The role of stimulus combinations in the repeated assessment of resurgence.

The current study examined the role of stimulus combinations on the repeated assessment of resurgence. Using a within-session resurgence procedure, rats were exposed to different conditions, each with distinct stimulus combinations (AAA, ABA, ABB, ABC and AAB). Two arrangements of stimulus changes were compared: Experiment 1 involved changes in stimulus combinations every five sessions, while Experiment 2 implemented changes every session. Resurgence was observed in both experiments; however, Experiment 2 demonstrated more consistent and repeated resurgence when stimulus combinations changed every session. Notably, the ABA, ABB and ABC conditions showed the highest percentage of sessions in which resurgence was observed. Lastly, the current study extends the application of the within-session resurgence procedure to rats and auditory stimuli, providing a reliable method for assessing resurgence in single subjects under different variable conditions.

Benchmarking assembly free nanopore read mappers to classify complex millipede gut microbiota via Oxford Nanopore Sequencing Technology.

Millipedes are key players in recycling leaf litter into soil in tropical ecosystems. To elucidate their gut microbiota, we collected millipedes from different municipalities of Puerto Rico. Here we aim to benchmark which method is best for metagenomic skimming of this highly complex millipede microbiome. We sequenced the gut DNA with Oxford Nanopore Technologies' (ONT) MinION sequencer, then analyzed the data using MEGAN-LR, Kraken2 protein mode, Kraken2 nucleotide mode, GraphMap, and Minimap2 to classify these long ONT reads. From our two samples, we obtained a total of 87,110 and 99,749 ONT reads, respectively. Kraken2 nucleotide mode classified the most reads compared to all other methods at the phylum and class taxonomic level, classifying 75% of the reads in the two samples, the other methods failed to assign enough reads to either phylum or class to yield asymptotes in the taxa rarefaction curves indicating that they required more sequencing depth to fully classify this community. The community is hyper diverse with all methods classifying 20-50 phyla in the two samples. There was significant overlap in the reads used and phyla classified between the five methods benchmarked. Our results suggest that Kraken2 nucleotide mode is the most appropriate tool for the application of metagenomic skimming of this highly complex community.

A Toxoplasma gondii lipoxygenase-like enzyme is necessary for virulence and changes localization associated with the host immune response.

IMPORTANCE: Lipoxygenases (LOXs) are enzymes that catalyze the deoxygenation of polyunsaturated fatty acids such as linoleic and arachidonic acid. These modifications create signaling molecules that are best characterized for modulating the immune response. Deletion of the first lipoxygenase-like enzyme characterized for Toxoplasma gondii (TgLOXL1) generated a less virulent strain, and infected mice showed a decreased immune response. This virulence defect was dependent on the mouse cytokine interferon gamma IFNγ. TgLOXL1 changes location from inside the parasite in tissue culture conditions to vesicular structures within the host immune cells during mouse infection. These results suggest that TgLOXL1 plays a role in the modification of the host immune response in mice.

Autochthonous transmission of the Indomalayan parasite, Transversotrema patialense, in the Caribbean: Molecular, morphological, and experimental evidence.

The Asian freshwater snail Melanoides tuberculata has been established since the 1960s in the Americas, where it transmits cercariae of a small number of digenetic trematode species from its native range. In 2021-2022, 24 M. tuberculata were discovered shedding transversotrematid cercariae in Puerto Rico, where parasites of this snail have not been previously studied. Adult transversotrematids (in some cases, gravid) were found on field-caught fish and on fish exposed to shedding snails, including on fish species native to Puerto Rico. Adults and cercariae were identified as Transversotrema patialense (Soparkar, 1924), a species native to the Indomalayan region. Morphological identification was supported with 28S rDNA sequences closely matching that from unidentified transversotrematid cercariae in Thailand. The absence of T. patialense in snails collected prior to 2021, increasing prevalence of infection in snails collected thereafter, and lack of variation in parasite DNA sequences (28S, internal transcribed spacer 2, cytochrome c oxidase I) from three isolates are consistent with a recently introduced and possibly expanding parasite population. Transversotrema patialense has been recorded outside its native range before, but most studies (including a prior record in the Americas) reported the parasite from captive hosts from commercial sources such as pet shops. The present results thus provide the first demonstration of natural transmission of T. patialense in the Americas. Phylogenetic analysis of 28S but not of ITS2 show the transversotrematid genus Transversotrema Witenberg, 1944 is paraphyletic, with Crusziella Cribb, Bray and Barker 1992 nested within it.

Comparative Analysis of the Chloroplast Genomes of Quercus × morehus and the Presumptive Parents Q. wislizeni and Q. kelloggii (Fagaceae) from California.

Here, we present the complete chloroplast genomes of Quercus × morehus, Q. wislizeni, and Q. kelloggii from California. The genomes are 161,119 to 161,130 bp and encode 132 genes. Quercus × morehus and Q. wislizeni are identical in sequence but differ from Q. kelloggii by three indels and eight SNPs.

Transcending Dimensions in Apicomplexan Research: from Two-Dimensional to Three-Dimensional In Vitro Cultures.

Parasites belonging to the Apicomplexa phylum are among the most successful pathogens known in nature. They can infect a wide range of hosts, often remain undetected by the immune system, and cause acute and chronic illness. In this phylum, we can find parasites of human and veterinary health relevance, such as Toxoplasma, Plasmodium, Cryptosporidium, and Eimeria. There are still many unknowns about the biology of these pathogens due to the ethical and practical issues of performing research in their natural hosts. Animal models are often difficult or nonexistent, and as a result, there are apicomplexan life cycle stages that have not been studied. One recent alternative has been the use of three-dimensional (3D) systems such as organoids, 3D scaffolds with different matrices, microfluidic devices, organs-on-a-chip, and other tissue culture models. These 3D systems have facilitated and expanded the research of apicomplexans, allowing us to explore life stages that were previously out of reach and experimental procedures that were practically impossible to perform in animal models. Human- and animal-derived 3D systems can be obtained from different organs, allowing us to model host-pathogen interactions for diagnostic methods and vaccine development, drug testing, exploratory biology, and other applications. In this review, we summarize the most recent advances in the use of 3D systems applied to apicomplexans. We show the wide array of strategies that have been successfully used so far and apply them to explore other organisms that have been less studied.

Proteomic characterization of the pellicle of Toxoplasma gondii.

Toxoplasma gondii is one of the most successful intracellular parasites in the world. The dynamic, adhesion, invasion, and even replication capabilities of Toxoplasma are based on dynamic machinery located in the pellicle, a three membrane complex that surrounds the parasite. Among the proteins that carry out these processes are inner membrane complex (IMC) proteins, gliding-associated proteins (GAP), diverse myosins, actin, tubulin, and SRS proteins. Despite the importance of the pellicle, the knowledge of its composition is limited. Broad protein identification from an enriched pellicle fraction was obtained by independent digestion with trypsin and chymotrypsin and quantified by mass spectrometry. By trypsin digestion, 548 proteins were identified, while by chymotrypsin digestion, additional 22 proteins were identified. Besides, a group of "sequences related to SAG1" proteins (SRS) were detected together with unidentified new proteins. From identified SRS proteins, SRS51 was chosen for analysis and modeling as its similarities with crystallized adhesion proteins, exhibiting the presence of a spatial groove that is apparently involved in adhesion and cell invasion. As SRS proteins have been reported to be involved in the activation of the host's immune response, further studies could consider them as targets in the design of vaccines or of drugs against Toxoplasma. SIGNIFICANCE: To date, the proteomic composition of the pellicle of Toxoplasma is unknown. Most proteins reported in Toxoplasma pellicle have been poorly studied, and many others remain unidentified. Herein, a group of new SRS proteins is described. Some SRS proteins previously described from pellicle fraction have adhesion properties to the host cell membrane, so their study would provide data related to invasion mechanism and to open possibilities for considering them as targets in the design of immunoprotective strategies or the design of new pharmacological treatments.

Toxoplasma gondii excreted/secreted proteases disrupt intercellular junction proteins in epithelial cell monolayers to facilitate tachyzoites paracellular migration.

Toxoplasma gondii shows high dissemination and migration properties across biological barriers infecting immunologically privileged organs. Toxoplasma uses different routes for dissemination; however, the mechanisms are not fully understood. Herein, we studied the effects of proteases present in excretion/secretion products (ESPs) of Toxoplasma on MDCK cell monolayers. Ultrastructural analysis showed that ESPs of Toxoplasma disrupt the intercellular junctions (IJ) of adjacent cells. The tight junction (TJ) proteins ZO-1, occludin, and claudin-1 suffered a progressive decrease in protein levels upon ESPs treatment. In addition, ESPs induced mislocalization of such TJ proteins, along with the adherent junction protein E-cadherin, and this was prevented by pre-treating the ESPs with protease inhibitors. Reorganisation of cytoskeleton proteins was also observed. Endocytosis inhibitors, Dyngo®-4a and Dynasore, impeded the modifications, suggesting that TJ proteins internalisation is triggered by the ESPs proteases hence contributing to the loss of IJ. The observed disruption in TJ proteins went in line with a decrease in the transepithelial electrical resistance of the monolayers, which was significantly blocked by pre-treating ESPs with metalloprotease and serine protease inhibitors. Moreover, exposure of cell monolayers to ESPs facilitated paracellular migration of tachyzoites. Our results demonstrate that Toxoplasma ESPs contain proteases that can disrupt the IJ of epithelial monolayers and this could facilitate the paracellular route for Toxoplasma tissue dissemination and migration.

Proteomic and structural characterization of self-assembled vesicles from excretion/secretion products of Toxoplasma gondii.

After the cell invasion, the parasite Toxoplasma gondii locates within a parasitophorous vacuole to proliferate. It continuously modifies the composition of the parasitophorous vacuole by the secretion of GRA and ROP proteins, some of which become inserted into the vacuole membrane, remain as soluble proteins or involved in the intravacuolar network. In this report, we analyze the excretion/secretion products and the vesicles released by extracellular tachyzoites, this structures were morphologically analyzed by electron microscopy and characterized by mass spectrometry. The structural analysis showed parasites secreting in vitro individual vesicles with similarities to ectosomes and exosomes and which characterized to self-assembly in vitro forming vesicle-tubular structures morphologically similar to the intravacuolar network from infected cells. The vesicle-tubular structures were recognized with antibodies against ROP2 and GRA2. In addition, analysis by Western blot evidenced proteins from the secretory organelles. A detailed proteomic analysis of exosomes, ectosomes and soluble proteins released in vitro is here reported. Presence of GRA proteins in secretions from resting extracellular parasites indicates that these molecules are not exclusively secreted within the parasitophorous vacuole of the infected cell as reported but they are constitutively excreted/secreted even in an extracellular condition. Data are available via ProteomeXchange with identifier PXD013767. SIGNIFICANCE: Extracellular tachyzoites constitutively secrete components that previously were considered be secreted only within the parasitophorous vacuole, suggesting that in the infected host these molecules are in direct interaction with cells and molecules of the host cell including those of the immune response.

Characterization of metalloproteases and serine proteases of Toxoplasma gondii tachyzoites and their effect on epithelial cells.

Toxoplasma gondii can infect all nucleated cells from warm-blooded organisms. After infection, Toxoplasma spreads throughout the body and migrates across biological barriers, such as the intestinal and blood-brain barriers, as well as the placenta in pregnant women. The mechanisms for parasite dissemination are still unknown; however, proteases could play a role as a virulence factor. The aim of this study was to detect and to characterize proteases in whole-cell extracts and in excretion/secretion products from tachyzoites of the RH strain isolated from infected mice. Both fractions were analyzed by gelatin and casein zymography and by azocasein degradation. The biochemical characterization of proteases included standardization of optimal conditions for their activation, such as pH, the presence of cofactors, and a reducing agent. In both fractions, we detected at least nine gelatin-degrading metalloproteases in the range of 50 to 290 kDa. The proteases present in the excretion/secretion products were found as soluble proteins and not associated with exosome-like vesicles or other secretory vesicles. Moreover, by using casein zymography, it was possible to detect three serine proteases. Exposure of MDCK cells to excretion/secretion products modified the organization of the cell monolayer, and this effect was reverted after washing thoroughly with PBS and inhibition by metalloprotease and serine protease inhibitors. Proteomic analysis of excretion/secretion products identified 19 proteases. These findings suggest that tachyzoites of a highly virulent strain of Toxoplasma use a battery of proteases to modify the epithelium, probably as a strategy to facilitate their tissue dissemination.

Mycophenolic acid induces differentiation of Toxoplasma gondii RH strain tachyzoites into bradyzoites and formation of cyst-like structure in vitro.

The biochemical and structural changes that occur during the conversion of Toxoplasma gondii tachyzoites to bradyzoites and the formation of tissue cyst are not well understood. Maintaining cells infected with T. gondii type II and III strains under stress conditions induces the tachyzoite-bradyzoite in vitro differentiation, along with the formation of cyst-like structures. However, due to the long exposure to such conditions required to induce the differentiation, the severe damages in the host cell and the low encystation frequency, it has been difficult to dissect in more detail these processes. Here, we successfully induced the in vitro formation of Toxoplasma cysts-like structures from tachyzoites of the type I RH strain by treating with mycophenolic acid, an inhibitor of the inosine monophosphate dehydrogenase. Mycophenolic acid is a drug widely used for HXGPRT positive selection of Toxoplasma mutant strains along with xanthine incubation in the culture medium; under such conditions, formation of tissue cysts has not been reported. We show that the exposure of extracellular tachyzoites to mycophenolic acid in absence of xanthine, followed by host cell invasion, triggered their differentiation into cyst-like structures. The differential expression of CST1, BAG1, and SAG1 molecules, as well as the structural modifications of infected cells, was characterized during the formation of cyst-like structures in vitro. These findings will allow the characterization of signaling pathways involved in tachyzoite to bradyzoite conversion and formation of tissue cysts.

A new type of quinoxalinone derivatives affects viability, invasion, and intracellular growth of Toxoplasma gondii tachyzoites in vitro.

Quinoxalinone derivatives, identified as VAM2 compounds (7-nitroquinoxalin-2-ones), were evaluated against Toxoplasma gondii tachyzoites of the RH strain. The VAM2 compounds were previously synthesized based on the design obtained from an in silico prediction with the software TOMOCOMD-CARDD. From the ten VAM2 drugs tested, several showed a deleterious effect on tachyzoites. However, VAM2-2 showed the highest toxoplasmicidal activity generating a remarkable decrease in tachyzoite viability (in about 91 %) and a minimal alteration in the host cell. An evident inhibition of host cell invasion by tachyzoites previously treated with VAM2-2 was observed in a dose-dependent manner. In addition, remarkable alterations were observed in the pellicle parasite, such as swelling, roughness, and blebbing. Toxoplasma motility was inhibited, and subpellicular cytoskeleton integrity was altered, inducing a release of its components to the soluble fraction. VAM2-2 showed a clear and specific deleterious effect on tachyzoites viability, structural integrity, and invasive capabilities with limited effects in host cells morphology and viability. VAM2-2 minimum inhibitory concentration (MIC50) was determined as 3.3 µM ± 1.8. Effects of quinoxalinone derivatives on T. gondii provide the basis for a future therapeutical alternative in the treatment of toxoplasmosis.