RNA-RNA interactions between respiratory syncytial virus and miR-26 and miR-27 are associated with regulation of cell cycle and antiviral immunity.

microRNAs (miRNAs) regulate nearly all physiological processes but our understanding of exactly how they function remains incomplete, particularly in the context of viral infections. Here, we adapt a biochemical method (CLEAR-CLIP) and analysis pipeline to identify targets of miRNAs in lung cells infected with Respiratory syncytial virus (RSV). We show that RSV binds directly to miR-26 and miR-27 through seed pairing and demonstrate that these miRNAs target distinct gene networks associated with cell cycle and metabolism (miR-27) and antiviral immunity (miR-26). Many of the targets are de-repressed upon infection and we show that the miR-27 targets most sensitive to miRNA inhibition are those associated with cell cycle. Finally, we demonstrate that high confidence chimeras map to long noncoding RNAs (lncRNAs) and pseudogenes in transcriptional regulatory regions. We validate that a proportion of miR-27 and Argonaute 2 (AGO2) is nuclear and identify a long non-coding RNA (lncRNA) as a miR-27 target that is linked to transcriptional regulation of nearby genes. This work expands the target networks of miR-26 and miR-27 to include direct interactions with RSV and lncRNAs and implicate these miRNAs in regulation of key genes that impact the viral life cycle associated with cell cycle, metabolism, and antiviral immunity.

Transverse Myelitis in the Setting of Enterobius vermicularis (Pinworm) Infection: Case Report.

Myelitis is a rare inflammatory myelopathy, and known associated etiologies only account for a small number of causes. A significant percentage of cases have an unknown etiology and are considered idiopathic. With 64% to 68% of cases fitting into the idiopathic category, helminth infections, and specifically pinworm parainfections, should be considered in cases that would otherwise be classified as idiopathic. This case report outlines a pediatric patient diagnosed with myelitis given her progressive weakness, fussiness, refusal to bear weight as well as magnetic resonance imaging (MRI) demonstrating T2-hyperintense signal and/or T1 gadolinium enhancement, and/or positive cerebrospinal fluid (CSF) inflammatory markers. This patient had a negative evaluation for typical known etiologies for myelitis including no signs of multiple sclerosis and neuromyelitis optica spectrum disorder on brain MRI, oligoclonal banding and aquaporin-4 autoantibodies, and no evidence of bacterial or viral meningitis given normal cell counts and cultures in CSF. She was found to have a pinworm infection, suggesting a parasitic parainfectious etiology of her myelitis. This case outlines the first case noting the correlation between myelitis and pinworm infection in a pediatric patient.

Nanophytomedicines as Therapeutic Agents for Parkinson's Disease.

Phytochemicals are promising therapeutics for various neurodegenerative disorders, including Parkinson's disease (PD). However, their efficacy, pharmacokinetic properties, and penetration across the blood-brain barrier can be improved using delivery systems such as nanoparticles. We reviewed recently published work in which nanoparticles were used to deliver phytochemicals toward PD treatment. The studies show that nanoparticles not only improve the pharmacological effect of the phytochemicals but also enable targeting to the brain and crossing of the blood-brain barrier. Various ligands were added to the nanoparticles to improve blood-brain barrier transportation. The promising findings from the published studies reveal that more research into nanophytomedicine approaches as therapeutic targets for PD is warranted, especially since they have the potential to protect against key features of PD, including α-synuclein aggregation, mitochondrial dysfunction, and dopaminergic neuronal death. Furthermore, future directions should involve smart designs to tailor nanoparticles for improved therapeutic delivery by modifying their features, such as architecture, surface and material properties, targeting ligands, and responsiveness.

Ancient diversity in host-parasite interaction genes in a model parasitic nematode.

Host-parasite interactions exert strong selection pressures on the genomes of both host and parasite. These interactions can lead to negative frequency-dependent selection, a form of balancing selection that is hypothesised to explain the high levels of polymorphism seen in many host immune and parasite antigen loci. Here, we sequence the genomes of several individuals of Heligmosomoides bakeri, a model parasite of house mice, and Heligmosomoides polygyrus, a closely related parasite of wood mice. Although H. bakeri is commonly referred to as H. polygyrus in the literature, their genomes show levels of divergence that are consistent with at least a million years of independent evolution. The genomes of both species contain hyper-divergent haplotypes that are enriched for proteins that interact with the host immune response. Many of these haplotypes originated prior to the divergence between H. bakeri and H. polygyrus, suggesting that they have been maintained by long-term balancing selection. Together, our results suggest that the selection pressures exerted by the host immune response have played a key role in shaping patterns of genetic diversity in the genomes of parasitic nematodes.

miR-7 is recruited to the high molecular weight RNA-induced silencing complex in CD8+ T cells upon activation and suppresses IL-2 signaling.

Increasing evidence suggests mammalian Argonaute (Ago) proteins partition into distinct complexes within cells, but there is still little biochemical or functional understanding of the miRNAs differentially associated with these complexes. In naïve T cells, Ago2 is found almost exclusively in low molecular weight (LMW) complexes which are associated with miRNAs but not their target mRNAs. Upon T-cell activation, a proportion of these Ago2 complexes move into a newly formed high molecular weight (HMW) RNA-induced silencing complex (RISC), which is characterized by the presence of the GW182 protein that mediates translational repression. Here, we demonstrate distinct partitioning of miRNAs and isomiRs in LMW versus HMW RISCs upon antigen-mediated activation of CD8+ T cells. We identify miR-7 as highly enriched in HMW RISC and demonstrate that miR-7 inhibition leads to increased production of IL-2 and up-regulation of the IL-2 receptor, the transferrin receptor, CD71 and the amino acid transporter, CD98. Our data support a model where recruitment of miR-7 to HMW RISC restrains IL-2 signaling and the metabolic processes regulated by IL-2.

Characterizing Twitter Content About HIV Pre-exposure Prophylaxis (PrEP) for Women: Qualitative Content Analysis.

BACKGROUND: HIV remains a persistent health problem in the United States, especially among women. Approved in 2012, HIV pre-exposure prophylaxis (PrEP) is a daily pill or bimonthly injection that can be taken by individuals at increased risk of contracting HIV to reduce their risk of new infection. Women who are at risk of HIV face numerous barriers to HIV services and information, underscoring the critical need for strategies to increase awareness of evidence-based HIV prevention methods, such as HIV PrEP, among women. OBJECTIVE: We aimed to identify historical trends in the use of Twitter hashtags specific to women and HIV PrEP and explore content about women and PrEP shared through Twitter. METHODS: This was a qualitative descriptive study using a purposive sample of tweets containing hashtags related to women and HIV PrEP from 2009 to 2022. Tweets were collected via Twitter's API. Each Twitter user profile, tweet, and related links were coded using content analysis, guided by the framework of the Health Belief Model (HBM) to generate results. We used a factor analysis to identify salient clusters of tweets. RESULTS: A total of 1256 tweets from 396 unique users were relevant to our study focus of content about PrEP specifically for women (1256/2908, 43.2% of eligible tweets). We found that this sample of tweets was posted mostly by organizations. The 2 largest groups of individual users were activists and advocates (61/396, 15.4%) and personal users (54/396, 13.6%). Among individual users, most were female (100/166, 60%) and American (256/396, 64.6%). The earliest relevant tweet in our sample was posted in mid-2014 and the number of tweets significantly decreased after 2018. We found that 61% (496/820) of relevant tweets contained links to informational websites intended to provide guidance and resources or promote access to PrEP. Most tweets specifically targeted people of color, including through the use of imagery and symbolism. In addition to inclusive imagery, our factor analysis indicated that more than a third of tweets were intended to share information and promote PrEP to people of color. Less than half of tweets contained any HBM concepts, and only a few contained cues to action. Lastly, while our sample included only tweets relevant to women, we found that the tweets directed to lesbian, gay, bisexual, transgender, queer (LGBTQ) audiences received the highest levels of audience engagement. CONCLUSIONS: These findings point to several areas for improvement in future social media campaigns directed at women about PrEP. First, future posts would benefit from including more theoretical constructs, such as self-efficacy and cues to action. Second, organizations posting on Twitter should continue to broaden their audience and followers to reach more people. Lastly, tweets should leverage the momentum and strategies used by the LGBTQ community to reach broader audiences and destigmatize PrEP use across all communities.

Special considerations for studies of extracellular vesicles from parasitic helminths: A community-led roadmap to increase rigour and reproducibility.

Over the last decade, research interest in defining how extracellular vesicles (EVs) shape cross-species communication has grown rapidly. Parasitic helminths, worm species found in the phyla Nematoda and Platyhelminthes, are well-recognised manipulators of host immune function and physiology. Emerging evidence supports a role for helminth-derived EVs in these processes and highlights EVs as an important participant in cross-phylum communication. While the mammalian EV field is guided by a community-agreed framework for studying EVs derived from model organisms or cell systems [e.g., Minimal Information for Studies of Extracellular Vesicles (MISEV)], the helminth community requires a supplementary set of principles due to the additional challenges that accompany working with such divergent organisms. These challenges include, but are not limited to, generating sufficient quantities of EVs for descriptive or functional studies, defining pan-helminth EV markers, genetically modifying these organisms, and identifying rigorous methodologies for in vitro and in vivo studies. Here, we outline best practices for those investigating the biology of helminth-derived EVs to complement the MISEV guidelines. We summarise community-agreed standards for studying EVs derived from this broad set of non-model organisms, raise awareness of issues associated with helminth EVs and provide future perspectives for how progress in the field will be achieved.

Organoids as tools to investigate gastrointestinal nematode development and host interactions.

Gastrointestinal nematodes are a diverse class of pathogens that colonise a quarter of the world's human population and nearly all grazing livestock. These macroparasites establish, and some migrate, within host gastrointestinal niches during their life cycles and release molecules that condition the host mucosa to enable chronic infections. Understanding how helminths do this, and defining the molecules and mechanisms involved in host modulation, holds promise for novel strategies of anthelmintics and vaccines, as well as new knowledge of immune regulation and tissue repair. Yet the size and complexity of these multicellular parasites, coupled with the reliance on hosts to maintain their life cycles, present obstacles to interrogate how they interact with the gastric and intestinal epithelium, stroma and immune cells during infection, and also to develop protocols to genetically modify these parasites. Gastrointestinal organoids have transformed research on gastric and gut physiology during homeostasis and disease, including investigations on host-pathogen interactions with viruses, bacteria, protozoa and more recently, parasitic nematodes. Here we outline applications and important considerations for the best use of organoids to study gastrointestinal nematode development and interactions with their hosts. The careful use of different organoid culture configurations in order to achieve a closer replication of the in vivo infection context will lead not only to new knowledge on gastrointestinal nematode infection biology, but also towards the replication of their life cycles in vitro, and the development of valuable experimental tools such as genetically modified parasites.

Extracellular vesicles from malaria-infected red blood cells: not all are secreted equal.

Extracellular vesicles (EVs) mediate the transfer of molecules between cells and play diverse roles in host-pathogen interactions. Malaria is an important disease caused by intracellular Plasmodium species that invade red blood cells and these red blood cells release EVs. The EVs from infected cells have diverse functions in the disease and an obstacle in understanding how they exert their functions is that multiple EV types exist. In this issue of EMBO reports, Abou Karam and colleagues use sophisticated biophysical techniques to isolate and characterize two EV subpopulations produced by red blood cells infected with Plasmodium falciparum (Abou Karam et al, 2022). The authors show that these EV subpopulations have distinct sizes, protein content, membrane packing, and fusion capabilities, suggesting that EV subpopulations from infected cells could target different cell types and subcellular locations. This work underscores the concept that understanding EV heterogeneity will go hand in hand with understanding EV functions.

Microfluidic system for near-patient extraction and detection of miR-122 microRNA biomarker for drug-induced liver injury diagnostics.

Drug-induced liver injury (DILI) results in over 100 000 hospital attendances per year in the UK alone and is a leading cause for the post-marketing withdrawal of new drugs, leading to significant financial losses. MicroRNA-122 (miR-122) has been proposed as a sensitive DILI marker although no commercial applications are available yet. Extracellular blood microRNAs (miRNAs) are promising clinical biomarkers but their measurement at point of care remains time-consuming, technically challenging, and expensive. For circulating miRNA to have an impact on healthcare, a key challenge to overcome is the development of rapid and reliable low-cost sample preparation. There is an acknowledged issue with miRNA stability in the presence of hemolysis and platelet activation, and no solution has been demonstrated for fast and robust extraction at the site of blood draw. Here, we report a novel microfluidic platform for the extraction of circulating miR-122 from blood enabled by a vertical approach and gravity-based bubble mixing. The performance of this disposable cartridge was verified by standard quantitative polymerase chain reaction analysis on extracted miR-122. The cartridge performed equivalently or better than standard bench extraction kits. The extraction cartridge was combined with electrochemical impedance spectroscopy to detect miR-122 as an initial proof-of-concept toward an application in point-of-care detection. This platform enables the standardization of sample preparation and the detection of miRNAs at the point of blood draw and in resource limited settings and could aid the introduction of miRNA-based assays into routine clinical practice.

Cells choose their words wisely.

In their recent Nature paper, Garcia-Martin et al. show that sequences within a microRNA influence how much of that microRNA is sent to another cell through extracellular vesicles. This supports a growing body of data demonstrating that cells use RNA to talk, but we know much less about how they listen.

Structural variation at the maize WUSCHEL1 locus alters stem cell organization in inflorescences.

Structural variation in plant genomes is a significant driver of phenotypic variability in traits important for the domestication and productivity of crop species. Among these are traits that depend on functional meristems, populations of stem cells maintained by the CLAVATA-WUSCHEL (CLV-WUS) negative feedback-loop that controls the expression of the WUS homeobox transcription factor. WUS function and impact on maize development and yield remain largely unexplored. Here we show that the maize dominant Barren inflorescence3 (Bif3) mutant harbors a tandem duplicated copy of the ZmWUS1 gene, ZmWUS1-B, whose novel promoter enhances transcription in a ring-like pattern. Overexpression of ZmWUS1-B is due to multimerized binding sites for type-B RESPONSE REGULATORs (RRs), key transcription factors in cytokinin signaling. Hypersensitivity to cytokinin causes stem cell overproliferation and major rearrangements of Bif3 inflorescence meristems, leading to the formation of ball-shaped ears and severely affecting productivity. These findings establish ZmWUS1 as an essential meristem size regulator in maize and highlight the striking effect of cis-regulatory variation on a key developmental program.

MicroRNAs and extracellular vesicles in the gut: new host modulators of the microbiome?

The gut microbiota plays an integral role in human health and its dysbiosis is associated with many chronic diseases. There are still large gaps in understanding the host and environmental factors that directly regulate the gut microbiota, and few effective strategies exist to modulate the microbiota in therapeutic applications. Recent reports suggest that certain microRNAs (miRNAs) released by mammalian cells can regulate bacterial gene expression to influence the microbiome composition and propose extracellular vesicles as one natural mechanism for miRNA transport in the gut. These new findings interface with a burgeoning body of data showing that miRNAs are present in a stable form in extracellular environments and can mediate cell-to-cell communication in mammals. Here, we review the literature on RNA-mediated modulation of the microbiome to bring cross-disciplinary perspective to this new type of interaction and its potential implications in biology and medicine.

Development of caecaloids to study host-pathogen interactions: new insights into immunoregulatory functions of Trichuris muris extracellular vesicles in the caecum.

The caecum, an intestinal appendage in the junction of the small and large intestines, displays a unique epithelium that serves as an exclusive niche for a range of pathogens including whipworms (Trichuris spp.). While protocols to grow organoids from small intestine (enteroids) and colon (colonoids) exist, the conditions to culture organoids from the caecum have yet to be described. Here, we report methods to grow, differentiate and characterise mouse adult stem cell-derived caecal organoids, termed caecaloids. We compare the cellular composition of caecaloids with that of enteroids, identifying differences in intestinal epithelial cell populations that mimic those found in the caecum and small intestine. The remarkable similarity in the intestinal epithelial cell composition and spatial conformation of caecaloids and their tissue of origin enables their use as an in vitro model to study host interactions with important caecal pathogens. Thus, exploiting this system, we investigated the responses of caecal intestinal epithelial cells to extracellular vesicles secreted/excreted by the intracellular helminth Trichuris muris. Our findings reveal novel immunoregulatory effects of whipworm extracellular vesicles on the caecal epithelium, including the downregulation of responses to nucleic acid recognition and type-I interferon signalling.

Extracellular vesicles from Heligmosomoides bakeri and Trichuris muris contain distinct microRNA families and small RNAs that could underpin different functions in the host.

Extracellular vesicles (EVs) have emerged as a ubiquitous component of helminth excretory-secretory products that can deliver parasite molecules to host cells to elicit immunomodulatory effects. RNAs are one type of cargo molecule that can underpin EV functions, hence there is extensive interest in characterising the RNAs that are present in EVs from different helminth species. Here we outline methods for identifying all of the small RNAs (sRNA) in helminth EVs and address how different methodologies may influence the sRNAs detected. We show that different EV purification methods introduce relatively little variation in the sRNAs that are detected, and that different RNA library preparation methods yielded larger differences. We compared the EV sRNAs in the gastrointestinal nematode Heligmosomoides bakeri with those in EVs from the distantly related gastrointestinal nematode Trichuris muris, and found that many of the sRNAs in both organisms derive from repetitive elements or intergenic regions. However, only in H. bakeri do these RNAs contain a 5' triphosphate, and Guanine (G) starting nucleotide, consistent with their biogenesis by RNA-dependent RNA polymerases (RdRPs). Distinct microRNA (miRNA) families are carried in EVs from each parasite, with H. bakeri EVs specific for miR-71, miR-49, miR-63, miR-259 and miR-240 gene families, and T. muris EVs specific for miR-1, miR-1822 and miR-252, and enriched for miR-59, miR-72 and miR-44 families, with the miR-9, miR-10, miR-80 and let-7 families abundant in both. We found a larger proportion of miRNA reads derive from the mouse host in T. muris EVs, compared with H. bakeri EVs. Our report underscores potential biases in the sRNAs sequenced based on library preparation methods, suggests specific nematode lineages have evolved distinct sRNA synthesis/export pathways, and highlights specific differences in EV miRNAs from H. bakeri and T. muris that may underpin functional adaptation to their host niches.

Disentangling sRNA-Seq data to study RNA communication between species.

Many organisms exchange small RNAs (sRNAs) during their interactions, that can target or bolster defense strategies in host-pathogen systems. Current sRNA-Seq technology can determine the sRNAs present in any symbiotic system, but there are very few bioinformatic tools available to interpret the results. We show that one of the biggest challenges comes from sequences that map equally well to the genomes of both interacting organisms. This arises due to the small size of the sRNAs compared to large genomes, and because a large portion of sequenced sRNAs come from genomic regions that encode highly conserved miRNAs, rRNAs or tRNAs. Here, we present strategies to disentangle sRNA-Seq data from samples of communicating organisms, developed using diverse plant and animal species that are known to receive or exchange RNA with their symbionts. We show that sequence assembly, both de novo and genome-guided, can be used for these sRNA-Seq data, greatly reducing the ambiguity of mapping reads. Even confidently mapped sequences can be misleading, so we further demonstrate the use of differential expression strategies to determine true parasite-derived sRNAs within host cells. We validate our methods on new experiments designed to probe the nature of the extracellular vesicle sRNAs from the parasitic nematode Heligmosomoides bakeri that get into mouse intestinal epithelial cells.

Comparative analysis of small RNAs released by the filarial nematode Litomosoides sigmodontis in vitro and in vivo.

BACKGROUND: The release of small non-coding RNAs (sRNAs) has been reported in parasitic nematodes, trematodes and cestodes of medical and veterinary importance. However, little is known regarding the diversity and composition of sRNAs released by different lifecycle stages and the portion of sRNAs that persist in host tissues during filarial infection. This information is relevant to understanding potential roles of sRNAs in parasite-to-host communication, as well as to inform on the location within the host and time point at which they can be detected. METHODOLOGY AND PRINCIPAL FINDINGS: We have used small RNA (sRNA) sequencing analysis to identify sRNAs in replicate samples of the excretory-secretory (ES) products of developmental stages of the filarial nematode Litomosoides sigmodontis in vitro and compare this to the parasite-derived sRNA detected in host tissues. We show that all L. sigmodontis developmental stages release RNAs in vitro, including ribosomal RNA fragments, 5'-derived tRNA fragments (5'-tRFs) and, to a lesser extent, microRNAs (miRNAs). The gravid adult females (gAF) produce the largest diversity and abundance of miRNAs in the ES compared to the adult males or microfilariae. Analysis of sRNAs detected in serum and macrophages from infected animals reveals that parasite miRNAs are preferentially detected in vivo, compared to their low levels in the ES products, and identifies miR-92-3p and miR-71-5p as L. sigmodontis miRNAs that are stably detected in host cells in vivo. CONCLUSIONS: Our results suggest that gravid adult female worms secrete the largest diversity of extracellular sRNAs compared to adult males or microfilariae. We further show differences in the parasite sRNA biotype distribution detected in vitro versus in vivo. We identify macrophages as one reservoir for parasite sRNA during infection, and confirm the presence of parasite miRNAs and tRNAs in host serum during patent infection.