Cloning, yeast expression, mutagenesis and phylogenetic analysis of a novel member of the Fasciola hepatica cathepsin L-like family

Cathepsin L2, a major cysteine proteinase secreted by adult Fasciola hepatica, differs from other reported cathepsin L-like enzymes in its’ ability to cleave peptide substrates that contain proline in the P2 position. In the present study we have isolated a cDNA clone encoding a complete cysteine proteinase precursor from a Fasciola hepatica cDNA library screened with anti-cathepsin L2 serum. The deduced amino acid sequence was compared with other cysteine proteinases of F. hepatica. This confirmed that it belongs to a gene family composed of at least five different cathepsin L-like genes, and is different from other F. hepatica secreted cathepsin L-like proteinases. The cloned gene was successfully expressed in yeast using the trafficking signals contained within its own propeptide, resulting in functionally active enzyme. Comparison of the yeast expressed enzyme and native liver fluke cathepsin L2 by immunological and biochemical analysis showed that the cloned zymogen encoded for the liver fluke cathepsin L2. Cathepsin L2 differs in substrate specificity from F. hepatica cathepsin LI. To test if this difference is due to a tyrosine in the active site, site directed mutagenesis was performed to convert the leucine present in cathepsin LI to the tyrosine present in cathepsin L2. The data obtained indicate that this substitution is not directly linked to the differences in substrate specificity observed between liver fluke cathepsin LI and cathepsin L2. The mutated purified enzyme was not capable of cleaving substrates with proline in the P2 position. Phylogenetic analysis of the papain superfamily indicated that at least four different types of cysteine proteinases of the papain superfamily exist in trematodes. The liver fluke enzymes cloned so far, constitute a cysteine proteinase family equally related to the vertebrate cathepsin Ls, cathepsin Ss and cathepsin Ks. Other relationships between cysteine proteinases of diverse origin were also detected, which allowed us to group them into families and classes

Editorial: Novel Frontiers in Helminth Genomics

No abstract available

Electroporation Facilitates Introduction of Reporter Transgenes and Virions into Schistosome Eggs

The genome sequences of two of the three major species of schistosomes are now available. Molecular tools are needed to determine the importance of these new genes. With this in mind, we investigated introduction of reporter transgenes into schistosome eggs, with the longer-term aim of manipulation of schistosome genes and gene functions. The egg is a desirable developmental stage for genome manipulation, not least because it contains apparently accessible germ cells. Introduction of transgenes into the germ cells of schistosome eggs might result in transgenic schistosomes. However, the egg is surrounded by a thick shell which might block access to entry of transgenes. We cultured eggs in the presence of three types of reporter transgenes of increasing molecular size, and in addition we tried to produce transient holes in the eggs by electroporation to investigate whether the transgenes would more easily enter the eggs. Electroporation of eggs appeared to allow entry of two larger types of transgenes into cultured schistosome eggs, messenger RNA encoding firefly luciferase, and retroviral virions. We anticipate that this approach, electroporation of transgenes into schistosome eggs, will facilitate genetic manipulation of schistosomes for investigating the importance of schistosome genes and gene products as new intervention targets

Materials Science Toolkit for Carbon Footprint Assessment: A Case Study for Endoscopic Accessories of Common Use

[EN] Ironically, healthcare systems are key agents in respiratory-related diseases and estimated deaths because of the high impact of their greenhouse gas emissions, along with industry, transportation, and housing. Based on safety requirements, hospitals and related services use an extensive number of consumables, most of which end up incinerated at the end of their life cycle. A thorough assessment of the carbon footprint of such devices typically requires knowing precise information about the manufacturing process, rarely available in detail because of the many materials, pieces and steps involved during the fabrication. And yet, tools most often used for determining the environmental impact of consumer goods just require a bunch of parameters, mainly based on the material composition of the device. Here we report a basic set of analytical methods that provide the information required by the software OpenLCA to calculate the main outcome related to environmental impact, the greenhouse gas emissions. Through thermogravimetry, calorimetry, infrared spectroscopy and elemental analysis we proved that obtaining relevant data for the calculator in the exemplifying case of endoscopy tooling or accessories is possible. This routine procedure opens the door to a broader, more accurate analysis of the environmental impact of everyday work at hospital services, offering potential alternatives to minimize it.This study has been funded by Instituto de Salud Carlos III (ISCIII) through the project PI21/00193 and cofunded by the European Union. Funding: Instituto de Salud Carlos III (ISCIII), PI21/00193, cofunded by the European Union. And through the project PI2023-6 from UPV-LaFe innovation projects.Martín-Cabezuelo, R.; Vilariño-Feltrer, G.; Campillo Fernandez, AJ.; Lorenzo-Zúñiga, V.; Pons, V.; López-Muñoz, P.; Tort-Ausina, I. (2023). Materials Science Toolkit for Carbon Footprint Assessment: A Case Study for Endoscopic Accessories of Common Use. ACS Environmental Au. https://doi.org/10.1021/acsenvironau.3c0004

Effects of thermal stress on the expression of glucocorticoid receptor complex linked genes in Senegalese sole (Solea senegalensis): Acute and adaptive stress responses

The present study examined the short and mid-term effects of a rise in temperature from 18 ºC to 24 ºC on the expression of genes related to the stress response regulation in juveniles of Senegalese sole, Solea senegalensis. The animals were exposed to a temperature increase of 6 °C, after 1 month of acclimation at 18 ºC. After this process, samples of different tissues were collected from a total of 96 fish at four sampling points: 1 hour, 24 hours, 3 days and 1 week. The transcript levels of a set of genes involved in the stress response such as glucocorticoid receptors 1 and 2, corticotrophin-releasing factor, corticotrophin-releasing factor binding proteins, proopiomelanocortin A and B, and cellular stress defense (heat shock protein 70, 90AA and 90AB) were quantified at these sampling points. Additionally, blood samples were also taken to measure the circulating plasma cortisol concentration.  Thermal stress induced by increasing temperature prompted an elevation of plasma cortisol levels in juvenile Senegalese sole after 1 h as a short-term response, and a consecutive increase after one week, as a mid-term response.. Senegalese sole seemed to respond positively in terms of adaptive mechanisms, with a rapid over-expression of grs and hsps in liver and brain, significantly higher after one hour post stress, denoting the fast and acute response of those tissues to a rapid change on temperature. The ratio hsp90/gr also increased 24 h after thermal shock, ratio proposed to be an adaptive mechanism to prevent proteosomal degradation of GR. As a mid-term response, the elevation of brain crfbp gene expression one week after thermal shock could be an adaptive mechanism of negative feedback on HPI axis Taken together, these data suggested an initial up-regulation of the glucocorticoid receptor complex linked genes in response to a temperature increase in Senegalese sole, with heat shock protein 90 potentially being a regulatory factor for the glucocorticoid receptor in the presence of cortisol

Genomes of Fasciola hepatica from the Americas Reveal Colonization with Neorickettsia Endobacteria Related to the Agents of Potomac Horse and Human Sennetsu Fevers.

Food borne trematodes (FBTs) are an assemblage of platyhelminth parasites transmitted through the food chain, four of which are recognized as neglected tropical diseases (NTDs). Fascioliasis stands out among the other NTDs due to its broad and significant impact on both human and animal health, as Fasciola sp., are also considered major pathogens of domesticated ruminants. Here we present a reference genome sequence of the common liver fluke, Fasciola hepatica isolated from sheep, complementing previously reported isolate from cattle. A total of 14,642 genes were predicted from the 1.14 GB genome of the liver fluke. Comparative genomics indicated that F. hepatica Oregon and related food-borne trematodes are metabolically less constrained than schistosomes and cestodes, taking advantage of the richer millieux offered by the hepatobiliary organs. Protease families differentially expanded between diverse trematodes may facilitate migration and survival within the heterogeneous environments and niches within the mammalian host. Surprisingly, the sequencing of Oregon and Uruguay F. hepatica isolates led to the first discovery of an endobacteria in this species. Two contigs from the F. hepatica Oregon assembly were joined to complete the 859,205 bp genome of a novel Neorickettsia endobacterium (nFh) closely related to the etiological agents of human Sennetsu and Potomac horse fevers. Immunohistochemical studies targeting a Neorickettsia surface protein found nFh in specific organs and tissues of the adult trematode including the female reproductive tract, eggs, the Mehlis\u27 gland, seminal vesicle, and oral suckers, suggesting putative routes for fluke-to-fluke and fluke-to-host transmission. The genomes of F. hepatica and nFh will serve as a resource for further exploration of the biology of F. hepatica, and specifically its newly discovered trans-kingdom interaction with nFh and the impact of both species on disease in ruminants and humans

Mycobacterioses in dogs and cats from Buenos Aires, Argentina

Mycobacterioses can produce nonspecific clinical signs in dogs and cats that make diagnosis difficult. Furthermore, the full characterization of mycobacterial agents is not always possible or practical. We characterized mycobacteria detected through cytology in 12 dogs and 7 cats with generalized clinical signs from the province of Buenos Aires in Argentina. In dogs, molecular testing confirmed the presence of Mycobacterium avium subsp. hominissuis (MAH) in 8 cases and M. fortuitum in 1 case. All dogs were Miniature Schnauzers, suggesting that this breed may be more susceptible to M. avium than other dog breeds. The cat isolates were 2 M. bovis, 1 M. fortuitum, and 1 MAH. Mycobacterial interspersed repetitive unit–variable-number tandem repeat patterns suggested possible links with cattle, swine, and humans studied previously in Argentina. The results show that pets may act as susceptible hosts with the potential risk of transmitting the infection to humans and other animals.Instituto de BiotecnologíaFil: Barandiaran, Soledad. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias; ArgentinaFil: Martinez Vivot, Marcela. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias; ArgentinaFil: Falzoni, Elvira. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias; ArgentinaFil: Marfil, Maria Jimena. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias; ArgentinaFil: Perez Tort, Gabriela. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias; Argentina. Virreyes Veterinary Hospital; ArgentinaFil: Rovatti, Paula. Private Veterinary Clinic; ArgentinaFil: Fernandez, Mónica. Zoonosis Luis Pasteur Institute; ArgentinaFil: Iachini, Ricardo. Zoonosis Luis Pasteur Institute; ArgentinaFil: Satek, Fernanda. Surgical Veterinary Clinic (EQVET); ArgentinaFil: Duchene, Adriana. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias; ArgentinaFil: Zumarraga, Martin Jose. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentin

Germline transgenesis and insertional mutagenesis in Schistosoma mansoni mediated by murine leukemia virus

Functional studies will facilitate characterization of role and essentiality of newly available genome sequences of the human schistosomes, Schistosoma mansoni, S. japonicum and S. haematobium. To develop transgenesis as a functional approach for these pathogens, we previously demonstrated that pseudotyped murine leukemia virus (MLV) can transduce schistosomes leading to chromosomal integration of reporter transgenes and short hairpin RNA cassettes. Here we investigated vertical transmission of transgenes through the developmental cycle of S. mansoni after introducing transgenes into eggs. Although MLV infection of schistosome eggs from mouse livers was efficient in terms of snail infectivity, \u3e10-fold higher transgene copy numbers were detected in cercariae derived from in vitro laid eggs (IVLE). After infecting snails with miracidia from eggs transduced by MLV, sequencing of genomic DNA from cercariae released from the snails also revealed the presence of transgenes, demonstrating that transgenes had been transmitted through the asexual developmental cycle, and thereby confirming germline transgenesis. High-throughput sequencing of genomic DNA from schistosome populations exposed to MLV mapped widespread and random insertion of transgenes throughout the genome, along each of the autosomes and sex chromosomes, validating the utility of this approach for insertional mutagenesis. In addition, the germline-transmitted transgene encoding neomycin phosphotransferase rescued cultured schistosomules from toxicity of the antibiotic G418, and PCR analysis of eggs resulting from sexual reproduction of the transgenic worms in mice confirmed that retroviral transgenes were transmitted to the next (F1) generation. These findings provide the first description of wide-scale, random insertional mutagenesis of chromosomes and of germline transmission of a transgene in schistosomes. Transgenic lines of schistosomes expressing antibiotic resistance could advance functional genomics for these significant human pathogens

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

A notable proportion of liver transplant candidates with alcohol-related cirrhosis can be delisted because of clinical improvement

Background & Aims To what extent patients with alcohol-related decompensated cirrhosis can improve until recovery from decompensation remains unclear. We aimed to investigate the probability of recovery and delisting due to improvement in patients with alcohol-related decompensated cirrhosis on the waiting list (WL) for liver transplantation (LT). Methods We conducted a registry-based, multicenter, retrospective study including all patients admitted to the LT WL in Catalonia (Spain) with the indication of alcohol-, HCV-, cholestasis- or non-alcoholic steatohepatitis-related decompensated cirrhosis between January 2007 and December 2018. Competing-risk analysis was used to investigate variables associated with delisting due to improvement in patients with alcohol-related decompensated cirrhosis. Criteria for delisting after improvement were not predefined. Outcomes of patients after delisting were also studied. Results One-thousand and one patients were included, 420 (37%) with alcohol-related decompensated cirrhosis. Thirty-six (8.6%) patients with alcohol-related decompensated cirrhosis were delisted after improvement at a median time of 29 months after WL admission. Lower model for end-stage liver disease (MELD) score, higher platelets and either female sex or lower height were independently associated with delisting due to improvement, while time of abstinence did not reach statistical significance in multivariate analysis (p = 0.055). Five years after delisting, the cumulative probability of remaining free from liver-related death or LT was 76%, similar to patients with HCV-related decompensated cirrhosis delisted after improvement. Conclusions A significant proportion of LT candidates with alcohol-related cirrhosis can be delisted due to improvement, which is predicted by low MELD score and higher platelet count at WL admission. Women also have a higher probability of being delisted after improvement, partially due to reduced early access to LT for height discrepancies. Early identification of patients with potential for improvement may avoid unnecessary transplants. Lay summary Patients with alcohol-related cirrhosis can improve until being delisted in approximately 9% of cases. Low model for end-stage liver disease score and high platelet levels at admission predict delisting after improvement, and women have higher probabilities of being delisted due to improvement. Long-term outcomes after delisting are generally favorable