Exploiting comparative omics to understand the pathogenic and virulence-associated protease: anti-protease relationships in the zoonotic parasites Fasciola hepatica and Fasciola gigantica.

The helminth parasites, Fasciola hepatica and Fasciola gigantica, are the causative agents of fasciolosis, a global and economically important disease of people and their livestock. Proteases are pivotal to an array of biological processes related to parasitism (development, feeding, immune evasion, virulence) and therefore their action requires strict regulation by parasite anti-proteases (protease inhibitors). By interrogating the current publicly available Fasciola spp. large sequencing datasets, including several genome assemblies and life cycle stage-specific transcriptome and proteome datasets, we reveal the complex profile and structure of proteases and anti-proteases families operating at various stages of the parasite’s life cycle. Moreover, we have discovered distinct profiles of peptidases and their cognate inhibitors expressed by the parasite stages in the intermediate snail host, reflecting the different environmental niches in which they move, develop and extract nutrients. Comparative genomics revealed a similar cohort of peptidase inhibitors in F. hepatica and F. gigantica but a surprisingly reduced number of cathepsin peptidases genes in the F. gigantica genome assemblies. Chromosomal location of the F. gigantica genes provides new insights into the evolution of these gene families, and critical data for the future analysis and interrogation of Fasciola spp. hybrids spreading throughout the Asian and African continents

A Sufficient Set of Experimentally Implementable Thermal Operations for Small Systems

Recent work using tools from quantum information theory has shown that for small systems where quantum effects become prevalent, there is not one thermodynamical second law but many. Derivations of these laws assume that an experimenter has very precise control of the system and heat bath. Here we show that these multitude of laws can be saturated using two very simple operations: changing the energy levels of the system and thermalizing over any two system energy levels. Using these two operations, one can distill the optimal amount of work from a system, as well as perform the reverse formation process. What is more, using only these two operations and one ancilla qubit in a thermal state, one can transform any state into any other state allowable by the second laws. We thus have the result that the second laws hold for fine-grained manipulation of system and bath, but can be achieved using very coarse control. This brings the full array of thermal operations towards a regime accessible by experiment, and establishes the physical relevance of these second laws, potentially opening a new direction of studies

NASA Light Emitting Diode Medical Applications from Deep Space to Deep Sea

This work is supported and managed through the NASA Marshall Space Flight Center-SBIR Program. LED-technology developed for NASA plant growth experiments in space shows promise for delivering light deep into tissues of the body to promote wound healing and human tissue growth. We present the results of LED-treatment of cells grown in culture and the effects of LEDs on patients’ chronic and acute wounds. LED-technology is also biologically optimal for photodynamic therapy of cancer and we discuss our successes using LEDs in conjunction with light-activated chemotherapeutic drugs

Tegument Glycoproteins and Cathepsins of Newly Excysted Juvenile Fasciola hepatica Carry Mannosidic and Paucimannosidic N-glycans

Recently, the prevalence of Fasciola hepatica in some areas has increased considerably and the availability of a vaccine to protect livestock from infection would represent a major advance in tools available for controlling this disease. To date, most vaccine-target discovery research on this parasite has concentrated on proteomic and transcriptomic approaches whereas little work has been carried out on glycosylation. As the F. hepatica tegument (Teg) may contain glycans potentially relevant to vaccine development and the Newly Excysted Juvenile (NEJ) is the first lifecycle stage in contact with the definitive host, our work has focused on assessing the glycosylation of the NEJTeg and identifying the NEJTeg glycoprotein repertoire. After in vitro excystation, NEJ were fixed and NEJTeg was extracted. Matrix-assisted laser desorption ionisation-time of flight-mass spectrometry (MALDI-TOF-MS) analysis of released N-glycans revealed that oligomannose and core-fucosylated truncated N-glycans were the most dominant glycan types. By lectin binding studies these glycans were identified mainly on the NEJ surface, together with the oral and ventral suckers. NEJTeg glycoproteins were affinity purified after targeted biotinylation of the glycans and identified using liquid chromatography and tandem mass spectrometry (LC-MS/MS). From the total set of proteins previously identified in NEJTeg, eighteen were also detected in the glycosylated fraction, including the F. hepatica Cathepsin B3 (FhCB3) and two of the Cathepsin L3 (FhCL3) proteins, among others. To confirm glycosylation of cathepsins, analysis at the glycopeptide level by LC-ESI-ion-trap-MS/MS with collision-induced dissociation (CID) and electron-transfer dissociation (ETD) was carried out. We established that cathepsin B1 (FhCB1) on position N80, and FhCL3 (BN1106_s10139B000014, scaffold10139) on position N153, carry unusual paucimannosidic Man2GlcNAc2 glycans. To our knowledge, this is the first description of F. hepatica NEJ glycosylation and the first report of N-glycosylation of F. hepatica cathepsins. The significance of these findings for immunological studies and vaccine development is discussed

Stage-specific miRNAs regulate gene expression associated with growth, development and parasite-host interaction during the intra-mammalian migration of the zoonotic helminth parasite Fasciola hepatica

Abstract Background MiRNAs are small non-coding RNAs that post-transcriptionally regulate gene expression in organisms ranging from viruses to mammals. There is great relevance in understanding how miRNAs regulate genes involved in the growth, development, and maturation of the many parasitic worms (helminths) that together afflict more than 2 billion people. Results Here, we describe the miRNAs expressed by each of the predominant intra-mammalian development stages of Fasciola hepatica, a foodborne flatworm that infects a wide range of mammals worldwide, most importantly humans and their livestock. A total of 124 miRNAs were profiled, 72 of which had been previously reported and three of which were conserved miRNA sequences described here for the first time. The remaining 49 miRNAs were novel sequences of which, 31 were conserved with F. gigantica and the remaining 18 were specific to F. hepatica. The newly excysted juveniles express 22 unique miRNAs while the immature liver and mature bile duct stages each express 16 unique miRNAs. We discovered several sequence variant miRNAs (IsomiRs) as well as miRNA clusters that exhibit strict temporal expression paralleling parasite development. Target analysis revealed the close association between miRNA expression and stage-specific changes in the transcriptome; for example, we identified specific miRNAs that target parasite proteases known to be essential for intestinal wall penetration (cathepsin L3). Moreover, we demonstrate that miRNAs fine-tune the expression of genes involved in the metabolic pathways that allow the parasites to move from an aerobic external environment to the anerobic environment of the host. Conclusions These results provide novel insight into the regulation of helminth parasite development and identifies new genes and miRNAs for therapeutic development to limit the virulence and pathogenesis caused by F. hepatica. </jats:sec

The Extracellular Vesicles of the Helminth Pathogen, Fasciola hepatica: Biogenesis Pathways and Cargo Molecules Involved in Parasite Pathogenesis

Extracellular vesicles (EVs) released by parasites have important roles in establishing and maintaining infection. Analysis of the soluble and vesicular secretions of adult Fasciola hepatica has established a definitive characterization of the total secretome of this zoonotic parasite. Fasciola secretes at least two subpopulations of EVs that differ according to size, cargo molecules and site of release from the parasite. The larger EVs are released from the specialized cells that line the parasite gastrodermus and contain the zymogen of the 37 kDa cathepsin L peptidase that performs a digestive function. The smaller exosome-like vesicle population originate from multivesicular bodies within the tegumental syncytium and carry many previously described immunomodulatory molecules that could be delivered into host cells. By integrating our proteomics data with recently available transcriptomic data sets we have detailed the pathways involved with EV biogenesis in F. hepatica and propose that the small exosome biogenesis occurs via ESCRT-dependent MVB formation in the tegumental syncytium before being shed from the apical plasma membrane. Furthermore, we found that the molecular "machinery" required for EV biogenesis is constitutively expressed across the intramammalian development stages of the parasite. By contrast, the cargo molecules packaged within the EVs are developmentally regulated, most likely to facilitate the parasites migration through host tissue and to counteract host immune attack

Exploring the utility of circulating miRNAs as diagnostic biomarkers of fasciolosis.

Effective management and control of parasitic infections on farms depends on their early detection. Traditional serological diagnostic methods for Fasciola hepatica infection in livestock are specific and sensitive, but currently the earliest detection of the parasite only occurs at approximately three weeks post-infection. At this timepoint, parasites have already entered the liver and caused the tissue damage and immunopathology that results in reduced body weight and loss in productivity. Here, we investigated whether the differential abundance of micro(mi)miRNAs in sera of F. hepatica-infected sheep has potential as a tool for the early diagnosis of infection. Using miRNA sequencing analysis, we discovered specific profiles of sheep miRNAs at both the pre-hepatic and hepatic infection phases in comparison to non-infected sheep. In addition, six F. hepatica-derived miRNAs were specifically identified in sera from infected sheep. Thus, a panel of differentially expressed miRNAs comprising four sheep (miR-3231-3p; miR133-5p; 3957-5p; 1197-3p) and two parasite miRNAs (miR-124-3p; miR-Novel-11-5p) were selected as potential biomarkers. The expression of these candidates in sera samples from longitudinal sheep infection studies collected between 7 days and 23 weeks was quantified using RT-qPCR and compared to samples from age-matched non-infected sheep. We identified oar-miR-133-5p and oar-miR-3957-5p as promising biomarkers of fasciolosis, detecting infection as early as 7 days. The differential expression of the other selected miRNAs was not sufficient to diagnose infection; however, our analysis found that the most abundant forms of fhe-miR-124-3p in sera were sequence variants (IsomiRs) of the canonical miRNA, highlighting the critical importance of primer design for accurate diagnostic RT-qPCR. Accordingly, this investigative study suggests that certain miRNAs are biomarkers of F. hepatica infection and validates miRNA-based diagnostics for the detection of fasciolosis in sheep

Fasciola hepatica is refractory to complement killing by preventing attachment of mannose binding lectin (MBL) and inhibiting MBL-associated serine proteases (MASPs) with serpins

The complement system is a first-line innate host immune defence against invading pathogens. It is activated via three pathways, termed Classical, Lectin and Alternative, which are mediated by antibodies, carbohydrate arrays or microbial liposaccharides, respectively. The three complement pathways converge in the formation of C3-convertase followed by the assembly of a lethal pore-like structure, the membrane attack complex (MAC), on the pathogen surface. We found that the infectious stage of the helminth parasite Fasciola hepatica, the newly excysted juvenile (NEJ), is resistant to the damaging effects of complement. Despite being coated with mannosylated proteins, the main initiator of the Lectin pathway, the mannose binding lectin (MBL), does not bind to the surface of live NEJ. In addition, we found that recombinantly expressed serine protease inhibitors secreted by NEJ (rFhSrp1 and rFhSrp2) selectively prevent activation of the complement via the Lectin pathway. Our experiments demonstrate that rFhSrp1 and rFhSrp2 inhibit native and recombinant MBL-associated serine proteases (MASPs), impairing the primary step that mediates C3b and C4b deposition on the NEJ surface. Indeed, immunofluorescence studies show that MBL, C3b, C4b or MAC are not deposited on the surface of NEJ incubated in normal human serum. Taken together, our findings uncover new means by which a helminth parasite prevents the activation of the Lectin complement pathway to become refractory to killing via this host response, in spite of presenting an assortment of glycans on their surface.</jats:p

Production of a functionally active recombinant SARS-CoV-2 (COVID-19) 3C-Like protease and a soluble inactive 3C-like protease-RBD chimeric in a prokaryotic expression system

During the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) intracellular life-cycle, two large polyproteins, pp1a and pp1ab, are produced. Processing of these by viral cysteine proteases, the papain-like protease (PLpro) and the chymotrypsin-like 3C-like protease (3CL-pro) release non-structural proteins necessary for the establishment of the viral replication and transcription complex (RTC), crucial for viral replication. Hence, these proteases are considered prime targets against which anti-coronavirus disease 2019 (COVID-19) drugs could be developed. Here, we describe the expression of a highly soluble and functionally active recombinant 3CL-pro using Escherichia coli BL21 cells. We show that the enzyme functions in a dimeric form and exhibits an unexpected inhibitory profile because its activity is potently blocked by serine rather than cysteine protease inhibitors. In addition, we assessed the ability of our 3CL-pro to function as a carrier for the receptor binding domain (RBD) of the Spike protein. The co-expressed chimeric protein, 3CLpro-RBD, did not exhibit 3CL-pro activity, but its enhanced solubility made purification easier and improved RBD antigenicity when tested against serum from vaccinated individuals in ELISAs. Chimeric proteins containing the 3CL-pro could represent an innovative approach to developing new COVID-19 vaccines