen expression was accompanied by increased interactions between previously silent antigen genes and the splicing locus. Our results reveal a mechanism to ensure monogenic expression, where antigen transcription and messenger RNA splicing occur in a specific nuclear compartment. These findings suggest a new means of post-transcriptional gene regulation.

Ingestion of anthropogenic materials by yellow-legged gulls (Larus michahellis) in natural, urban, and landfill sites along Portugal in relation to diet composition.

Pollution is a global concern, increasing rapidly throughout marine and terrestrial ecosystems, and affecting many species. Urbanization enhances waste production, leading to the opening of landfills that constitute a spatially and temporally predictable food source for opportunistic species. Several species of gulls are known to exploit and breed in urban areas, taking advantage of accessible and diverse food resources. The exploitation of anthropogenic food subsidies at sea (e.g. fishery discards), urban sites, and landfills leads to debris ingestion by gulls with potential negative effects. Here we characterize anthropogenic debris ingested by yellow-legged gulls (Larus michahellis) along Portugal, by analysing the content of pellets collected in (1) natural and urban breeding locations, and in (2) urban and landfill resting sites, to assess seasonal patterns in the ingestion of anthropogenic debris. We also relate diet with the presence of anthropogenic debris. Debris materials were found in 28.8% of pellets from breeding locations (natural and urban) and in 89.7% of pellets from resting sites (urban and landfill). Gulls from the most urbanized breeding location exhibited higher levels of ingested materials during the entire breeding cycle, however, gulls from a natural breeding site also ingested high levels of debris during the pre-breeding season. At resting sites, small seasonal differences were detected in the number and mass of debris items ingested, which were both higher during spring and summer. Gulls that typically fed on pelagic fish had significantly less sheet and fragment plastics in their pellets. The presence of certain debris categories in gull pellets was positively related to the presence of some prey items, suggesting that gulls may accidentally ingest debris while foraging at multiple habitats. The quantity of anthropogenic materials ingested by gulls from urban locations and landfills indicates a need for improved waste management.

A nuclear enterprise: zooming in on nuclear organization and gene expression control in the African trypanosome.

African trypanosomes are early divergent protozoan parasites responsible for high mortality and morbidity as well as a great economic burden among the world's poorest populations. Trypanosomes undergo antigenic variation in their mammalian hosts, a highly sophisticated immune evasion mechanism. Their nuclear organization and mechanisms for gene expression control present several conventional features but also a number of striking differences to the mammalian counterparts. Some of these unorthodox characteristics, such as lack of controlled transcription initiation or enhancer sequences, render their monogenic antigen transcription, which is critical for successful antigenic variation, even more enigmatic. Recent technological developments have advanced our understanding of nuclear organization and gene expression control in trypanosomes, opening novel research avenues. This review is focused on Trypanosoma brucei nuclear organization and how it impacts gene expression, with an emphasis on antigen expression. It highlights several dedicated sub-nuclear bodies that compartmentalize specific functions, whilst outlining similarities and differences to more complex eukaryotes. Notably, understanding the mechanisms underpinning antigen as well as general gene expression control is of great importance, as it might help designing effective control strategies against these organisms.

Characterization of anthropogenic materials on yellow-legged gull (Larus michahellis) nests breeding in natural and urban sites along the coast of Portugal.

Anthropogenic materials are a persistent pressure on ecosystems, affecting many species. Seabirds can collect these materials to construct their nests, which may modify nest characteristics and cause entanglement of chicks and adults, with possible consequences on breeding success. The incorporation of anthropogenic materials in nests of seabird species that breed in both natural and urban environments, such as gulls, is poorly known. Here, we characterize and compare anthropogenic materials incorporated in yellow-legged gull (Larus michahellis) nests from two natural and two urban breeding sites across their Portuguese breeding range and during 2 consecutive years. Anthropogenic materials were found in 2.6% and 15.4% of gull nests from natural locations and in 47.6% and 95.7% of nests from urban breeding sites. No differences were found on hatching success between urban and natural breeding colonies. A significantly higher number of anthropogenic materials were found in the largest and more populated urban breeding colony, which on average included items of a greater mass but smaller size than items from the other three colonies. The higher incorporation of anthropogenic materials in urban locations could be a consequence of a lower access to natural nest construction materials and higher availability of anthropogenic debris. The quantity and diversity of anthropogenic materials incorporated in gull nests from urban locations indicate a need for improved debris management in urban settlements.

Septic shock following hysteroscopy - A case report.

INTRODUCTION: Minimally invasive gynecological surgery such as hysteroscopy has a small risk of complications. These include uterine perforation (with or without adjacent pelvic organ lesion), bleeding and infection, and are more common in the presence of risk factors such as smoking, history of pelvic inflammatory disease (PID) and endometriosis. CASE PRESENTATION: A patient submitted to a diagnostic hysteroscopy with no immediate complications was admitted five days later to the emergency department in septic shock. The diagnosis of ruptured tubal abscess was made, requiring emergency laparotomy with sub-total hysterectomy and bilateral adnexectomy. Despite multiple organ failure requiring admission to the intensive care unit, the patient made a full recovery. CONCLUSION: Ascending infection can be a life-threatening complication of hysteroscopy, even in the absence of previously known risk factors.

Monoallelic expression and epigenetic inheritance sustained by a Trypanosoma brucei variant surface glycoprotein exclusion complex.

The largest gene families in eukaryotes are subject to allelic exclusion, but mechanisms underpinning single allele selection and inheritance remain unclear. Here, we describe a protein complex sustaining variant surface glycoprotein (VSG) allelic exclusion and antigenic variation in Trypanosoma brucei parasites. The VSG-exclusion-1 (VEX1) protein binds both telomeric VSG-associated chromatin and VEX2, an ortholog of nonsense-mediated-decay helicase, UPF1. VEX1 and VEX2 assemble in an RNA polymerase-I transcription-dependent manner and sustain the active, subtelomeric VSG-associated transcription compartment. VSG transcripts and VSG coats become highly heterogeneous when VEX proteins are depleted. Further, the DNA replication-associated chromatin assembly factor, CAF-1, binds to and specifically maintains VEX1 compartmentalisation following DNA replication. Thus, the VEX-complex controls VSG-exclusion, while CAF-1 sustains VEX-complex inheritance in association with the active-VSG. Notably, the VEX2-orthologue and CAF-1 in mammals are also implicated in exclusion and inheritance functions. In trypanosomes, these factors sustain a highly effective and paradigmatic immune evasion strategy.

Consensus Recommendations for the Use of Hyperdiluted Calcium Hydroxyapatite (Radiesse) as a Face and Body Biostimulatory Agent.

BACKGROUND: Calcium hydroxyapatite (CaHA) is a well-established collagen stimulator. In recent years, it has been increasingly used in hyperdiluted form as a biostimulatory agent rather than a volumizing filler to improve skin quality and firmness in both facial and corporal areas. However, guidelines on the techniques required to achieve optimal results are still lacking. The objective of this study was to develop a consensus recommendation for the safe and effective use of hyperdiluted CaHA for face and body biostimulation. METHODS: A team of 10 experts with extensive experience in dermal fillers and biostimulatory treatments for facial and body rejuvenation convened for a live meeting. Consensus was defined as approval by 70%-89% of all participants, whereas agreement of ≥90% denoted strong consensus. RESULTS: For most items, the group achieved a majority consensus. Recommendations have been provided for the face, neck, décolletage, buttocks, thighs, arms, abdomen, knees, and elbows with detailed injection techniques, providing information on insertion points, dosages, and volumes for both needle and cannula injections as well as the number of treatment sessions and intervals. CONCLUSIONS: The expert consensus supports and provides guidance for the use of CaHA as a biostimulatory agent for face and body rejuvenation. However, further clinical studies are necessary to provide physicians with the best evidence for the best treatment practices.

Evolvability of the actin cytoskeleton in oligodendrocytes during central nervous system development and aging.

The organization of actin filaments into a wide range of subcellular structures is a defining feature of cell shape and dynamics, important for tissue development and homeostasis. Nervous system function requires morphological and functional plasticity of neurons and glial cells, which is largely determined by the dynamic reorganization of the actin cytoskeleton in response to intrinsic and extracellular signals. Oligodendrocytes are specialized glia that extend multiple actin-based protrusions to form the multilayered myelin membrane that spirally wraps around axons, increasing conduction speed and promoting long-term axonal integrity. Myelination is a remarkable biological paradigm in development, and maintenance of myelin is essential for a healthy adult nervous system. In this review, we discuss how structure and dynamics of the actin cytoskeleton is a defining feature of myelinating oligodendrocytes' biology and function. We also review "old and new" concepts to reflect on the potential role of the cytoskeleton in balancing life and death of myelin membranes and oligodendrocytes in the aging central nervous system.

Gluconeogenesis using glycerol as a substrate in bloodstream-form Trypanosoma brucei.

Bloodstream form African trypanosomes are thought to rely exclusively upon glycolysis, using glucose as a substrate, for ATP production. Indeed, the pathway has long been considered a potential therapeutic target to tackle the devastating and neglected tropical diseases caused by these parasites. However, plasma membrane glucose and glycerol transporters are both expressed by trypanosomes and these parasites can infiltrate tissues that contain glycerol. Here, we show that bloodstream form trypanosomes can use glycerol for gluconeogenesis and for ATP production, particularly when deprived of glucose following hexose transporter depletion. We demonstrate that Trypanosoma brucei hexose transporters 1 and 2 (THT1 and THT2) are localized to the plasma membrane and that knockdown of THT1 expression leads to a growth defect that is more severe when THT2 is also knocked down. These data are consistent with THT1 and THT2 being the primary routes of glucose supply for the production of ATP by glycolysis. However, supplementation of the growth medium with glycerol substantially rescued the growth defect caused by THT1 and THT2 knockdown. Metabolomic analyses with heavy-isotope labelled glycerol demonstrated that trypanosomes take up glycerol and use it to synthesize intermediates of gluconeogenesis, including fructose 1,6-bisphosphate and hexose 6-phosphates, which feed the pentose phosphate pathway and variant surface glycoprotein biosynthesis. We used Cas9-mediated gene knockout to demonstrate a gluconeogenesis-specific, but fructose-1,6-bisphosphatase (Tb927.9.8720)-independent activity, converting fructose 1,6-bisphosphate into fructose 6-phosphate. In addition, we observed increased flux through the tricarboxylic acid cycle and the succinate shunt. Thus, contrary to prior thinking, gluconeogenesis can operate in bloodstream form T. brucei. This pathway, using glycerol as a physiological substrate, may be required in mammalian host tissues.

Codon usage bias controls mRNA and protein abundance in trypanosomatids.

Protein abundance differs from a few to millions of copies per cell. Trypanosoma brucei presents an excellent model for studies on codon bias and differential gene expression because transcription is broadly unregulated and uniform across the genome. T. brucei is also a major human and animal protozoal pathogen. Here, an experimental assessment, using synthetic reporter genes, revealed that GC3 codons have a major positive impact on both mRNA and protein abundance. Our estimates of relative expression, based on coding sequences alone (codon usage and sequence length), are within 2-fold of the observed values for the majority of measured cellular mRNAs (n > 7000) and proteins (n > 2000). Our estimates also correspond with expression measures from published transcriptome and proteome datasets from other trypanosomatids. We conclude that codon usage is a key factor affecting global relative mRNA and protein expression in trypanosomatids and that relative abundance can be effectively estimated using only protein coding sequences.