Search

This study examined nine prominent commercially available single-cell RNA sequencing (scRNA-seq) kits across four technology groups. Each kit was characterized using peripheral blood mononuclear cells (PBMCs) from a single donor, which enabled consistent assessment of factors such as analytical performance, protocol duration and cost. The Chromium Fixed RNA Profiling kit from 10× Genomics, with its probe-based RNA detection method, demonstrated the best overall performance. The Rhapsody WTA kit from Becton Dickinson exhibited a balance between performance and cost. Importantly, we introduce the read utilization metric, which differentiates scRNA-seq kits based on the efficiency of converting sequencing reads into usable counts. Thus, read utilization is an important feature that substantially impacts sensitivity and cost. With data from 169, 262 cells, our work provides a comprehensive comparison of commercial scRNA-seq technologies to facilitate the effective implementation of single-cell studies., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Multiomic profiling of single cells by sequencing is a powerful technique for investigating cellular diversity. Existing droplet-based microfluidic methods produce many cell-free droplets, underutilizing bead barcodes and reagents. Combinatorial indexing on microplates is more efficient for barcoding but labor-intensive. Here we present Overloading And unpacKing (OAK), which uses a droplet-based barcoding system for initial compartmentalization followed by a second aliquoting round to achieve combinatorial indexing. We demonstrate OAK's versatility with single-cell RNA sequencing as well as paired single-nucleus RNA sequencing and accessible chromatin profiling. We further showcase OAK's performance on complex samples, including differentiated bronchial epithelial cells and primary retinal tissue. Finally, we examine transcriptomic responses of over 400,000 melanoma cells to a RAF inhibitor, belvarafenib, discovering a rare resistant cell population (0.12%). OAK's ultra-high throughput, broad compatibility, high sensitivity, and simplified procedures make it a powerful tool for large-scale molecular analysis, even for rare cells., (© 2024. The Author(s).)

A Caribbean spiny lobster, Panulirus argus , was submitted for necropsy after a number of species-specific fatalities in a public aquarium. The hemolymph was opaque and did not clot and the hepatopancreas had multiple foci of necrosis centered on gram-negative bacteria. Pure cultures of Vibrio harveyi were isolated, identified initially by matrix laser desorption ionization-time of flight mass spectrometry, and confirmed by multilocus sequencing of the gyrB , recA , rpoA , and pyrH genes. As Caribbean spiny lobsters continue to be used for consumption and displayed in public aquariums, chronicling potential pathogens is warranted to inform differential diagnoses and to develop management strategies to reduce incidence of infectious disease in captive populations.

In the last decade, single-cell RNA sequencing routinely performed on large numbers of single cells has greatly advanced our understanding of the underlying heterogeneity of complex biological systems. Technological advances have also enabled protein measurements, further contributing to the elucidation of cell types and states present in complex tissues. Recently, there have been independent advances in mass spectrometric techniques bringing us one step closer to characterizing single-cell proteomes. Here we discuss the challenges of detecting proteins in single cells by both mass spectrometry and sequencing-based methods. We review the state of the art for these techniques and propose that there is a space for technological advancements and complementary approaches that maximize the advantages of both classes of technologies., (© 2023. Springer Nature America, Inc.)

Whipworms are large metazoan parasites that inhabit multi-intracellular epithelial tunnels in the large intestine of their hosts, causing chronic disease in humans and other mammals. How first-stage larvae invade host epithelia and establish infection remains unclear. Here we investigate early infection events using both Trichuris muris infections of mice and murine caecaloids, the first in-vitro system for whipworm infection and organoid model for live helminths. We show that larvae degrade mucus layers to access epithelial cells. In early syncytial tunnels, larvae are completely intracellular, woven through multiple live dividing cells. Using single-cell RNA sequencing of infected mouse caecum, we reveal that progression of infection results in cell damage and an expansion of enterocytes expressing of Isg15, potentially instigating the host immune response to the whipworm and tissue repair. Our results unravel intestinal epithelium invasion by whipworms and reveal specific host-parasite interactions that allow the whipworm to establish its multi-intracellular niche., (© 2022. The Author(s).)

Over 250 million people suffer from schistosomiasis, a tropical disease caused by parasitic flatworms known as schistosomes. Humans become infected by free-swimming, water-borne larvae, which penetrate the skin. The earliest intra-mammalian stage, called the schistosomulum, undergoes a series of developmental transitions. These changes are critical for the parasite to adapt to its new environment as it navigates through host tissues to reach its niche, where it will grow to reproductive maturity. Unravelling the mechanisms that drive intra-mammalian development requires knowledge of the spatial organisation and transcriptional dynamics of different cell types that comprise the schistomulum body. To fill these important knowledge gaps, we perform single-cell RNA sequencing on two-day old schistosomula of Schistosoma mansoni. We identify likely gene expression profiles for muscle, nervous system, tegument, oesophageal gland, parenchymal/primordial gut cells, and stem cells. In addition, we validate cell markers for all these clusters by in situ hybridisation in schistosomula and adult parasites. Taken together, this study provides a comprehensive cell-type atlas for the early intra-mammalian stage of this devastating metazoan parasite.

Background: Guinea worm-Dracunculus medinensis-was historically one of the major parasites of humans and has been known since antiquity. Now, Guinea worm is on the brink of eradication, as efforts to interrupt transmission have reduced the annual burden of disease from millions of infections per year in the 1980s to only 54 human cases reported globally in 2019. Despite the enormous success of eradication efforts to date, one complication has arisen. Over the last few years, hundreds of dogs have been found infected with this previously apparently anthroponotic parasite, almost all in Chad. Moreover, the relative numbers of infections in humans and dogs suggests that dogs are currently the principal reservoir on infection and key to maintaining transmission in that country., Principal Findings: In an effort to shed light on this peculiar epidemiology of Guinea worm in Chad, we have sequenced and compared the genomes of worms from dog, human and other animal infections. Confirming previous work with other molecular markers, we show that all of these worms are D. medinensis, and that the same population of worms are causing both infections, can confirm the suspected transmission between host species and detect signs of a population bottleneck due to the eradication efforts. The diversity of worms in Chad appears to exclude the possibility that there were no, or very few, worms present in the country during a 10-year absence of reported cases., Conclusions: This work reinforces the importance of adequate surveillance of both human and dog populations in the Guinea worm eradication campaign and suggests that control programs aiming to interrupt disease transmission should stay aware of the possible emergence of unusual epidemiology as pathogens approach elimination., Competing Interests: The authors have declared that no competing interests exist. Author Ouakou Tchindebet was unable to confirm their authorship contributions. On their behalf, the corresponding author has reported their contributions to the best of their knowledge.

Anthropogenic stressors are impacting ecological systems across the world. Of particular concern are the recent rapid changes occurring in coral reef systems. With ongoing degradation from both local and global stressors, future reefs are likely to function differently from current coral-dominated ecosystems. Determining key attributes of future reef states is critical to reliably predict outcomes for ecosystem service provision. Here we explore the impacts of changing sponge dominance on coral reefs. Qualitative modelling of reef futures suggests that changing sponge dominance due to increased sponge abundance will have different outcomes for other trophic levels compared with increased sponge dominance as a result of declining coral abundance. By exploring uncertainty in the model outcomes we identify the need to (1) quantify changes in carbon flow through sponges, (2) determine the importance of food limitation for sponges, (3) assess the ubiquity of the recently described "sponge loop," (4) determine the competitive relationships between sponges and other benthic taxa, particularly algae, and (5) understand how changing dominance of other organisms alters trophic pathways and energy flows through ecosystems. Addressing these knowledge gaps will facilitate development of more complex models that assess functional attributes of sponge-dominated reef ecosystems., (© 2018 by the Ecological Society of America.)

Despite improvements in the CRISPR molecular toolbox, identifying and purifying properly edited clones remains slow, laborious, and low-yield. Here, we establish a method to enable clonal isolation, selection, and expansion of properly edited cells, using OptoElectroPositioning technology for single-cell manipulation on a nanofluidic device. Briefly, after electroporation of primary T cells with CXCR4 -targeting Cas9 ribonucleoproteins, single T cells are isolated on a chip and expanded into colonies. Phenotypic consequences of editing are rapidly assessed on-chip with cell-surface staining for CXCR4. Furthermore, individual colonies are identified based on their specific genotype. Each colony is split and sequentially exported for on-target sequencing and further off-chip clonal expansion of the validated clones. Using this method, single-clone editing efficiencies, including the rate of mono- and bi-allelic indels or precise nucleotide replacements, can be assessed within 10 days from Cas9 ribonucleoprotein introduction in cells., Competing Interests: The authors declare the following competing interests: An.M., H.M.B., M.S., A.S., K.C., and G.L. are or were fulltime employees of Berkeley Lights, Inc, a private company. Al.M. is a founder of Spotlight Therapeutics and serves as an advisor to Juno Therapeutics and PACT Pharma and the Marson lab has received sponsored research support from Juno Therapeutics, Epinomics, and Sanofi. The remaining authors declare no competing interests.

Single-cell RNA-sequencing is revolutionising our understanding of seemingly homogeneous cell populations but has not yet been widely applied to single-celled organisms. Transcriptional variation in unicellular malaria parasites from the Plasmodium genus is associated with critical phenotypes including red blood cell invasion and immune evasion, yet transcriptional variation at an individual parasite level has not been examined in depth. Here, we describe the adaptation of a single-cell RNA-sequencing (scRNA-seq) protocol to deconvolute transcriptional variation for more than 500 individual parasites of both rodent and human malaria comprising asexual and sexual life-cycle stages. We uncover previously hidden discrete transcriptional signatures during the pathogenic part of the life cycle, suggesting that expression over development is not as continuous as commonly thought. In transmission stages, we find novel, sex-specific roles for differential expression of contingency gene families that are usually associated with immune evasion and pathogenesis., Competing Interests: AR, AT, HB, AG, MS, CI, OB, MB, ML No competing interests declared, (© 2018, Reid et al.)

Schistosomes infect more than 200 million people. These parasitic flatworms rely on a syncytial outer coat called the tegument to survive within the vasculature of their host. Although the tegument is pivotal for their survival, little is known about maintenance of this tissue during the decades schistosomes survive in the bloodstream. Here, we demonstrate that the tegument relies on stem cells (neoblasts) to specify fusogenic progenitors that replace tegumental cells lost to turnover. Molecular characterization of neoblasts and tegumental progenitors led to the discovery of two flatworm-specific zinc finger proteins that are essential for tegumental cell specification. These proteins are homologous to a protein essential for neoblast-driven epidermal maintenance in free-living flatworms. Therefore, we speculate that related parasites (i.e., tapeworms and flukes) employ similar strategies to control tegumental maintenance. Since parasitic flatworms infect every vertebrate species, understanding neoblast-driven tegumental maintenance could identify broad-spectrum therapeutics to fight diseases caused by these parasites., Competing Interests: GW, JC, JP, MP, HB, AL, MB, NG, JC No competing interests declared, (© 2018, Wendt et al.)

We examined if 12 weeks of capsaicinoid (CAP) supplementation affected appetite, body composition and metabolic health markers. Seventy seven healthy male and female volunteers (30 ± 1 y, 171.2 ± 9.8 cm, 81.0 ± 2.2 kg, 27.5 ± 0.6 kg/m 2 ) were randomly assigned to ingest either low-dose CAP (2 mg/d; L-CAP, n = 27), high-dose CAP (4 mg/d; H-CAP, n = 22) from Capsimax or placebo (corn starch; PLA, n = 28) for 12 weeks. At baseline (0 WK), 6 weeks (6 WK) and 12 weeks (12 WK) waist: hip ratio, body composition via dual energy x-ray absorptiometry (DEXA, 0 WK and 12 WK only), self-reported Calorie intakes, appetite levels via Council on Nutrition Appetite Questionnaire (CNAQ) and serum metabolic health markers (0 WK and 12 WK only) were analyzed. Moreover, an oral glucose tolerance test (OGTT) was administered at 0 WK and 12 WK, and serum glucose and insulin responses were examined 30-120 min post test-drink consumption. Waist: hip ratio significantly decreased in L-CAP from 0 WK to 6 WK (p < 0.05), although supplementation did not significantly affect body composition. H-CAP consumed less kcal/d compared to PLA at 12 WK (difference = 257 kcal/d, p < 0.05) and L-CAP participants at 12 WK (difference = 247, p < 0.05). Twenty-three percent (9/39) of the originally-enrolled H-CAP participants reported GI distress, although no participants in the L-CAP group reported such adverse events. Interestingly, H-CAP participants presented significant increases in serum insulin as well as significant decreases in serum HDL cholesterol levels from WK0 to WK12. However, supplementation did not affect the insulin response to the administered OGTT and/or other indices of insulin sensitivity. These data suggest that H-CAP supplementation reduces self-reported energy intake after 12 weeks of supplementation, and L-CAP supplementation also reduces waist: hip ratio. Longer-term effects of capsaicinoid supplementation on basal insulin and cholesterol levels warrant further investigation., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

As atmospheric CO 2 concentrations rise, associated ocean warming (OW) and ocean acidification (OA) are predicted to cause declines in reef-building corals globally, shifting reefs from coral-dominated systems to those dominated by less sensitive species. Sponges are important structural and functional components of coral reef ecosystems, but despite increasing field-based evidence that sponges may be 'winners' in response to environmental degradation, our understanding of how they respond to the combined effects of OW and OA is limited. To determine the tolerance of adult sponges to climate change, four abundant Great Barrier Reef species were experimentally exposed to OW and OA levels predicted for 2100, under two CO 2 Representative Concentration Pathways (RCPs). The impact of OW and OA on early life-history stages was also assessed for one of these species to provide a more holistic view of species impacts. All species were generally unaffected by conditions predicted under RCP6.0, although environmental conditions projected under RCP8.5 caused significant adverse effects: with elevated temperature decreasing the survival of all species, increasing levels of tissue necrosis and bleaching, elevating respiration rates and decreasing photosynthetic rates. OA alone had little adverse effect, even under RCP8.5 concentrations. Importantly, the interactive effect of OW and OA varied between species with different nutritional modes, with elevated pCO 2 exacerbating temperature stress in heterotrophic species but mitigating temperature stress in phototrophic species. This antagonistic interaction was reflected by reduced mortality, necrosis and bleaching of phototrophic species in the highest OW/OA treatment. Survival and settlement success of Carteriospongia foliascens larvae were unaffected by experimental treatments, and juvenile sponges exhibited greater tolerance to OW than their adult counterparts. With elevated pCO 2 providing phototrophic species with protection from elevated temperature, across different life stages, climate change may ultimately drive a shift in the composition of sponge assemblages towards a dominance of phototrophic species., (© 2016 John Wiley & Sons Ltd.)

We present case histories of four patients treated with artemether-lumefantrine for falciparum malaria in UK hospitals in 2015 to 2016. Each subsequently presented with recurrent symptoms and Plasmodium falciparum parasitemia within 6 weeks of treatment with no intervening travel to countries where malaria is endemic. Parasite isolates, all of African origin, harbored variants at some candidate resistance loci. No evidence of pfk13 -mediated artemisinin resistance was found. Vigilance for signs of unsatisfactory antimalarial efficacy among imported cases of malaria is recommended., (Copyright © 2017 Sutherland et al.)

Soil-transmitted nematodes, including the Strongyloides genus, cause one of the most prevalent neglected tropical diseases. Here we compare the genomes of four Strongyloides species, including the human pathogen Strongyloides stercoralis, and their close relatives that are facultatively parasitic (Parastrongyloides trichosuri) and free-living (Rhabditophanes sp. KR3021). A significant paralogous expansion of key gene families--families encoding astacin-like and SCP/TAPS proteins--is associated with the evolution of parasitism in this clade. Exploiting the unique Strongyloides life cycle, we compare the transcriptomes of the parasitic and free-living stages and find that these same gene families are upregulated in the parasitic stages, underscoring their role in nematode parasitism.

Background: Sparganosis is an infection with a larval Diphyllobothriidea tapeworm. From a rare cerebral case presented at a clinic in the UK, DNA was recovered from a biopsy sample and used to determine the causative species as Spirometra erinaceieuropaei through sequencing of the cox1 gene. From the same DNA, we have produced a draft genome, the first of its kind for this species, and used it to perform a comparative genomics analysis and to investigate known and potential tapeworm drug targets in this tapeworm., Results: The 1.26 Gb draft genome of S. erinaceieuropaei is currently the largest reported for any flatworm. Through investigation of β-tubulin genes, we predict that S. erinaceieuropaei larvae are insensitive to the tapeworm drug albendazole. We find that many putative tapeworm drug targets are also present in S. erinaceieuropaei, allowing possible cross application of new drugs. In comparison to other sequenced tapeworm species we observe expansion of protease classes, and of Kuntiz-type protease inhibitors. Expanded gene families in this tapeworm also include those that are involved in processes that add post-translational diversity to the protein landscape, intracellular transport, transcriptional regulation and detoxification., Conclusions: The S. erinaceieuropaei genome begins to give us insight into an order of tapeworms previously uncharacterized at the genome-wide level. From a single clinical case we have begun to sketch a picture of the characteristics of these organisms. Finally, our work represents a significant technological achievement as we present a draft genome sequence of a rare tapeworm, and from a small amount of starting material.

Tapeworms (Cestoda) cause neglected diseases that can be fatal and are difficult to treat, owing to inefficient drugs. Here we present an analysis of tapeworm genome sequences using the human-infective species Echinococcus multilocularis, E. granulosus, Taenia solium and the laboratory model Hymenolepis microstoma as examples. The 115- to 141-megabase genomes offer insights into the evolution of parasitism. Synteny is maintained with distantly related blood flukes but we find extreme losses of genes and pathways that are ubiquitous in other animals, including 34 homeobox families and several determinants of stem cell fate. Tapeworms have specialized detoxification pathways, metabolism that is finely tuned to rely on nutrients scavenged from their hosts, and species-specific expansions of non-canonical heat shock proteins and families of known antigens. We identify new potential drug targets, including some on which existing pharmaceuticals may act. The genomes provide a rich resource to underpin the development of urgently needed treatments and control.

RIC-3 enhances the functional expression of certain nicotinic acetylcholine receptors (nAChRs) in vertebrates and invertebrates and increases the availability of functional receptors in cultured cells and Xenopus laevis oocytes. Maximal activity of RIC-3 may be cell-type dependent, so neither mammalian nor invertebrate proteins is optimal in amphibian oocytes. We cloned the X. laevis ric-3 cDNA and tested the frog protein in oocyte expression studies. X. laevis RIC-3 shares 52% amino acid identity with human RIC-3 and only 17% with that of Caenorhabditis elegans. We used the C. elegans nicotinic receptor, ACR-16, to compare the ability of RIC-3 from three species to enhance receptor expression. In the absence of RIC-3, the proportion of oocytes expressing detectable nAChRs was greatly reduced. Varying the ratio of acr-16 to X. laevis ric-3 cRNAs injected into oocytes had little impact on the total cell current. When X. laevis, human or C. elegans ric-3 cRNAs were co-injected with acr-16 cRNA (1 : 1 ratio), 100 μM acetylcholine induced larger currents in oocytes expressing X. laevis RIC-3 compared with its orthologues. This provides further evidence for a species-specific component of RIC-3 activity, and suggests that X. laevis RIC-3 is useful for enhancing the expression of invertebrate nAChRs in X. laevis oocytes., (© 2012 The Authors Journal of Neurochemistry © 2012 International Society for Neurochemistry.)

Nematode nicotinic acetylcholine receptors are the targets for many effective anthelmintics, including those recently introduced into the market. We have identified a novel nicotinic receptor subunit sequence, acr-26, that is expressed in all the animal parasitic nematodes we examined from clades III, IV and V, but is not present in the genomes of Trichinella spiralis, Caenorhabditis elegans, Pristionchus pacificus and Meloidogyne spp. In Ascaris suum, ACR-26 is expressed on muscle cells isolated from the head, but not from the mid-body region. Sequence comparisons with other vertebrate and nematode subunits suggested that ACR-26 may be capable of forming a functional homomeric receptor; when acr-26 cRNA was injected into Xenopus oocytes along with Xenopus laevis ric-3 cRNA we occasionally observed the formation of acetylcholine- and nicotine-sensitive channels. The unreliable expression of ACR-26 in vitro may suggest that additional subunits or chaperones may be required for efficient formation of the functional receptors. ACR-26 may represent a novel target for the development of cholinergic anthelmintics specific for animal parasites., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: Cervical spine clearance in the very young child is challenging. Radiographic imaging to diagnose cervical spine injuries (CSI) even in the absence of clinical findings is common, raising concerns about radiation exposure and imaging-related complications. We examined whether simple clinical criteria can be used to safely rule out CSI in patients younger than 3 years., Methods: The trauma registries from 22 level I or II trauma centers were reviewed for the 10-year period (January 1995 to January 2005). Blunt trauma patients younger than 3 years were identified. The measured outcome was CSI. Independent predictors of CSI were identified by univariate and multivariate analysis. A weighted score was calculated by assigning 1, 2, or 3 points to each independent predictor according to its magnitude of effect. The score was established on two thirds of the population and validated using the remaining one third., Results: Of 12,537 patients younger than 3 years, CSI was identified in 83 patients (0.66%), eight had spinal cord injury. Four independent predictors of CSI were identified: Glasgow Coma Score <14, GCSEYE = 1, motor vehicle crash, and age 2 years or older. A score of <2 had a negative predictive value of 99.93% in ruling out CSI. A total of 8,707 patients (69.5% of all patients) had a score of <2 and were eligible for cervical spine clearance without imaging. There were no missed CSI in this study., Conclusions: CSI in patients younger than 3 years is uncommon. Four simple clinical predictors can be used in conjunction to the physical examination to substantially reduce the use of radiographic imaging in this patient population.

A new method of acute urography suitable for use in any general hospital is described. It takes only 30 minutes and can be performed by the duty radiographer.The method has proved invaluable in the diagnosis and differentiation of renal colic. With radiation safeguards, it may be of value in pregnancy. It has been found useful in renal trauma and in the differential diagnosis of haematuria.