Immune response profiles from humans experimentally exposed to Phlebotomus duboscqi bites.

Introduction: Cutaneous leishmaniasis is a neglected vector-borne parasitic disease prevalent in 92 countries with approximately one million new infections annually. Interactions between vector saliva and the human host alter the response to infection and outcome of disease. Methods: To characterize the human immunological responses developed against saliva of Phlebotomus duboscqi, a Leishmania major (L. major) vector, we repeatedly exposed the arms of 14 healthy U.S volunteers to uninfected P. duboscqi bites. Blood was collected a week after each exposure and used to assess total IgG antibodies against the proteins of P. duboscqi salivary gland homogenate (SGH) and the levels of IFN-gamma and IL-10 from peripheral blood mononuclear cells (PBMCs) stimulated with SGH or recombinant sand fly proteins. We analyzed skin punch biopsies of the human volunteer arms from the insect bite site and control skin site after multiple P. duboscqi exposures (four volunteers) using immunohistochemical staining. Results: A variety of immediate insect bite skin reactions were observed. Late skin reactions to insect bites were characterized by macular hyperpigmentation and/or erythematous papules. Hematoxylin and eosin staining showed moderate mononuclear skin infiltrate with eosinophils in those challenged recently (within 2 months), eosinophils were not seen in biopsies with recall challenge (6 month post bites). An increase in plasma antigen-specific IgG responses to SGH was observed over time. Western Blot results showed strong plasma reactivity to five P. duboscqi salivary proteins. Importantly, volunteers developed a cellular immunity characterized by the secretion of IFN-gamma upon PBMC stimulation with P. duboscqi SGH and recombinant antigens. Discussion: Our results demonstrate that humans mounted a local and systemic immune response against P. duboscqi salivary proteins. Specifically, PduM02/SP15-like and PduM73/adenosine deaminase recombinant salivary proteins triggered a Th1 type immune response that might be considered in future development of a potential Leishmania vaccine.

Weighted gene co-expression network analysis reveals immune evasion related genes in Echinococcus granulosus sensu stricto.

Cystic echinococcosis (CE) is a zoonotic disease caused by the tapeworm Echinococcus granulosus sensu lato (s.l). In the intermediate host, this disease is characterized by the growth of cysts in viscera such as liver and lungs, inside of which the parasite develops to the next infective stage known as protoscoleces. There are records that the infected viscera affect the development and morphology of E. granulosus s.l. protoscolex in hosts such as buffalo or humans. However, the molecular mechanisms that drive these differences remains unknown. Weighted gene co-expression network analysis (WGCNA) using a set of RNAseq data obtained from E. granulosus sensu stricto (s.s.) protoscoleces found in liver and lung cysts reveals 34 modules in protoscoleces of liver origin, of which 12 have differential co-expression from protoscoleces of lung origin. Three of these twelve modules contain hub genes related to immune evasion: tegument antigen, tegumental protein, ubiquitin hydrolase isozyme L3, COP9 signalosome complex subunit 3, tetraspanin CD9 antigen, and the methyl-CpG-binding protein Mbd2. Also, two of the twelve modules contain only hypothetical proteins with unknown orthology, which means that there are a group of unknown function proteins co-expressed inside the protoscolex of liver CE cyst origin. This is the first evidence of gene expression differences in protoscoleces from CE cysts found in different viscera, with co-expression networks that are exclusive to protoscoleces from liver CE cyst samples. This should be considered in the control strategies of CE, as intermediate hosts can harbor CE cysts in liver, lungs, or both organs simultaneously.

Structural insights into a functional unit from an immunogenic mollusk hemocyanin.

Mollusk hemocyanins, among the largest known proteins, are used as immunostimulants in biomedical and clinical applications. The hemocyanin of the Chilean gastropod Concholepas concholepas (CCH) exhibits unique properties, which makes it safe and effective for human immunotherapy, as observed in animal models of bladder cancer and melanoma, and dendritical cell vaccine trials. Despite its potential, the structure and amino acid sequence of CCH remain unknown. This study reports two sequence fragments of CCH, representing three complete functional units (FUs). We also determined the high-resolution (1.5 Å) X-ray crystal structure of an "FU-g type" from the CCHB subunit. This structure enables in-depth analysis of chemical interactions at the copper-binding center and unveils an unusual, truncated N-glycosylation pattern. These features are linked to eliciting more robust immunological responses in animals, offering insights into CCH's enhanced immunostimulatory properties and opening new avenues for its potential applications in biomedical research and therapies.

A First Omics Data Integration Approach in Hass Avocados to Evaluate Rootstock-Scion Interactions: From Aerial and Root Plant Growth to Fruit Development.

Grafting, the careful selection of rootstocks and scions, has played a crucial role maintaining Chilean avocado fruit quality standards in a scenario in which climate change and drought-related issues have considerably decreased avocado fruit production in the last fifteen years. The historical use of seedling rootstocks in Chile has experienced a recent shift towards clonal rootstocks, driven by the potential to produce more consistent and predictable crops. This research aims to compare Hass avocado plants grafted on Mexicola seedling and Dusa® clonal rootstocks in a soilless and protected system using (i) a differential expression analysis of root and leaf samples and (ii) a fruit transcriptomic and metabolomic integration analysis to improve our understanding of rootstock-scion interaction and its impact on avocado tree performance and fruit quality. The results demonstrated that no significant transcriptomic and metabolomic differences were identified at fruit level in the ready-to-eat (RTE) stage for Hass avocado fruit from both rootstocks. However, Hass avocados grafted on the clonal rootstock showed greater aerial growth and slightly increased fruit size than the seedling rootstock due to the enrichment of cell wall-remodeling genes as revealed in leaves and fruit at harvest stage.

Disentangling detrimental sand fly-mite interactions in a closed laboratory sand fly colony: implications for vector-borne disease studies and guidelines for overcoming severe mite infestations.

BACKGROUND: Vector sand fly colonies are a critical component of studies aimed at improving the understanding of the neglected tropical disease leishmaniasis and alleviating its global impact. However, among laboratory-colonized arthropod vectors of infectious diseases, the labor-intensive nature of sand fly rearing coupled with the low number of colonies worldwide has generally discouraged the widespread use of sand flies in laboratory settings. Among the different factors associated with the low productivity of sand fly colonies, mite infestations are a significant factor. Sand fly colonies are prone to infestation by mites, and the physical interactions between sand flies and mites and metabolites have a negative impact on sand fly larval development. METHODS: Mites were collected from sand fly larval rearing pots and morphologically identified using taxonomic keys. Upon identification, they were photographed with a scanning electron microscope. Several mite control measures were adopted in two different laboratories, one at the Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases-National Institutes of Health (Rockville, MD, USA), and the other at the University of Calgary (Calgary, AB, Canada). RESULTS: The mite species associated with sand fly colonies in the two laboratories were morphologically identified as Tyrophagus sp. and Stratiolaelaps scimitus. While complete eradication of mites in sand fly colonies is considered unrealistic, drastically reducing their population has been associated with higher sand fly productivity. CONCLUSIONS: We report a case of detrimental interaction between sand flies and Tyrophagus sp. and S. scimitus in a closed laboratory sand fly colony, discuss their impact on sand fly production and provide guidelines for limiting the mite population size in a closed laboratory colony leading to improved sand fly yields.

A novel pectin methylesterase inhibitor, PMEI3, in common bean suggests a key role of pectin methylesterification in Pseudomonas resistance.

The mechanisms underlying susceptibility to and defense against Pseudomonas syringae (Pph) of the common bean (Phaseolus vulgaris) have not yet been clarified. To investigate these, 15-day-old plants of the variety Riñón were infected with Pph and the transcriptomic changes at 2 h and 9 h post-infection were analysed. RNA-seq analysis showed an up-regulation of genes involved in defense/signaling at 2 h, most of them being down-regulated at 9 h, suggesting that Pph inhibits the transcriptomic reprogramming of the plant. This trend was also observed in the modulation of 101 cell wall-related genes. Cell wall composition changes at early stages of Pph infection were associated with homogalacturonan methylation and the formation of egg boxes. Among the cell wall genes modulated, a pectin methylesterase inhibitor 3 (PvPMEI3) gene, closely related to AtPMEI3, was detected. PvPMEI3 protein was located in the apoplast and its pectin methylesterase inhibitory activity was demonstrated. PvPMEI3 seems to be a good candidate to play a key role in Pph infection, which was supported by analysis of an Arabidopsis pmei3 mutant, which showed susceptibility to Pph, in contrast to resistant Arabidopsis Col-0 plants. These results indicate a key role of the degree of pectin methylesterification in host resistance to Pph during the first steps of the attack.

Proteomic and Low-Polar Metabolite Profiling Reveal Unique Dynamics in Fatty Acid Metabolism during Flower and Berry Development of Table Grapes.

Grapevine development and ripening are complex processes that involve several biochemical pathways, including fatty acid and lipid metabolism. Fatty acids are essential components of lipids, which play crucial roles in fruit maturation and flavor development. However, the dynamics of fatty acid metabolism in grape flowers and berries are poorly understood. In this study, we present those dynamics and investigate the mechanisms of fatty acid homeostasis on 'Thompson Seedless' berries using metabolomic and proteomic analyses. Low-polar metabolite profiling indicated a higher abundance of fatty acids at the pre-flowering and pre-veraison stages. Proteomic analyses revealed that grape flowers and berries display unique profiles of proteins involved in fatty acid biosynthesis, triacylglycerol assembly, fatty acid ß-oxidation, and lipid signaling. These findings show, for the first time, that fatty acid metabolism also plays an important role in the development of non-oil-rich tissues, opening new perspectives about lipid function and its relation to berry quality.

Leishmania genetic exchange is mediated by IgM natural antibodies.

Host factors that mediate Leishmania genetic exchange are not well defined. Here we demonstrate that natural IgM (IgMn)1-4 antibodies mediate parasite genetic exchange by inducing the transient formation of a spherical parasite clump that promotes parasite fusion and hybrid formation. We establish that IgMn from Leishmania-free animals binds to the surface of Leishmania parasites to induce significant changes in the expression of parasite transcripts and proteins. Leishmania binding to IgMn is partially lost after glycosidase treatment, although parasite surface phosphoglycans, including lipophosphoglycan, are not required for IgMn-induced parasite clumping. Notably, the transient formation of parasite clumps is essential for Leishmania hybridization in vitro. In vivo, we observed a 12-fold increase in hybrid formation in sand flies provided a second blood meal containing IgMn compared with controls. Furthermore, the generation of recombinant progeny from mating hybrids and parental lines were only observed in sand flies provided with IgMn. Both in vitro and in vivo IgM-induced Leishmania crosses resulted in full genome hybrids that show equal patterns of biparental contribution. Leishmania co-option of a host natural antibody to facilitate mating in the insect vector establishes a new paradigm of parasite-host-vector interdependence that contributes to parasite diversity and fitness by promoting genetic exchange.

Optimizing tick artificial membrane feeding for Ixodes scapularis.

Artificial membrane feeding (AMF) is a powerful and versatile technique with a wide range of applications in the study of disease vectors species. Since its first description, AMF has been under constant optimization and standardization for different tick species and life stages. In the USA, Ixodes scapularis is the main vector of tick-borne zoonoses including the pathogens causing Lyme disease in humans and animals. Seeking to improve the overall fitness of I. scapularis adult females fed artificially, here, we have optimized the AMF technique, considerably enhancing attachment rate, engorgement success, egg laying, and egg hatching compared to those described in previous studies. Parameters such as the membrane thickness and the light/dark cycle to which the ticks were exposed were refined to more closely reflect the tick's natural behavior and life cycle. Additionally, ticks were fed on blood only, blood + ATP or blood + ATP + gentamicin. The artificial feeding of ticks on blood only was successful and generated a progeny capable of feeding naturally on a host, i.e., mice. Adding ATP as a feeding stimulant did not improve tick attachment or engorgement. Notably, the administration of gentamicin, an antibiotic commonly used in tick AMF to prevent microbial contamination, negatively impacted Rickettsia buchneri endosymbiont levels in the progeny of artificially fed ticks. In addition, gentamicin-fed ticks showed a reduction in oviposition success compared to ticks artificially fed on blood only, discouraging the use of antibiotics in AMF. Overall, our data suggest that the AMF of adult females on blood only, in association with the natural feeding of their progeny on mice, might be used as an integrated approach in tick rearing, eliminating the use of protected species under the Animal Welfare Act (AWA). Of note, although optimized for I. scapularis adult ticks, I. scapularis nymphs, other tick species, and sand flies could also be fed using the membrane described in this study, indicating that it might be a suitable alternative for the artificial feeding of a variety of hematophagous species.

Ontogenetically distinct neutrophils differ in function and transcriptional profile in zebrafish.

The current view of hematopoiesis considers leukocytes on a continuum with distinct developmental origins, and which exert non-overlapping functions. However, there is less known about the function and phenotype of ontogenetically distinct neutrophil populations. In this work, using a photoconvertible transgenic zebrafish line; Tg(mpx:Dendra2), we selectively label rostral blood island-derived and caudal hematopoietic tissue-derived neutrophils in vivo during steady state or upon injury. By comparing the migratory properties and single-cell expression profiles of both neutrophil populations at steady state we show that rostral neutrophils show higher csf3b expression and migration capacity than caudal neutrophils. Upon injury, both populations share a core transcriptional profile as well as subset-specific transcriptional signatures. Accordingly, both rostral and caudal neutrophils are recruited to the wound independently of their distance to the injury. While rostral neutrophils respond uniformly, caudal neutrophils respond heterogeneously. Collectively, our results reveal that co-existing neutrophils populations with ontogenically distinct origin display functional differences.

Identification of grapevine clones via high-throughput amplicon sequencing: a proof-of-concept study.

Wine cultivars are available to growers in multiple clonal selections with agronomic and enological differences. Phenotypic differences between clones originated from somatic mutations that accrued over thousands of asexual propagation cycles. Genetic diversity between grape cultivars remains unexplored, and tools to discriminate unequivocally clones have been lacking. This study aimed to uncover genetic variations among a group of clonal selections of 4 important Vitis vinifera cultivars: Cabernet sauvignon, Sauvignon blanc, Chardonnay, and Merlot, and use this information to develop genetic markers to discriminate the clones of these cultivars. We sequenced with short-read sequencing technology the genomes of 18 clones, including biological replicates for a total of 46 genomes. Sequences were aligned to their respective cultivar's reference genome for variant calling. We used reference genomes of Cabernet sauvignon, Chardonnay, and Merlot and developed a de novo genome assembly of Sauvignon blanc using long-read sequencing. On average, 4 million variants were detected for each clone, with 74.2% being single nucleotide variants and 25.8% being small insertions or deletions (InDel). The frequency of these variants was consistent across all clones. From these variants, we validated 46 clonal markers using high-throughput amplicon sequencing for 77.7% of the evaluated clones, most of them small InDel. These results represent an advance in grapevine genotyping strategies and will benefit the viticulture industry for the characterization and identification of the plant material.

Drosophila DAxud1 Has a Repressive Transcription Activity on Hsp70 and Other Heat Shock Genes.

Drosophila melanogaster DAxud1 is a transcription factor that belongs to the Cysteine Serine Rich Nuclear Protein (CSRNP) family, conserved in metazoans, with a transcriptional transactivation activity. According to previous studies, this protein promotes apoptosis and Wnt signaling-mediated neural crest differentiation in vertebrates. However, no analysis has been conducted to determine what other genes it might control, especially in connection with cell survival and apoptosis. To partly answer this question, this work analyzes the role of Drosophila DAxud1 using Targeted-DamID-seq (TaDa-seq), which allows whole genome screening to determine in which regions it is most frequently found. This analysis confirmed the presence of DAxud1 in groups of pro-apoptotic and Wnt pathway genes, as previously described; furthermore, stress resistance genes that coding heat shock protein (HSP) family genes were found as hsp70, hsp67, and hsp26. The enrichment of DAxud1 also identified a DNA-binding motif (AYATACATAYATA) that is frequently found in the promoters of these genes. Surprisingly, the following analyses demonstrated that DAxud1 exerts a repressive role on these genes, which are necessary for cell survival. This is coupled with the pro-apoptotic and cell cycle arrest roles of DAxud1, in which repression of hsp70 complements the maintenance of tissue homeostasis through cell survival modulation.

A First Insight into the Microbial and Viral Communities of Comau Fjord-A Unique Human-Impacted Ecosystem in Patagonia (42∘ S).

While progress has been made in surveying the oceans to understand microbial and viral communities, the coastal ocean and, specifically, estuarine waters, where the effects of anthropogenic activity are greatest, remain partially understudied. The coastal waters of Northern Patagonia are of interest since this region experiences high-density salmon farming as well as other disturbances such as maritime transport of humans and cargo. Here, we hypothesized that viral and microbial communities from the Comau Fjord would be distinct from those collected in global surveys yet would have the distinctive features of microbes from coastal and temperate regions. We further hypothesized that microbial communities will be functionally enriched in antibiotic resistance genes (ARGs) in general and in those related to salmon farming in particular. Here, the analysis of metagenomes and viromes obtained for three surface water sites showed that the structure of the microbial communities was distinct in comparison to global surveys such as the Tara Ocean, though their composition converges with that of cosmopolitan marine microbes belonging to Proteobacteria, Bacteroidetes, and Actinobacteria. Similarly, viral communities were also divergent in structure and composition but matched known viral members from North America and the southern oceans. Microbial communities were functionally enriched in ARGs dominated by beta-lactams and tetracyclines, bacitracin, and the group macrolide-lincosamide-streptogramin (MLS) but were not different from other communities from the South Atlantic, South Pacific, and Southern Oceans. Similarly, viral communities were characterized by exhibiting protein clusters similar to those described globally (Tara Oceans Virome); however, Comau Fjord viromes displayed up to 50% uniqueness in their protein content. Altogether, our results indicate that microbial and viral communities from the Comau Fjord are a reservoir of untapped diversity and that, given the increasing anthropogenic impacts in the region, they warrant further study, specifically regarding resilience and resistance against antimicrobials and hydrocarbons.

Leishmania major-Infected Phlebotomus duboscqi Sand Fly Bites Enhance Mast Cell Degranulation.

Leishmania parasites infect mammalian hosts through the bites of sand fly vectors. The response by mast cells (MC) to the parasite and vector-derived factors, delivered by sand fly bites, has not been characterized. We analyzed MC numbers and their mediators in BALB/c mice naturally infected in the ear with Leishmania major through the bite of the sand fly vector Phlebotomus duboscqi and compared them to non-infected sand fly bites. MC were found at the bite sites of infective and non-infected sand flies throughout 48 h, showing the release of granules with intense TNF-α, histamine, and tryptase staining. At 30 min and 48 h, the MC numbers were significantly higher (p < 0.001) in infected as compared to non-infected bites or controls. Neutrophil recruitment was intense during the first 6 h in the skin of infected and non-infected sand fly bites and decreased thereafter. An influx of neutrophils also occurred in lymph nodes, where a strong TNF-α stain was observed in mononuclear cells. Our data show that MC orchestrate an early inflammatory response after infected and non-infected sand fly bites, leading to neutrophilic recruitment, which potentially provides a safe passage for the parasite within the mammalian host.

Metabolite Profiling Reveals the Effect of Cold Storage on Primary Metabolism in Nectarine Varieties with Contrasting Mealiness.

Chilling injury is a physiological disorder caused by cold storage in peaches and nectarines. The main symptom of chilling injury is mealiness/wooliness, described as a lack of juice in fruit flesh. In this work, we studied two nectarine varieties (Andes Nec-2 and Andes Nec-3) with contrasting susceptibility to mealiness after cold storage. A non-targeted metabolomic analysis was conducted by GC-MS to understand if changes in metabolite abundance are associated with nectarine mealiness induced by cold storage. Multivariate analyses indicated that in unripe nectarines, cold storage promoted a higher accumulation of amino acids in both varieties. Interestingly, for ripe nectarines, cold storage induced an accumulation of fewer amino acids in both varieties and showed an increased abundance of sugars and organic acids. A pathway reconstruction of primary metabolism revealed that in ripe nectarines, cold storage disrupted metabolite abundance in sugar metabolism and the TCA cycle, leading to a differential accumulation of amino acids, organic acids, and sugars in mealy and juicy nectarines.

Computer-related ophthalmic syndrome in teachers of a University of the Province of Cañete.

Virtual education has impacted the vision of people during the coronavirus pandemic, as by spending more time on the computer, it compromises the eye health of the person causing long-term visual problems. So the objective of this investigation is to assess computer-related ophthalmic syndrome in teachers of a University of the Province of Cañete. Methods: This is a quantitative, nonexperimental, descriptive, cross-sectional study on a total population of 63 teachers, who answered a digital survey using the sociodemographic data and the Computer Vision Syndrome Questionnaire. Clinical Discussion: From the results it can be observed that the results of computer ophthalmic syndrome in the university teachers of the province of Cañete, where 51 (81%) of the teachers do not present the computer vision syndrome and 12 (19%) presented with the computer vision syndrome. Conclusion: The population conducting virtual education as well as the students should be educated on the measures to be taken to prevent computer ophthalmic syndrome and its consequences.

Proteomic and metabolomic integration reveals the effects of pre-flowering cytokinin applications on central carbon metabolism in table grape berries.

Consumers around the world prefer high quality table grapes. To achieve higher quality traits at ripening, grapevine producers apply different plant growth regulators. The synthetic cytokinin forchlorfenuron N-(2-chloro-4-pyridinyl)-N'-phenylurea (CPPU) is widely used, its effect on grape quality is poorly understood. We hypothesized that the use of CPPU in pre-flowering can lead to changes in the metabolism that affects grape quality at harvest. Therefore, we investigated the role of CPPU applications on the quality of grapes by integrating proteomics and metabolomics. CPPU-treated grapevines showed a significant increase in berry size and firmness. Proteomic analyses indicated that CPPU-treated berries accumulated enzymes associated with carbohydrate metabolism, glycolysis, and tricarboxylic acid (TCA) cycle at harvest. Metabolomic analyses showed shifts in the abundance of compounds associated with carbohydrate metabolism and TCA cycle in CPPU-treated grapes. These findings suggest that CPPU applications modulate central carbon metabolism, improving grape berry quality.

A multiomics integrative analysis of color de-synchronization with softening of 'Hass' avocado fruit: A first insight into a complex physiological disorder.

Exocarp color de-synchronization with softening of 'Hass' avocado is a relevant recurrent problem for the avocado supply chain. This study aimed to unravel the mechanisms driving this de-synchronization integrating omics datasets from avocado exocarp of different storage conditions and color phenotypes. In addition, we propose potential biomarkers to predict color synchronized/de-synchronized fruit. Integration of transcriptomics, proteomics and metabolomics and network analysis revealed eight transcription factors associated with differentially regulated genes between regular air (RA) and controlled atmosphere (CA) and twelve transcription factors related to avocado fruit color de-synchronization control in ready-to-eat stage. CA was positively correlated to auxins, ethylene, cytokinins and brassinosteroids-related genes, while RA was characterized by enrichment of cell wall remodeling and abscisic acid content associated genes. At ready-to-eat higher contents of flavonoids, abscisic acid and brassinosteroids were associated with color-softening synchronized avocados. In contrast, de-synchronized fruit revealed increases of jasmonic acid, salicylic acid and auxin levels.

Transcriptome and Gene Regulatory Network Analyses Reveal New Transcription Factors in Mature Fruit Associated with Harvest Date in Prunus persica.

Harvest date is a critical parameter for producers and consumers regarding agro-industrial performance. It involves a pleiotropic effect controlling the development of other fruit quality traits through finely controlling regulatory mechanisms. Fruit ripening is a process in which various signals and biological events co-occur and are regulated by hormone signaling that produces the accumulation/degradation of multiple compounds. However, the regulatory mechanisms that control the hormone signaling involved in fruit development and ripening are still unclear. To investigate the issue, we used individuals with early, middle and late harvest dates from a peach segregating population to identify regulatory candidate genes controlling fruit quality traits at the harvest stage and validate them in contrasting peach varieties for this trait. We identified 467 and 654 differentially expressed genes for early and late harvest through a transcriptomic approach. In addition, using the Arabidopsis DAP-seq database and network analysis, six transcription factors were selected. Our results suggest significant hormonal balance and cell wall composition/structure differences between early and late harvest samples. Thus, we propose that higher expression levels of the transcription factors HB7, ERF017 and WRKY70 in early harvest individuals would induce the expression of genes associated with the jasmonic acid pathway, photosynthesis and gibberellins inhibition. While on the other hand, the high expression levels of LHY, CDF3 and NAC083 in late harvest individuals would promote the induction of genes associated with abscisic acid biosynthesis, auxins and cell wall remodeling.