Structure, Hydration, and Interactions of Native and Hydrophobically Modified Phytoglycogen Nanoparticles.

Phytoglycogen is a highly branched polymer of glucose produced as soft, compact nanoparticles by sweet corn. Properties such as softness, porosity, and mechanical integrity, combined with nontoxicity and biodegradability, make phytoglycogen nanoparticles ideal for applications involving the human body, ranging from skin moisturizing and rejuvenation agents in personal care formulations to functional therapeutics in biomedicine. To further broaden the range of applications, phytoglycogen nanoparticles can be chemically modified with hydrophobic species such as octenyl succinic anhydride (OSA). Here, we present a self-consistent model of the particle structure, water content, and degree of chemical modification of the particles, as well as the emergence of well-defined interparticle spacings in concentrated dispersions, based on small-angle neutron scattering (SANS) measurements of aqueous dispersions of native phytoglycogen nanoparticles and particles that were hydrophobically modified using octenyl succinic anhydride (OSA) in both its protiated (pOSA) and deuterated (dOSA) forms. Measurements on native particles with reduced polydispersity have allowed us to refine the particle morphology, which is well described by a hairy particle (core-chain) geometry with short chains decorating the surface of the particles. The isotopic variants of OSA-modified particles enhanced the scattering contrast for neutrons, revealing lightly modified hairy chains for small degrees of substitution (DS) of OSA, and a raspberry particle geometry for the largest DS value, where the OSA-modified hairy chains collapse to form small seeds on the surface of the particles. This refined model of native and OSA-modified phytoglycogen nanoparticles establishes a quantitative basis for the development of new applications of this promising sustainable nanotechnology.

Tissue-specific transcriptomics and proteomics of a filarial nematode and its Wolbachia endosymbiont.

BACKGROUND: Filarial nematodes cause debilitating human diseases. While treatable, recent evidence suggests drug resistance is developing, necessitating the development of novel targets and new treatment options. Although transcriptomic and proteomic studies around the nematode life cycle have greatly enhanced our knowledge, whole organism approaches have not provided spatial resolution of gene expression, which can be gained by examining individual tissues. Generally, due to their small size, tissue dissection of human-infecting filarial nematodes remains extremely challenging. However, canine heartworm disease is caused by a closely related and much larger filarial nematode, Dirofilaria immitis. As with many other filarial nematodes, D. immitis contains Wolbachia, an obligate bacterial endosymbiont present in the hypodermis and developing oocytes within the uterus. Here, we describe the first concurrent tissue-specific transcriptomic and proteomic profiling of a filarial nematode (D. immitis) and its Wolbachia (wDi) in order to better understand tissue functions and identify tissue-specific antigens that may be used for the development of new diagnostic and therapeutic tools. METHODS: Adult D. immitis worms were dissected into female body wall (FBW), female uterus (FU), female intestine (FI), female head (FH), male body wall (MBW), male testis (MT), male intestine (MI), male head (MH) and 10.1186/s12864-015-2083-2 male spicule (MS) and used to prepare transcriptomic and proteomic libraries. RESULTS: Transcriptomic and proteomic analysis of several D. immitis tissues identified many biological functions enriched within certain tissues. Hierarchical clustering of the D. immitis tissue transcriptomes, along with the recently published whole-worm adult male and female D. immitis transcriptomes, revealed that the whole-worm transcriptome is typically dominated by transcripts originating from reproductive tissue. The uterus appeared to have the most variable transcriptome, possibly due to age. Although many functions are shared between the reproductive tissues, the most significant differences in gene expression were observed between the uterus and testis. Interestingly, wDi gene expression in the male and female body wall is fairly similar, yet slightly different to that of Wolbachia gene expression in the uterus. Proteomic methods verified 32 % of the predicted D. immitis proteome, including over 700 hypothetical proteins of D. immitis. Of note, hypothetical proteins were among some of the most abundant Wolbachia proteins identified, which may fulfill some important yet still uncharacterized biological function. CONCLUSIONS: The spatial resolution gained from this parallel transcriptomic and proteomic analysis adds to our understanding of filarial biology and serves as a resource with which to develop future therapeutic strategies against filarial nematodes and their Wolbachia endosymbionts.

Concurrent transcriptional profiling of Dirofilaria immitis and its Wolbachia endosymbiont throughout the nematode life cycle reveals coordinated gene expression.

BACKGROUND: Dirofilaria immitis, or canine heartworm, is a filarial nematode parasite that infects dogs and other mammals worldwide. Current disease control relies on regular administration of anthelmintic preventives, however, relatively poor compliance and evidence of developing drug resistance could warrant alternative measures against D. immitis and related human filarial infections be taken. As with many other filarial nematodes, D. immitis contains Wolbachia, an obligate bacterial endosymbiont thought to be involved in providing certain critical metabolites to the nematode. Correlations between nematode and Wolbachia transcriptomes during development have not been examined. Therefore, we detailed the developmental transcriptome of both D. immitis and its Wolbachia (wDi) in order to gain a better understanding of parasite-endosymbiont interactions throughout the nematode life cycle. RESULTS: Over 215 million single-end 50 bp reads were generated from total RNA from D. immitis adult males and females, microfilariae (mf) and third and fourth-stage larvae (L3 and L4). We critically evaluated the transcriptomes of the various life cycle stages to reveal sex-biased transcriptional patterns, as well as transcriptional differences between larval stages that may be involved in larval maturation. Hierarchical clustering revealed both D. immitis and wDi transcriptional activity in the L3 stage is clearly distinct from other life cycle stages. Interestingly, a large proportion of both D. immitis and wDi genes display microfilarial-biased transcriptional patterns. Concurrent transcriptome sequencing identified potential molecular interactions between parasite and endosymbiont that are more prominent during certain life cycle stages. In support of metabolite provisioning between filarial nematodes and Wolbachia, the synthesis of the critical metabolite, heme, by wDi appears to be synchronized in a stage-specific manner (mf-specific) with the production of heme-binding proteins in D. immitis. CONCLUSIONS: Our integrated transcriptomic study has highlighted interesting correlations between Wolbachia and D. immitis transcription throughout the life cycle and provided a resource that may be used for the development of novel intervention strategies, not only for the treatment and prevention of D. immitis infections, but of other closely related human parasites as well.

Intraperitoneal development of the filarial nematode Brugia malayi in the Mongolian jird (Meriones unguiculatus).

In the present study, we describe intraperitoneal development of the FR3 strain of Brugia malayi in Mongolian jirds (Meriones unguiculatus). The third molt for male worms occurred between 4 and 7 days postinfection (dpi) and between 4 and 8 dpi for females. The fourth and final molt occurred between days 21 and 29 for males and 25 and 34 for females, considerably earlier than the times reported for subcutaneous infection models using cats and jirds. The timing of the third molt coincided largely with reports for subcutaneous Brugia pahangi infections of cats and jirds, but the final molt occurred considerably later and lasted longer than those reported for subcutaneous B. pahangi models. Spermatogenesis occurred by at least 50 dpi in adult males, and insemination of females likely occurred between 50 and 60 dpi. Microfilariae were observed in the uteri and ovejectors of adult females at 65 dpi.

Description of Ornithodoros (Pavlovskyella) tartakovskyi using scanning electron microscopy, with notes on the morphology of Pavlovskyella sensu stricto and Theriodoros subgenera.

Accumulation of DNA sequence data and its use in systematics of the family Argasidae reveals new incongruencies between genera and subgenera, since several groups defined by classical taxonomy appear to be paraphyletic, which is the case of the subgenus Pavlovskyella. In order to identify morphological characters unique to one of the monophyletic groupings within Pavlovskyella and improve its system, we describe all active stages of Ornithodoros (Pavlovskyella) tartakovskyi, a species with an incomplete original description. Larvae, nymphs, males and females from Iran were examined with scanning electron microscopy (SEM). The larva of O. (P.) tartakovskyi lacks dorsal plate, posteromedian seta, postcoxal setae and a spinose area in palpal article I; the dorsal surface has 13 pairs of setae and the hypostome has two short rows of denticles in the apex only. The first nymphal instar (N1) has a micromammillated body with faint dorsal and ventral disks. The second nymphal instar (N2) shows mammillated body with an incipient hood, and dorsal disks outlined as in subsequent instars. Both N1 and N2 have a small patch of glabrous surface in the anterior margin of preanal groove, absent in posterior stages. Third nymphal instar (N3) has a small hood and lack cheeks, which are incipient in the fourth nymphal instar (N4). The four nymphal instars (N1-N4) have blunt hypostomes, striated capitula, non-mammillated legs, and 3 humps on tarsi I. Females and males of O. (P.) tartakovskyi have mammillated bodies, small cheeks not covering the capitulum, the anteromedian disk is placed anteriorly to anterior central disks, and a posterior median file of disks is merged with the median disk. We describe all postembryonic stages of O. (P.) tartakovskyi providing morphological characters that define Pavlovskyella sensu stricto and Theriodoros, a sister subgenus that includes similarly shaped species.

Common analysis of direct RNA sequencinG CUrrently leads to misidentification of m5C at GCU motifs.

RNA modifications, such as methylation, can be detected with Oxford Nanopore Technologies direct RNA sequencing. One commonly used tool for detecting 5-methylcytosine (m5C) modifications is Tombo, which uses an "Alternative Model" to detect putative modifications from a single sample. We examined direct RNA sequencing data from diverse taxa including viruses, bacteria, fungi, and animals. The algorithm consistently identified a m5C at the central position of a GCU motif. However, it also identified a m5C in the same motif in fully unmodified in vitro transcribed RNA, suggesting that this is a frequent false prediction. In the absence of further validation, several published predictions of m5C in a GCU context should be reconsidered, including those from human coronavirus and human cerebral organoid samples.

Common Analysis of Direct RNA SequencinG CUrrently Leads to Misidentification of 5-Methylcytosine Modifications at GCU Motifs.

RNA modifications, such as méthylation, can be detected with Oxford Nanopore Technologies direct RNA sequencing. One commonly used tool for detecting 5-methylcytosine (m5C) modifications is Tombo, which uses an "Alternative Model" to detect putative modifications from a single sample. We examined direct RNA sequencing data from diverse taxa including virus, bacteria, fungi, and animals. The algorithm consistently identified a 5-methylcytosine at the central position of a GCU motif. However, it also identified a 5-methylcytosine in the same motif in fully unmodified in vitro transcribed RNA, suggesting that this a frequent false prediction. In the absence of further validation, several published predictions of 5-methylcytosine in human coronavirus and human cerebral organoid RNA in a GCU context should be reconsidered.

DIPLOTRIAENA OBTUSA (NEMATODA: DIPLOTRIAENIDAE) FROM BARN SWALLOWS (HIRUNDO RUSTICA) AND CLIFF SWALLOWS (PETROCHELIDON PYRRHONOTA) COLLECTED DURING MORTALITY EVENTS IN THE UPPER MIDWEST, USA.

Several mortality events involving barn swallows (Hirundo rustica) and cliff swallows (Petrochelidon pyrrhonota) were reported in the Upper Midwestern states in 2017 and 2018. Barn swallow mortality followed unseasonal cold snaps, with the primary cause of death being emaciation with concurrent air sac nematodiasis. Lesions in cliff swallows were consistent with blunt force trauma from suspected car impacts. Examination of air sac nematodes from both bird species revealed morphological characters consistent with Diplotriaena obtusa. Sequence analysis of the partial 18S rRNA gene indicated the samples clustered with other species in the genus Diplotriaena. These nematodes provide a link between morphological specimens and DNA sequence data for D. obtusa.

X-treme loss of sequence diversity linked to neo-X chromosomes in filarial nematodes.

The sequence diversity of natural and laboratory populations of Brugia pahangi and Brugia malayi was assessed with Illumina resequencing followed by mapping in order to identify single nucleotide variants and insertions/deletions. In natural and laboratory Brugia populations, there is a lack of sequence diversity on chromosome X relative to the autosomes (πX/πA = 0.2), which is lower than the expected (πX/πA = 0.75). A reduction in diversity is also observed in other filarial nematodes with neo-X chromosome fusions in the genera Onchocerca and Wuchereria, but not those without neo-X chromosome fusions in the genera Loa and Dirofilaria. In the species with neo-X chromosome fusions, chromosome X is abnormally large, containing a third of the genetic material such that a sizable portion of the genome is lacking sequence diversity. Such profound differences in genetic diversity can be consequential, having been associated with drug resistance and adaptability, with the potential to affect filarial eradication.

Using an intravenous catheter to carry out abdominal lavage in the gerbil.

Abdominal lavage is used in laboratory rodents for a variety of applications but carries an inherent risk of abdominal organ laceration; therefore, personnel carrying out this procedure must have considerable expertise. In this paper, the authors describe an improved method for delivering sterile media to and collecting peritoneal fluids from dark-clawed Mongolian gerbils (Meriones unguiculatus) that had been peritoneally infected with filarial nematode parasites (genus Brugia). To carry out this gravity-assisted technique, the authors used a catheter to introduce sterile media into the peritoneal cavity of each gerbil and then to passively drain peritoneal fluid and larval worms for collection. Average fluid recovery was consistently greater when using this gravity-assisted method than when using aspiration. Larval parasites were recovered by both methods. To recover large volumes of fluid using the standard method of abdominal lavage, personnel typically must euthanize rodents. This gravity-assisted technique allows researchers to collect large numbers of parasite larvae without euthanizing gerbils.

A Meta-Analysis of Wolbachia Transcriptomics Reveals a Stage-Specific Wolbachia Transcriptional Response Shared Across Different Hosts.

Wolbachia is a genus containing obligate, intracellular endosymbionts with arthropod and nematode hosts. Numerous studies have identified differentially expressed transcripts in Wolbachia endosymbionts that potentially inform the biological interplay between these endosymbionts and their hosts, albeit with discordant results. Here, we re-analyze previously published Wolbachia RNA-Seq transcriptomics data sets using a single workflow consisting of the most up-to-date algorithms and techniques, with the aim of identifying trends or patterns in the pan-Wolbachia transcriptional response. We find that data from one of the early studies in filarial nematodes did not allow for robust conclusions about Wolbachia differential expression with these methods, suggesting the original interpretations should be reconsidered. Across datasets analyzed with this unified workflow, there is a general lack of global gene regulation with the exception of a weak transcriptional response resulting in the upregulation of ribosomal proteins in early larval stages. This weak response is observed across diverse Wolbachia strains from both nematode and insect hosts suggesting a potential pan-Wolbachia transcriptional response during host development that diverged more than 700 million years ago.

Nearly Complete Genome Sequence of Brugia pahangi FR3.

Brugia pahangi is a zoonotic parasite that is closely related to human-infecting filarial nematodes. Here, we report the nearly complete genome of Brugia pahangi, including assemblies of four autosomes and an X chromosome, with only seven gaps. The Y chromosome is still not completely assembled.

Complete Genome Sequence of wBp, the Wolbachia Endosymbiont of Brugia pahangi FR3.

Lymphatic filariasis is a devastating disease caused by filarial nematode roundworms, which contain obligate Wolbachia endosymbionts. Here, we assembled the genome of wBp, the Wolbachia endosymbiont of the filarial nematode Brugia pahangi, from Illumina, Pacific Biosciences, and Oxford Nanopore data. The complete, circular genome is 1,072,967 bp.

Sex chromosome evolution in parasitic nematodes of humans.

Sex determination mechanisms often differ even between related species yet the evolution of sex chromosomes remains poorly understood in all but a few model organisms. Some nematodes such as Caenorhabditis elegans have an XO sex determination system while others, such as the filarial parasite Brugia malayi, have an XY mechanism. We present a complete B. malayi genome assembly and define Nigon elements shared with C. elegans, which we then map to the genomes of other filarial species and more distantly related nematodes. We find a remarkable plasticity in sex chromosome evolution with several distinct cases of neo-X and neo-Y formation, X-added regions, and conversion of autosomes to sex chromosomes from which we propose a model of chromosome evolution across different nematode clades. The phylum Nematoda offers a new and innovative system for gaining a deeper understanding of sex chromosome evolution.

Nearly Complete Genome Sequence of Brugia malayi Strain FR3.

Lymphatic filariasis affects ∼120 million people and can result in elephantiasis and hydrocele. Here, we report the nearly complete genome sequence of the best-studied causative agent of lymphatic filariasis, Brugia malayi The assembly contains four autosomes, an X chromosome, and only eight gaps but lacks a contiguous sequence for the known Y chromosome.

Use of microarray hybridization to identify Brugia genes involved in mosquito infectivity.

Brugia malayi and Brugia pahangi microfilariae (mf) require a maturation period of at least 5 days in the mammalian host to successfully infect laboratory mosquitoes. This maturation process coincides with changes in the surface composition of mf that likely are associated with changes in gene expression. To test this hypothesis, we verified the differential infectivity of immature (< or =3 day) and mature (>30 day) Brugia mf for black-eyed Liverpool strain of Aedes aegypti and then assessed transcriptome changes associated with microfilarial maturation by competitively hybridizing microfilarial cDNAs to the B. malayi oligonucleotide microarray. We identified transcripts differentially abundant in immature (94 in B. pahangi and 29 in B. malayi) and mature (64 in B. pahangi and 14 in B. malayi) mf. In each case, >40% of Brugia transcripts shared no similarity to known genes or were similar to genes with unknown function; the remaining transcripts were categorized by putative function based on sequence similarity to known genes/proteins. Microfilarial maturation was not associated with demonstrable changes in the abundance of transmembrane or secreted proteins; however, immature mf expressed more transcripts associated with immune modulation, neurotransmission, transcription, and cellular cytoskeleton elements, while mature mf displayed increased transcripts potentially encoding hypodermal/muscle and surface molecules, e.g., cuticular collagens and sheath components. The results of the homologous B. malayi microarray hybridization were validated by quantitative reverse transcriptase polymerase chain reaction. These findings preliminarily lend support to the underlying hypothesis that changes in microfilarial gene expression drive maturation-associated changes that influence the parasite to develop in compatible vectors.

Drug Repurposing of Bromodomain Inhibitors as Potential Novel Therapeutic Leads for Lymphatic Filariasis Guided by Multispecies Transcriptomics.

To better understand the transcriptomic interplay of organisms associated with lymphatic filariasis, we conducted multispecies transcriptome sequencing (RNA-Seq) on the filarial nematode Brugia malayi, its Wolbachia endosymbiont wBm, and its laboratory vector Aedes aegypti across the entire B. malayi life cycle. In wBm, transcription of the noncoding 6S RNA suggests that it may be a regulator of bacterial cell growth, as its transcript levels correlate with bacterial replication rates. For A. aegypti, the transcriptional response reflects the stress that B. malayi infection exerts on the mosquito with indicators of increased energy demand. In B. malayi, expression modules associated with adult female samples consistently contained an overrepresentation of genes involved in chromatin remodeling, such as the bromodomain-containing proteins. All bromodomain-containing proteins encoded by B. malayi were observed to be upregulated in the adult female, embryo, and microfilaria life stages, including 2 members of the bromodomain and extraterminal (BET) protein family. The BET inhibitor JQ1(+), originally developed as a cancer therapeutic, caused lethality of adult worms in vitro, suggesting that it may be a potential therapeutic that can be repurposed for treating lymphatic filariasis.IMPORTANCE The current treatment regimen for lymphatic filariasis is mostly microfilaricidal. In an effort to identify new drug candidates for lymphatic filariasis, we conducted a three-way transcriptomics/systems biology study of one of the causative agents of lymphatic filariasis, Brugia malayi, its Wolbachia endosymbiont wBm, and its vector host Aedes aegypti at 16 distinct B. malayi life stages. B. malayi upregulates the expression of bromodomain-containing proteins in the adult female, embryo, and microfilaria stages. In vitro, we find that the existing cancer therapeutic JQ1(+), which is a bromodomain and extraterminal protein inhibitor, has adulticidal activity in B. malayi.

Multispecies Transcriptomics Data Set of Brugia malayi, Its Wolbachia Endosymbiont wBm, and Aedes aegypti across the B. malayi Life Cycle.

Here, we present a comprehensive transcriptomics data set of Brugia malayi, its Wolbachia endosymbiont wBm, and its vector host. This study samples from 16 stages across the entire B. malayi life cycle, including stage 1 through 4 larvae, adult males and females, embryos, immature microfilariae, and mature microfilariae.

Targeted enrichment outperforms other enrichment techniques and enables more multi-species RNA-Seq analyses.

Enrichment methodologies enable the analysis of minor members in multi-species transcriptomic data. We compared the standard enrichment of bacterial and eukaryotic mRNA to a targeted enrichment using an Agilent SureSelect (AgSS) capture for Brugia malayi, Aspergillus fumigatus, and the Wolbachia endosymbiont of B. malayi (wBm). Without introducing significant systematic bias, the AgSS quantitatively enriched samples, resulting in more reads mapping to the target organism. The AgSS-enriched libraries consistently had a positive linear correlation with their unenriched counterparts (r2 = 0.559-0.867). Up to a 2,242-fold enrichment of RNA from the target organism was obtained following a power law (r2 = 0.90), with the greatest fold enrichment achieved in samples with the largest ratio difference between the major and minor members. While using a single total library for prokaryote and eukaryote enrichment from a single RNA sample could be beneficial for samples where RNA is limiting, we observed a decrease in reads mapping to protein coding genes and an increase in multi-mapping reads to rRNAs in AgSS enrichments from eukaryotic total RNA libraries compared to eukaryotic poly(A)-enriched libraries. Our results support a recommendation of using AgSS targeted enrichment on poly(A)-enriched libraries for eukaryotic captures, and total RNA libraries for prokaryotic captures, to increase the robustness of multi-species transcriptomic studies.

Neospora caninum antibodies detected in Midwestern white-tailed deer (Odocoileus virginianus) by Western blot and ELISA.

White-tailed deer (Odocoileus virginianus) serve to maintain the Neospora caninum life cycle in the wild. Sera from white-tailed deer from south central Wisconsin and southeastern Missouri, USA were tested for antibodies to N. caninum by Western blot analyses and two indirect ELISAs. Seroreactivity against N. caninum surface antigens was observed in 30 of 147 (20%) of WI deer and 11 of 23 (48%) of MO deer using Western blot analysis. Compared to Western blot, the two indirect ELISAs were found to be uninformative due to degradation of the field-collected samples. The results indicate the existence of N. caninum antibodies in MO and WI deer, and that Western blot is superior to ELISA for serologic testing when using degraded blood samples collected from deer carcasses.