Annelid adult cell type diversity and their pluripotent cellular origins.

Many annelids can regenerate missing body parts or reproduce asexually, generating all cell types in adult stages. However, the putative adult stem cell populations involved in these processes, and the diversity of cell types generated by them, are still unknown. To address this, we recover 75,218 single cell transcriptomes of the highly regenerative and asexually-reproducing annelid Pristina leidyi. Our results uncover a rich cell type diversity including annelid specific types as well as novel types. Moreover, we characterise transcription factors and gene networks that are expressed specifically in these populations. Finally, we uncover a broadly abundant cluster of putative stem cells with a pluripotent signature. This population expresses well-known stem cell markers such as vasa, piwi and nanos homologues, but also shows heterogeneous expression of differentiated cell markers and their transcription factors. We find conserved expression of pluripotency regulators, including multiple chromatin remodelling and epigenetic factors, in piwi+ cells. Finally, lineage reconstruction analyses reveal computational differentiation trajectories from piwi+ cells to diverse adult types. Our data reveal the cell type diversity of adult annelids by single cell transcriptomics and suggest that a piwi+ cell population with a pluripotent stem cell signature is associated with adult cell type differentiation.

Annelid adult cell type diversity and their pluripotent cellular origins.

Annelids are a broadly distributed, highly diverse, economically and environmentally important group of animals. Most species can regenerate missing body parts, and many are able to reproduce asexually. Therefore, many annelids can generate all adult cell types in adult stages. However, the putative adult stem cell populations involved in these processes, as well as the diversity of adult cell types generated by them, are still unknown. Here, we recover 75,218 single cell transcriptomes of Pristina leidyi, a highly regenerative and asexually-reproducing freshwater annelid. We characterise all major annelid adult cell types, and validate many of our observations by HCR in situ hybridisation. Our results uncover complex patterns of regionally expressed genes in the annelid gut, as well as neuronal, muscle and epidermal specific genes. We also characterise annelid-specific cell types such as the chaetal sacs and globin+ cells, and novel cell types of enigmatic affinity, including a vigilin+ cell type, a lumbrokinase+ cell type, and a diverse set of metabolic cells. Moreover, we characterise transcription factors and gene networks that are expressed specifically in these populations. Finally, we uncover a broadly abundant cluster of putative stem cells with a pluripotent signature. This population expresses well-known stem cell markers such as vasa, piwi and nanos homologues, but also shows heterogeneous expression of differentiated cell markers and their transcription factors. In these piwi+ cells, we also find conserved expression of pluripotency regulators, including multiple chromatin remodelling and epigenetic factors. Finally, lineage reconstruction analyses reveal the existence of differentiation trajectories from piwi+ cells to diverse adult types. Our data reveal the cell type diversity of adult annelids for the first time and serve as a resource for studying annelid cell types and their evolution. On the other hand, our characterisation of a piwi+ cell population with a pluripotent stem cell signature will serve as a platform for the study of annelid stem cells and their role in regeneration.

A mosaic of conserved and novel modes of gene expression and morphogenesis in mesoderm and muscle formation of a larval bivalve.

The mesoderm gives rise to several key morphological features of bilaterian animals including endoskeletal elements and the musculature. A number of regulatory genes involved in mesoderm and/or muscle formation (e.g., Brachyury (Bra), even-skipped (eve), Mox, myosin II heavy chain (mhc)) have been identified chiefly from chordates and the ecdysozoans Drosophila and Caenorhabditis elegans, but data for non-model protostomes, especially those belonging to the ecdysozoan sister clade, Lophotrochozoa (e.g., flatworms, annelids, mollusks), are only beginning to emerge. Within the lophotrochozoans, Mollusca constitutes the most speciose and diverse phylum. Interestingly, however, information on the morphological and molecular underpinnings of key ontogenetic processes such as mesoderm formation and myogenesis remains scarce even for prominent molluscan sublineages such as the bivalves. Here, we investigated myogenesis and developmental expression of Bra, eve, Mox, and mhc in the quagga mussel Dreissena rostriformis, an invasive freshwater bivalve and an emerging model in invertebrate evodevo. We found that all four genes are expressed during mesoderm formation, but some show additional, individual sites of expression during ontogeny. While Mox and mhc are involved in early myogenesis, eve is also expressed in the embryonic shell field and Bra is additionally present in the foregut. Comparative analysis suggests that Mox has an ancestral role in mesoderm and possibly muscle formation in bilaterians, while Bra and eve are conserved regulators of mesoderm development of nephrozoans (protostomes and deuterostomes). The fully developed Dreissena veliger larva shows a highly complex muscular architecture, supporting a muscular ground pattern of autobranch bivalve larvae that includes at least a velum muscle ring, three or four pairs of velum retractors, one or two pairs of larval retractors, two pairs of foot retractors, a pedal plexus, possibly two pairs of mantle retractors, and the muscles of the pallial line, as well as an anterior and a posterior adductor. As is typical for their molluscan kin, remodelling and loss of prominent larval features such as the velum musculature and various retractor systems appear to be also common in bivalves. Supplementary information: The online version contains supplementary material available at 10.1007/s13127-022-00569-5.

Comparative Single-Cell Transcriptomics Reveals Novel Genes Involved in Bivalve Embryonic Shell Formation and Questions Ontogenetic Homology of Molluscan Shell Types.

Mollusks are known for their highly diverse repertoire of body plans that often includes external armor in form of mineralized hardparts. Representatives of the Conchifera, one of the two major lineages that comprises taxa which originated from a uni-shelled ancestor (Monoplacophora, Gastropoda, Cephalopoda, Scaphopoda, Bivalvia), are particularly relevant regarding the evolution of mollusk shells. Previous studies have found that the shell matrix of the adult shell (teleoconch) is rapidly evolving and that the gene set involved in shell formation is highly taxon-specific. However, detailed annotation of genes expressed in tissues involved in the formation of the embryonic shell (protoconch I) or the larval shell (protoconch II) are currently lacking. Here, we analyzed the genetic toolbox involved in embryonic and larval shell formation in the quagga mussel Dreissena rostriformis using single cell RNA sequencing. We found significant differences in genes expressed during embryonic and larval shell secretion, calling into question ontogenetic homology of these transitory bivalve shell types. Further ortholog comparisons throughout Metazoa indicates that a common genetic biomineralization toolbox, that was secondarily co-opted into molluscan shell formation, was already present in the last common metazoan ancestor. Genes included are engrailed, carbonic anhydrase, and tyrosinase homologs. However, we found that 25% of the genes expressed in the embryonic shell field of D. rostriformis lack an ortholog match with any other metazoan. This indicates that not only adult but also embryonic mollusk shells may be fast-evolving structures. We raise the question as to what degree, and on which taxonomic level, the gene complement involved in conchiferan protoconch formation may be lineage-specific or conserved across taxa.

Non-collinear Hox gene expression in bivalves and the evolution of morphological novelties in mollusks.

Hox genes are key developmental regulators that are involved in establishing morphological features during animal ontogeny. They are commonly expressed along the anterior-posterior axis in a staggered, or collinear, fashion. In mollusks, the repertoire of body plans is widely diverse and current data suggest their involvement during development of landmark morphological traits in Conchifera, one of the two major lineages that comprises those taxa that originated from a uni-shelled ancestor (Monoplacophora, Gastropoda, Cephalopoda, Scaphopoda, Bivalvia). For most clades, and bivalves in particular, data on Hox gene expression throughout ontogeny are scarce. We thus investigated Hox expression during development of the quagga mussel, Dreissena rostriformis, to elucidate to which degree they might contribute to specific phenotypic traits as in other conchiferans. The Hox/ParaHox complement of Mollusca typically comprises 14 genes, 13 of which are present in bivalve genomes including Dreissena. We describe here expression of 9 Hox genes and the ParaHox gene Xlox during Dreissena development. Hox expression in Dreissena is first detected in the gastrula stage with widely overlapping expression domains of most genes. In the trochophore stage, Hox gene expression shifts towards more compact, largely mesodermal domains. Only few of these domains can be assigned to specific developing morphological structures such as Hox1 in the shell field and Xlox in the hindgut. We did not find traces of spatial or temporal staggered expression of Hox genes in Dreissena. Our data support the notion that Hox gene expression has been coopted independently, and to varying degrees, into lineage-specific structures in the respective conchiferan clades. The non-collinear mode of Hox expression in Dreissena might be a result of the low degree of body plan regionalization along the bivalve anterior-posterior axis as exemplified by the lack of key morphological traits such as a distinct head, cephalic tentacles, radula apparatus, and a simplified central nervous system.

Effects of albumin, transferrin and humic-like substances on iron-mediated OH radical formation in human lung fluids.

Inhalation of particulate matter is hypothesized to contribute to health effects by overproducing reactive oxygen species (ROS) and inducing oxidative stress. Fe(II) has been shown to contribute to ROS generation in acellular simulated lung fluids. Atmospheric humic-like substances (HULIS) have been shown to chelate Fe(II) and significantly enhance this ROS generation. Here, we investigate Fe(II)-mediated .OH generation from the iron active proteins in lung fluid, albumin and transferrin, and fulvic acid, a surrogate for HULIS, in human bronchoalveolar lavage fluid (BALF). We find that albumin enhances .OH generation from inorganic Fe(II) and that transferrin attenuates this enhancement. We estimate the rate constants for albumin-Fe(II) and fulvic acid-Fe(II) mediated O2.- reduction (1.9 ± 0.3) M-1 s-1 and (2.7 ± 0.3) M-1s-1 (pH = 5.5, T = 37 °C), 17-25 times the rate for free iron, which we measured to be (110 ± 20) × 10-3 M-1s-1, in agreement with the literature. .OH generation measured from fulvic acid-Fe(II) in BALF from 8 individuals with added fulvic acid is successfully predicted rates of .OH generation by mixtures of Fe(II), albumin, transferrin, fulvic acid, and ascorbate in saline solution. This indicates that fulvic acid enhances .OH formation in BALF, and that albumin and transferrin in BALF moderate the effect. We propose that fulvic acid, and thereby HULIS, is capable of mobilizing Fe(II) away from albumin and transferrin and this increases the formation rate of O2.- and ultimately of .OH. Furthermore, we find that albumin and transferrin have significantly different impacts on Fe(II)-mediated .OH than citrate, a common component of simulated lung fluids, a factor that should be considered carefully in the interpretation of results obtained from solutions containing citrate.

¿Te Recurseas? Mapping, Enumerating, and Describing Male and Transwomen Sex Workers in Venue-Based and Virtual Spaces in Lima, Peru.

HIV disproportionately affects men who have sex with men and transwomen in Latin America. Globally, efforts to map, enumerate, and describe male and transwomen sex workers (MTSWs) are limited. We mapped and described venue- and non-venue-based MTSWs and enumerated venuebased MTSWs in Lima, Peru's capital. With venue-based MTSWs, we identified and described the venues, SWs, and clients and enumerated the SWs. With non-venue-based MTSWs, we described SW offerings and SWs. Male SWs (MSWs) are concentrated downtown, with many moving online. Transwomen SWs (TSWs) are spread across metropolitan Lima, with fewer online. At venues, there are more TSWs than MSWs, TSWs reported more HIV and sexual risk behaviors, and MSWs had more female partners. Non-venue-based MSWs used condoms less than venue-based MSWs. Results support systematic efforts to describe hard-to-reach MTSWs and their different types of partners, all of whom have unique, differing needs for HIV/ STI education, prevention, testing, and care services.

Sexual Risk, Behavior, and HIV Testing and Status among Male and Transgender Women Sex Workers and their Clients in Lima, Peru.

OBJECTIVES ­: To determine the socio-demographics, sexual practices, and HIV prevalence of male sex workers (MSWs), transwomen sex workers (TSWs), and their male clients in Lima, Peru. METHODS ­: Following ethnographic mapping of sex work venues, we revisited randomly selected venues to survey MSWs, TSWs and their clients. RESULTS ­: MSWs and TSW clients are more educated than MSW clients and TSWs. Only 50% of TSW clients have received HIV testing. Self-reported HIV positivity is highest among MSW clients and TSWs. CONCLUSION ­: Notable differences exist between MSWs and TSWs and their clients that can affect the health of these groups and warrant intervention.

'Just getting by': a cross-sectional study of male sex workers as a key population for HIV/STIs among men who have sex with men in Peru.

OBJECTIVES: To describe the sociodemographic and sex work characteristics, recent sexual practices, HIV risk perception and testing history, and HIV and syphilis prevalence of Cercado (downtown) and non-Cercado (close urban) male sex workers (MSWs) in Lima, Peru. METHODS: Eighty-nine MSWs completed a self-administered survey and participated in HIV and syphilis testing. RESULTS: Cercado MSWs had significantly lower median weekly earnings than non-Cercado MSWs (US$43 vs US$72, p=0.04). Most non-Cercado MSWs (81%) reported only insertive anal intercourse with male/transgender partners, while Cercado MSWs primarily reported only insertive (52%) or insertive and receptive (45%) anal intercourse (p=0.03). Consistent condom use was low with insertive and receptive anal intercourse in both subgroups. Among MSWs with recent female partners, condom use with the last partner was lower among Cercado versus non-Cercado MSWs for vaginal sex (37% vs 65%, p=0.04) and anal sex (27% vs 80%, p=0.01). More Cercado than non-Cercado MSWs (57% vs 42%) perceived high HIV risk (p=0.05) and Cercado MSWs had a much higher prevalence than non-Cercado MSWs of HIV (23% vs 4%, p=0.04) and syphilis (22% vs 0%, p=0.02) infections. CONCLUSIONS: MSWs in Lima are diverse and Cercado MSWs are 'just getting by,' engaging in more risk behaviours, and more likely to have HIV/STIs. Future research should identify, describe and carry out HIV/STI testing with broader groups of MSWs and their client and non-client partners. Prevention efforts should provide HIV/STI risk reduction education for MSWs and related subgroups who are currently not targeted such as female partners.

When Sex Work Becomes Your Everything: The Complex Linkages Between Economy and Affection Among Male Sex Workers in Peru.

In Peru, there are few studies on male sex workers (MSWs), and existing studies explore limited subgroups or offer limited information about MSWs' perspectives. This study provides in-depth perspectives from 40 MSWs who work in downtown Lima (Cercado) and in surrounding urban neighborhoods (non-Cercado) through interviews on their identities, lives, and HIV/STI (sexually transmitted infection) risks and vulnerabilities. Findings are that entry into sex work links economy and affection, particularly among Cercado MSWs. Continued sex work cements this link, making it difficult to exit sex work and establish goals. Ties between economics and affections influence MSWs' perceived HIV/STI risks, vulnerabilities, and prevention practices. Although Cercado MSWs report higher HIV/STI risks and vulnerabilities than non-Cercado peers, they report fewer prevention practices given inability to buy condoms and acceptance of client offers of higher payment, especially clients they feel affection for. MSWs need support to strengthen their self-perceptions and define and pursue their goals in order to improve their HIV/STI prevention practices, health, and well-being.

Spectrum of immune-mediated inner ear disease and cochlear implant results.

OBJECTIVES/HYPOTHESIS: To characterize the progression of hearing loss in patients with immune-mediated inner ear disease (IMIED), and to identify disease- and patient-specific factors associated with cochlear implant (CI) performance. STUDY DESIGN: Retrospective cohort study. METHODS: Subjects consisted of CI patients suspected to have lost their hearing due to IMIED. The primary dependent variable for functional decline was time to deafness, whereas for CI benefit it was post-CI speech perception scores. Independent variables included presence or absence of systemic autoimmune disease, age at CI, and insertion depth of the cochlear electrode. RESULTS: A transient favorable response to immunosuppressive therapy was reported in 16 of 26 patients (66.67%). The time to deafness differed between an organ (ear)-specific immune-mediated group, a systemic immune-mediated group including Cogan syndrome and relapsing polychondritis (subgroup A), and a systemic immune-mediated group associated with other autoimmune diseases (subgroup B; P = .001). Disease group (-15.52; P = .04), insertion depth of the CI electrode (40.71; P = .01), and the age at CI (-0.48, P = .05) were associated with speech perception results. CONCLUSIONS: Triaging IMIED cases based on presence and type of systemic autoimmune disease may aid in selecting a management strategy. Knowledge about the predictors of CI outcome will help clinicians select appropriate patients for CIs. In the setting of significant and irreversible hearing deficit, the restoration of hearing using a cochlear prosthesis may be appropriate earlier rather than later.