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Wild animals host a wide variety of parasites, and the disorders caused by these parasites are among the most prevalent and significant infectious diseases affecting wildlife. The investigation of parasitic fauna is crucial for the conservation of wild animals, domestic populations, and humans. Therefore, the aim of the study was to survey endo- and ectoparasites in free-living wildlife in southern Brazil. Carcasses of road-killed wildlife were collected from highways in the Pelotas microregion, Rio Grande do Sul. All carcasses were necropsied, and endo- and ectoparasites were collected and identified. A total of 82 animals were examined; 87.80% were infected by helminths, 51.21% infested by ectoparasites, and 48.78% were affected by both types of parasites. This study presents the first records of parasitism by Rhipicephalus microplus in Ozotoceros bezoarticus in southern Brazil, and by Ancylostoma caninum in Leopardus geoffroyi worldwide. The study contributes to the knowledge about the parasitic fauna in wild animals of the Pampa biome. The presence of parasites in these threatened species underscores the need for further research into parasitism, which is crucial for their effective management and conservation., Competing Interests: Declarations Ethics approval The animals in this study are part of a scheduled research project on protozoa in wild animals from southern Brazil (Cobalto/UFPel registration 5604). The collection and transportation of carcasses of wild animals killed by vehicles were authorized by the Biodiversity Authorization and Information System of the Ministry of the Environment under registration 82632-3, based on Normative Instruction number 03/2014. This work does not require approval from the Ethics Committee on Animal Use of UFPel (process number 23110.046990/2022-02). Competing interests The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Patients with peripheral artery disease and generalized atherosclerosis are at high risk of cardiovascular and limb complications, affecting both quality of life and longevity. Lower limb atherosclerotic disease is associated with high cardiovascular morbidity and mortality and adequate management is founded on treatments involving patient-dependent factors, such as lifestyle changes, and physician-dependent factors, such as clinical treatment, endovascular treatment, or conventional surgery. Medical management of peripheral artery disease is multifaceted, and its most important elements are reduction of cholesterol level, antithrombotic therapy, control of arterial blood pressure, control of diabetes, and smoking cessation. Adhesion to this regime can reduce complications related to the limbs, such as chronic limb-threatening ischemia, that can result in amputation, and the systemic complications of atherosclerosis, such as stroke and myocardial infarction., Competing Interests: Conflicts of interest: No conflicts of interest declared concerning the publication of this article., (Copyright© 2024 The authors.)

Phalacrocorax brasilianus (Gmelin, 1789) (Aves: Phalacrocoracidae) is one of the few piscivorous birds inhabiting freshwater and saline environments, being considered one of the most abundant aquatic species in Rio Grande do Sul, Brazil, especially along the state's coastline. It is known that birds are hosts to a wide variety of disease-causing agents, among them, nematodes of the Contracaecum (Anisakidae) have a large number of recognized species. However, little is still known about the occurrence of these parasites in the Southern region of Brazil. Herein we identified for the first time Contracaecum australe Garbin, Mattiucci, Paoletti, González-Acuña, and Nascetti, 2011 (Nematoda: Anisakidae) parasitizing P. brasilianus in Southern Brazil. Nematodes found in the bird's proventriculus were subjected to morphometric analyses, by optical microscopy and scanning electron microscopy, and molecular analyses. Molecular phylogeny based on the analysis of the 18S, ITS-1, 5.8S and ITS-2 genes showed our sequences identical to those of C. australe. Therefore, this is the first record of C. australe in southern Brazil, expanding the geographical distribution of the parasite species in the country. Additionally, new molecular sequences are being provided, contributing to the knowledge of Contracaecum species parasitizing cormorants., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Leopardus geoffroyi (Geoffroy's cat) is a neotropical feline considered globally threatened. In Brazil, it occurs exclusively in the Pampa biome. Its predatory habits contribute to the infection, dispersion, and continuation of the life cycle of various pathogens, including helminths, within ecosystems. However, few studies involving cestodes in wild felines are found in the literature, especially in Brazil. Therefore, we aimed to report the first case of parasitism by Hydatigera taeniaeformis in L. geoffroyi. The helminths were found in the small intestine of the necropsied feline. Specimens were analyzed morphometrically and subjected to molecular analyses for taxonomic identification. The molecular phylogeny based on the analysis of the mitochondrial gene (COX1) allowed the identification of these parasites. Thus, this is the first description of H. taeniaeformis parasitizing L. geoffroyi in Brazil. Consequently, the number of known host species parasitized by this helminth in the country and the world is increased. Additionally, a new molecular sequence is being provided, contributing to the knowledge of Hydatigera in South America., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest., (Copyright © 2024. Published by Elsevier B.V.)

Sheep farming has been growing in Brazil, driven by an expanding consumer market due to greater acceptance of its meat and derivatives. There are several factors that limit sheep production, and one of them is infestation by ectoparasites, which cause stress in animals, weight loss, poor development, low productivity, low quality wool and reduced fertility. Chrysomya albiceps is a species of blowfly belonging to the Calliphoridae family that occurs in neotropical regions, where it causes secondary myiasis. We identified here a rare case of cutaneous myiasis with the presence of tissue lesions caused by C. albiceps in sheep in southern Brazil. We highlight the need to carry out more in-depth studies regarding the biology of these insects, with the aim of proving this atypical behavior for Brazil., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Leishmaniasis are neglected diseases transmitted by vectors that affect domestic and wild animals, including humans. Due to its incidence and lethality, this zoonosis is a worrying public health problem, making it essential to identify all links in the transmission chain. Infection of wild mammals by Leishmania spp. remains poorly understood, especially in southern Brazil. Therefore, the objective was to research, using the PCR technique, the presence of Leishmania spp. DNA in road-killed wild mammals in Southern Brazil. Carcasses of 96 animals were collected from highways in the Pelotas microregion, Rio Grande do Sul, southern Brazil and subjected to necropsies. Tissue fragments (spleen, skin, liver, kidney, heart, lung, lymph nodes, bone marrow and blood) were collected and genomic DNA was extracted. PCR protocols targeting the ITS1, kDNA and 18S genes were tested. We found no evidence of Leishmania spp. circulation in the studied population. However, epidemiological studies like this one are of great relevance, as they allow monitoring of the occurrence of pathogens and help identify possible risk areas. As these animals act as epidemiological markers for the presence of the microorganism, studies must be carried out continuously to understand whether there are sources of infection in the region.

Chimeric antigen receptor (CAR) T cell therapies targeting B cell-restricted antigens CD19, CD20, or CD22 can produce potent clinical responses for some B cell malignancies, but relapse remains common. Camelid single-domain antibodies (sdAbs or nanobodies) are smaller, simpler, and easier to recombine than single-chain variable fragments (scFvs) used in most CARs, but fewer sdAb-CARs have been reported. Thus, we sought to identify a therapeutically active sdAb-CAR targeting human CD22. Immunization of an adult Llama glama with CD22 protein, sdAb-cDNA library construction, and phage panning yielded >20 sdAbs with diverse epitope and binding properties. Expressing CD22-sdAb-CAR in Jurkat cells drove varying CD22-specific reactivity not correlated with antibody affinity. Changing CD28- to CD8-transmembrane design increased CAR persistence and expression in vitro . CD22-sdAb-CAR candidates showed similar CD22-dependent CAR-T expansion in vitro, although only membrane-proximal epitope targeting CD22-sdAb-CARs activated direct cytolytic killing and extended survival in a lymphoma xenograft model. Based on enhanced survival in blinded xenograft studies, a lead CD22sdCAR-T was selected, achieving comparable complete responses to a benchmark short linker m971-scFv CAR-T in high-dose experiments. Finally, immunohistochemistry and flow cytometry confirm tissue and cellular-level specificity of the lead CD22-sdAb. This presents a complete report on preclinical development of a novel CD22sdCAR therapeutic., Competing Interests: CD22-nanobody binding elements used in this work were disclosed in provisional patent filing US 2023/0265185 A1 and other provisional filings by the NRC. The authors declare no competing interests., (Crown Copyright © 2024.)

Metabolomics is the study of small molecules, primarily metabolites, that are produced during metabolic processes. Analysis of the composition of an organism's metabolome can yield useful information about an individual's health status at any given time. In recent years, the development of large-scale, targeted metabolomic methods has allowed for the analysis of biological samples using analytical techniques such as LC-MS/MS. This paper presents a large-scale metabolomics method for analysis of biological samples, with a focus on quantification of metabolites found in blood plasma. The method comprises a 10-min chromatographic separation using HILIC and RP stationary phases combined with positive and negative electrospray ionization in order to maximize metabolome coverage. Complete analysis of a single sample can be achieved in as little as 40 min using the two columns and dual modes of ionization. With 540 metabolites and the inclusion of over 200 analytical standards, this method is comprehensive and quantitatively robust when compared to current targeted metabolomics methods. This study uses a large-scale evaluation of metabolite recovery from plasma that enables absolute quantification of metabolites by correcting for analyte loss throughout processes such as extraction, handling, or storage. In addition, the method was applied to plasma collected from adjuvant breast cancer patients to confirm the suitability of the method to clinical samples., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Cutaneous melanoma (CM) patients respond better to immune checkpoint inhibitors (ICI) than mucosal and uveal melanoma patients (MM/UM). Aiming to explore these differences and understand the distinct response to ICI, we evaluated the serum metabolome of advanced CM, MM, and UM patients. Levels of 115 metabolites were analyzed in samples collected before ICI, using a targeted metabolomics platform. In our analysis, molecules involved in the tryptophan-kynurenine axis distinguished UM/MM from CM. UM/MM patients had higher levels of 3-hydroxykynurenine (3-HKyn), whilst patients with CM were found to have higher levels of kynurenic acid (KA). The KA/3-HKyn ratio was significantly higher in CM versus the other subtypes. UM, the most ICI-resistant subtype, was also associated with higher levels of sphingomyelin-d18:1/22:1 and the polyamine spermine (SPM). Overall survival was prolonged in a cohort of CM patients with lower SPM levels, suggesting there are also conserved metabolic factors promoting ICI resistance across melanoma subtypes. Our study revealed a distinct metabolomic profile between the most resistant melanoma subtypes, UM and MM, compared to CM. Alterations within the kynurenine pathway, polyamine metabolism, and sphingolipid metabolic pathway may contribute to the poor response to ICI. Understanding the different metabolomic profiles introduces opportunities for novel therapies with potential synergic activity to ICI, to improve responses of UM/MM.

Osteopontin (OPN) is a secreted glycophosphoprotein that derives its name from its high abundance in bone and secretion by osteoblasts. It is also secreted by a number of immune cells and, therefore, is present in human plasma at nanogram per millilitre levels where it affects cell adhesion and motility. OPN is involved in several normal physiological processes; however, OPN dyregulation leads to overexpression by tumor cells leading to immune evasion and increased metastasis. Plasma OPN is primarily measured by enzyme-linked immunosorbent assay (ELISA). However, due to the complexity of the various OPN isoforms, conflicting results have been obtained on the use of OPN as a biomarker even in the same disease condition. These discrepant results may result from the difficulty in comparing ELISA results obtained with different antibodies that target unique OPN epitopes. Mass spectrometry can be used to quantify proteins in plasma and, by targeting OPN regions that do not bear post-translational modifications, may provide more consistent quantification. However, the low (ng/mL) levels in plasma present a significant analytical challenge. In order to develop a sensitive assay for plasma OPN, we explored a single-step precipitation method using a recently developed spin-tube format. Quantification was performed using isotope-dilution mass spectrometry. The concentration detection limit of this assay was 39 ± 15 ng/mL. The assay was applied to the analysis of plasma OPN in metastatic breast cancer patients, where levels from 17 to 53 ng/mL were detected. The sensitivity of the method is higher than previously published methods and sufficient for OPN detection in large, high grade tumors but still requires improvement in sensitivity to be widely applicable., Competing Interests: The authors have declared no competing interests exist., (Copyright: © 2023 Leslie et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

While post-transcriptional control is thought to be required at the periphery of neurons and glia, its extent is unclear. Here, we investigate systematically the spatial distribution and expression of mRNA at single molecule sensitivity and their corresponding proteins of 200 YFP trap lines across the intact Drosophila nervous system. 97.5% of the genes studied showed discordance between the distribution of mRNA and the proteins they encode in at least one region of the nervous system. These data suggest that post-transcriptional regulation is very common, helping to explain the complexity of the nervous system. We also discovered that 68.5% of these genes have transcripts present at the periphery of neurons, with 9.5% at the glial periphery. Peripheral transcripts include many potential new regulators of neurons, glia, and their interactions. Our approach is applicable to most genes and tissues and includes powerful novel data annotation and visualization tools for post-transcriptional regulation., (© 2023 Titlow et al.)

Background and Aims: Nutritional therapy with the Crohn's Disease Exclusion Diet + Partial Enteral Nutrition [CDED+PEN] or Exclusive Enteral Nutrition [EEN] induces remission and reduces inflammation in mild-to-moderate paediatric Crohn's disease [CD]. We aimed to assess if reaching remission with nutritional therapy is mediated by correcting compositional or functional dysbiosis., Methods: We assessed metagenome sequences, short chain fatty acids [SCFA] and bile acids [BA] in 54 paediatric CD patients reaching remission after nutritional therapy [with CDED + PEN or EEN] [NCT01728870], compared to 26 paediatric healthy controls., Results: Successful dietary therapy decreased the relative abundance of Proteobacteria and increased Firmicutes towards healthy controls. CD patients possessed a mixture of two metabotypes [M1 and M2], whereas all healthy controls had metabotype M1. M1 was characterised by high Bacteroidetes and Firmicutes, low Proteobacteria, and higher SCFA synthesis pathways, and M2 was associated with high Proteobacteria and genes involved in SCFA degradation. M1 contribution increased during diet: 48%, 63%, up to 74% [Weeks 0, 6, 12, respectively.]. By Week 12, genera from Proteobacteria reached relative abundance levels of healthy controls with the exception of E. coli. Despite an increase in SCFA synthesis pathways, remission was not associated with increased SCFAs. Primary BA decreased with EEN but not with CDED+PEN, and secondary BA did not change during diet., Conclusion: Successful dietary therapy induced correction of both compositional and functional dysbiosis. However, 12 weeks of diet was not enough to achieve complete correction of dysbiosis. Our data suggests that composition and metabotype are important and change quickly during the early clinical response to dietary intervention. Correction of dysbiosis may therefore be an important future treatment goal for CD., (© The Author(s) 2022. Published by Oxford University Press on behalf of European Crohn’s and Colitis Organisation.)

Culicoides have medical and veterinary importance, as they play a role as vectors of viruses, protozoa, and nematodes that cause diseases. Despite the relevance to public health, greater attention has been given to other insect vectors. Therefore, the objective of this study was to evaluate the efficiency of the Captor ® light trap in capturing Culicoides that could be examined for the presence of Leishmania DNA. The insects were captured in a rural area of Santa Maria, Rio Grande do Sul, Brazil, where canine and human visceral leishmaniasis have been diagnosed. Adult insects were collected weekly, from 6:00 pm to 6:00 am, for a 12 month period using a Captor ® brand suction light trap. All Culicoides were identified at species level. Pools of Culicoides were tested using the Polymerase Chain Reaction (PCR) technique for the detection of Leishmania DNA. A total of 16,016 specimens were collected (71.54% females and 28.39% males), divided among seven species. In the DNA analysis, none of the pools showed a positive result for Leishmania . The data presented demonstrate that the trap is efficient and can be an alternative for use in entomological research. They also demonstrate that, despite the females having hematophagous habits, similar to other vector insects, they did not have contact with Leishmania in the studied locality., Competing Interests: Conflicts of interest: JSL, DMP and SGM – The authors declare that they have no conflict of interest., (Copyright Lignon et al.)

Background & Aims: The Crohn's disease (CD) exclusion diet (CDED) plus partial enteral nutrition (PEN) and exclusive enteral nutrition (EEN) both induce remission in pediatric CD. CDED+PEN is better tolerated and able to sustain remission. We characterized the changes in fecal metabolites induced by CDED+PEN and EEN and their relationship with remission., Methods: A total of 216 fecal metabolites were measured in 80 fecal samples at week (W) 0, W6, and W12, of children with mild to moderate CD in a prospective randomized trial comparing CDED+PEN vs EEN. The metabolites were measured using liquid chromatography coupled to mass spectrometry. Metagenome Kyoto Encyclopedia of Genes and Genomes Orthology analysis was performed to investigate the differential functional gene abundance involved in specific metabolic pathways. Data were analyzed according to clinical outcome of remission (W6_rem), no remission (W6_nr), sustained remission (W12_sr), and nonsustained (W12_nsr) remission., Results: A decrease in kynurenine and succinate synthesis and an increase in N-α-acetyl-arginine characterized CDED+PEN W6_rem, whereas changes in lipid metabolism characterized EEN W6_rem, especially reflected by lower levels in ceramides. In contrast, fecal metabolites in EEN W6_nr were comparable to baseline/W0 samples. CDED+PEN W6_rem children maintained metabolome changes through W12. In contrast, W12_nsr children in the EEN group, who resumed a free diet after week 6, did not. The metabolome of CDED+PEN differed from EEN in the purine, pyrimidine, and sphingolipid pathways. A significant differential abundance in several genes involved in these pathways was detected., Conclusion: CDED+PEN- and EEN-induced remission are associated with significant changes in inflammatory bowel disease-associated metabolites such as kynurenine, ceramides, amino acids, and others. Sustained remission with CDED+PEN, but not EEN, was associated with persistent changes in metabolites., Clinicaltrials: gov, Number NCT01728870., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The aryl hydrocarbon receptor (AhR) is a sensor of products of tryptophan metabolism and a potent modulator of immunity. Here, we examined the impact of AhR in tumor-associated macrophage (TAM) function in pancreatic ductal adenocarcinoma (PDAC). TAMs exhibited high AhR activity and Ahr-deficient macrophages developed an inflammatory phenotype. Deletion of Ahr in myeloid cells or pharmacologic inhibition of AhR reduced PDAC growth, improved efficacy of immune checkpoint blockade, and increased intra-tumoral frequencies of IFNγ + CD8 + T cells. Macrophage tryptophan metabolism was not required for this effect. Rather, macrophage AhR activity was dependent on Lactobacillus metabolization of dietary tryptophan to indoles. Removal of dietary tryptophan reduced TAM AhR activity and promoted intra-tumoral accumulation of TNFα + IFNγ + CD8 + T cells; provision of dietary indoles blocked this effect. In patients with PDAC, high AHR expression associated with rapid disease progression and mortality, as well as with an immune-suppressive TAM phenotype, suggesting conservation of this regulatory axis in human disease., Competing Interests: Declaration of interests The authors have no conflicting interests to declare., (Copyright © 2022 Elsevier Inc. All rights reserved.)

We have developed "Microscope-Cockpit" (Cockpit), a highly adaptable open source user-friendly Python-based Graphical User Interface (GUI) environment for precision control of both simple and elaborate bespoke microscope systems. The user environment allows next-generation near instantaneous navigation of the entire slide landscape for efficient selection of specimens of interest and automated acquisition without the use of eyepieces. Cockpit uses "Python-Microscope" (Microscope) for high-performance coordinated control of a wide range of hardware devices using open source software. Microscope also controls complex hardware devices such as deformable mirrors for aberration correction and spatial light modulators for structured illumination via abstracted device models. We demonstrate the advantages of the Cockpit platform using several bespoke microscopes, including a simple widefield system and a complex system with adaptive optics and structured illumination. A key strength of Cockpit is its use of Python, which means that any microscope built with Cockpit is ready for future customisation by simply adding new libraries, for example machine learning algorithms to enable automated microscopy decision making while imaging., Competing Interests: Competing interests: Martin Booth declares a significant interest in Aurox Ltd., whose microscopes were used in this work., (Copyright: © 2022 Phillips MA et al.)

Introduction: Aldehyde dehydrogenase 1A3 (ALDH1A3) is a cancer stem cell (CSC) marker and in breast cancer it is associated with triple-negative/basal-like subtypes and aggressive disease. Studies on the mechanisms of ALDH1A3 in cancer have primarily focused on gene expression changes induced by the enzyme; however, its effects on metabolism have thus far been unstudied and may reveal novel mechanisms of pathogenesis., Objective: Determine how ALDH1A3 alters the metabolite profile in breast cancer cells and assess potential impacts., Method: Triple-negative MDA-MB-231 tumors and cells with manipulated ALDH1A3 levels were assessed by HPLC-MS metabolomics and metabolite data was integrated with transcriptome data. Mice harboring MDA-MB-231 tumors with or without altered ALDH1A3 expression were treated with γ-aminobutyric acid (GABA) or placebo. Effects on tumor growth, and lungs and brain metastasis were quantified by staining of fixed thin sections and quantitative PCR. Breast cancer patient datasets from TCGA, METABRIC and GEO were used to assess the co-expression of GABA pathway genes with ALDH1A3., Results: Integrated metabolomic and transcriptome data identified GABA metabolism as a primary dysregulated pathway in ALDH1A3 expressing breast tumors. Both ALDH1A3 and GABA treatment enhanced metastasis. Patient dataset analyses revealed expression association between ALDH1A3 and GABA pathway genes and corresponding increased risk of metastasis., Conclusion: This study revealed a novel pathway affected by ALDH1A3, GABA metabolism. Like ALDH1A3 expression, GABA treatment promotes metastasis. Given the clinical use of GABA mimics to relieve chemotherapy-induced peripheral nerve pain, further study of the effects of GABA in breast cancer progression is warranted., (© 2022. The Author(s).)

Comparing cancer proteomes across many samples offers a window into cancer cell biology and may reveal new treatment options for specific subsets of cancer. Here we describe a method using tandem mass tag (TMT) technology to multiplex up to 18 samples in a single analysis, paving the way for the analysis of large cohorts of tumors, cell lines, and perturbations thereof. The procedure we describe will result in samples ready for in-depth LC-MS/MS analysis in 3-4 days., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: The management of locally advanced rectal cancer (LARC) requires a multidisciplinary approach, with an increasing interest for non-operative strategies. Liquid biopsy for obtaining circulating tumor DNA (ctDNA) can provide information on neoadjuvant chemoradiotherapy (nCRT) pathological response and cancer-specific prognosis, and therefore might be a promising guide for these treatments., Methods: A systematic review of the studies available in literature has been performed to assess the role of ctDNA as a predictive and prognostic biomarker in LARC patients., Results: We retrieved 21 publications, of which 17 full-text articles and 4 abstracts. Results have been labelled into two groups: predictive and prognostic. Data about the usefulness of liquid biopsy in this setting is still inconclusive. However, baseline higher levels of longer fragments of cell-free DNA and integrity index, tumor-specific mutations and certain methylated genes could predict non-responders. Also, undetectable baseline ctDNA and decrease of common rectal cancer mutations throughout treatment (dynamic monitoring) were predictive factors of pathological complete response. The continuous detection of ctDNA in different timepoints of treatment (minimal residual disease) was consistently associated with worse prognosis., Conclusions: ctDNA is a promising biomarker that could assist predicting treatment response to nCRT and prognosis in patients with LARC. The ideal methods and timings for the liquid biopsy still have to be defined., (Copyright © 2021 Elsevier Ltd, BASO ~ The Association for Cancer Surgery, and the European Society of Surgical Oncology. All rights reserved.)

Metabolic programming is intricately linked to the anti-tumor properties of T cells. To study the metabolic pathways associated with increased anti-tumor T cell function, we utilized a metabolomics approach to characterize three different CD8 + T cell subsets with varying degrees of anti-tumor activity in murine models, of which IL-22-producing Tc22 cells displayed the most robust anti-tumor activity. Tc22s demonstrated upregulation of the pantothenate/coenzyme A (CoA) pathway and a requirement for oxidative phosphorylation (OXPHOS) for differentiation. Exogenous administration of CoA reprogrammed T cells to increase OXPHOS and adopt the CD8 + Tc22 phenotype independent of polarizing conditions via the transcription factors HIF-1α and the aryl hydrocarbon receptor (AhR). In murine tumor models, treatment of mice with the CoA precursor pantothenate enhanced the efficacy of anti-PDL1 antibody therapy. In patients with melanoma, pre-treatment plasma pantothenic acid levels were positively correlated with the response to anti-PD1 therapy. Collectively, our data demonstrate that pantothenate and its metabolite CoA drive T cell polarization, bioenergetics, and anti-tumor immunity., Competing Interests: Declaration of interests M.S.P., S.D.S., and P.S.O. have filed for a patent pertaining to this work., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Custom-built microscopes often require control of multiple hardware devices and precise hardware coordination. It is also desirable to have a solution that is scalable to complex systems and that is translatable between components from different manufacturers. Here we report Python-Microscope, a free and open-source Python library for high-performance control of arbitrarily complex and scalable custom microscope systems. Python-Microscope offers simple to use Python-based tools, abstracting differences between physical devices by providing a defined interface for different device types. Concrete implementations are provided for a range of specific hardware, and a framework exists for further expansion. Python-Microscope supports the distribution of devices over multiple computers while maintaining synchronisation via highly precise hardware triggers. We discuss the architectural features of Python-Microscope that overcome the performance problems often raised against Python and demonstrate the different use cases that drove its design: integration with user-facing projects, namely the Microscope-Cockpit project; control of complex microscopes at high speed while using the Python programming language; and use as a microscope simulation tool for software development., Competing Interests: Competing interests M.J.B. declares a significant interest in Aurox Ltd, whose microscopes were used in this work., (© 2021. Published by The Company of Biologists Ltd.)

The cervical rib syndrome occurs when the interscalene triangle is occupied by a cervical rib, displacing the brachial plexus and the subclavian artery forward, which can cause pain and muscle spasms. The objective of this study is to discuss diagnosis of the cervical rib syndrome and treatment possibilities. This therapeutic challenge describes clinical and surgical management of a 37-year-old female patient with upper limb arterial occlusion caused by a cervical rib., Competing Interests: Conflicts of interest: No conflicts of interest declared concerning the publication of this article., (Copyright© 2021 The authors.)

Background: Patients with advanced chronic venous disease are more likely to need additional procedures for relapsed varicose veins. It has not yet been established whether severity of venous insufficiency is a factor that influences the occlusion rate of saphenous veins treated with endolasers., Objectives: To analyze occlusion rate of venous segments treated with endolaser and correlate it with patients' Venous Clinical Severity Score (VCSS) and Clinical-Etiological-Anatomical-Pathological (CEAP) classification., Methods: Retrospective analysis of a cohort of patients operated using a 1,470 nm endolaser from November 2012 to March 2020. Descriptive statistics were calculated and Kaplan-Meier survival curves were plotted with Cox regression for groups stratified by VCSS and CEAP., Results: A total of 180 venous segments were analyzed in 170 patients. Mean age was 44.3 ± 9.2 and the majority of patients were female (71%). Mean energy density used in the great saphenous vein was 49.2 ± 8.3 J/cm. The most common complications were pain along the course of the saphenous vein (12.2%) and paresthesias at 6 months (17.2%). There was no difference in venous occlusion rate between groups with VCSS ≤ 7 and VCSS > 7 (p = 0.067). A group of patients classified as CEAP classes C4, C5, or C6 had a lower occlusion rate than a group at classes C2 or C3 (hazard ratio [HR] = 3.22; confidence interval [CI] 1.85, 5.61; p = 0.001]., Conclusions: The occlusion rates of venous segments treated with endolaser were lower in patients with higher CEAP classes. It is probably necessary to use more energy in these patients to achieve effective treatment of saphenous veins., Competing Interests: Conflicts of interest: No conflicts of interest declared concerning the publication of this article., (Copyright© 2021 The authors.)

The isolation and analysis of circulating tumor cells (CTC) has the potential to provide minimally invasive diagnostic, prognostic and predictive information. Widespread clinical implementation of CTC analysis has been hampered by a lack of comparative investigation between different analytic methodologies in clinically relevant settings. The objective of this study was to evaluate four different CTC isolation techniques-those that rely on surface antigen expression (EpCAM or CD45 using DynaBeads® or EasySep™ systems) or the biophysical properties (RosetteSep™ or ScreenCell®) of CTCs. These were evaluated using cultured cells in order to calculate isolation efficiency at various levels including; inter-assay and inter-operator variability, protocol complexity and turn-around time. All four techniques were adequate at levels above 100 cells/mL which is commonly used for the evaluation of new isolation techniques. Only the RosetteSep™ and ScreenCell® techniques were found to provide adequate sensitivity at a level of 10 cells/mL. These techniques were then applied to the isolation and analysis of circulating tumor cells blood drawn from metastatic breast cancer patients where CTCs were detected in 54% (15/28) of MBC patients using the RosetteSep™ and 75% (6/8) with ScreenCell®. Overall, the ScreenCell® method had better sensitivity., Competing Interests: Funding for this study was provided in part from a Pfizer Investigator Initiated Research Grant (WS575460). This does not alter our adherence to PLOS ONE policies on sharing data and materials. We have no further relevant declarations (such as consultancies, patents, employment, product development) related to this funder or any other commercial entities.

All land plants (embryophytes) share a common ancestor that likely evolved from a filamentous freshwater alga. Elucidating the transition from algae to embryophytes - and the eventual conquering of Earth's surface - is one of the most fundamental questions in plant evolutionary biology. Here, we investigated one of the organismal properties that might have enabled this transition: resistance to drastic temperature shifts. We explored the effect of heat stress in Mougeotia and Spirogyra, two representatives of Zygnematophyceae - the closest known algal sister lineage to land plants. Heat stress induced pronounced phenotypic alterations in their plastids, and high-performance liquid chromatography-tandem mass spectroscopy-based profiling of 565 transitions for the analysis of main central metabolites revealed significant shifts in 43 compounds. We also analyzed the global differential gene expression responses triggered by heat, generating 92.8 Gbp of sequence data and assembling a combined set of 8905 well-expressed genes. Each organism had its own distinct gene expression profile; less than one-half of their shared genes showed concordant gene expression trends. We nevertheless detected common signature responses to heat such as elevated transcript levels for molecular chaperones, thylakoid components, and - corroborating our metabolomic data - amino acid metabolism. We also uncovered the heat-stress responsiveness of genes for phosphorelay-based signal transduction that links environmental cues, calcium signatures and plastid biology. Our data allow us to infer the molecular heat stress response that the earliest land plants might have used when facing the rapidly shifting temperature conditions of the terrestrial habitat., (© 2020 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Rapid cryopreservation of biological specimens is the gold standard for visualizing cellular structures in their true structural context. However, current commercial cryo-fluorescence microscopes are limited to low resolutions. To fill this gap, we have developed cryoSIM, a microscope for 3D super-resolution fluorescence cryo-imaging for correlation with cryo-electron microscopy or cryo-soft X-ray tomography. We provide the full instructions for replicating the instrument mostly from off-the-shelf components and accessible, user-friendly, open-source Python control software. Therefore, cryoSIM democratizes the ability to detect molecules using super-resolution fluorescence imaging of cryopreserved specimens for correlation with their cellular ultrastructure., Competing Interests: The authors declare no conflicts of interest., (Published by The Optical Society under the terms of the Creative Commons Attribution 4.0 License. Further distribution of this work must maintain attribution to the author(s) and the published article’s title, journal citation, and DOI.)

Aims: To understand the psychological and behavioural impact of type 2 diabetes in the Portuguese Primary Care setting., Methods: Observational, cross-sectional study of a convenience sample of portuguese people diagnosed with type 2 diabetes mellitus. The Diabetes Health Profile (DHP-18) and the Four Item Morisky Medication Adherence Scale (MMAS-4) were applied, as well as socio-demographic and disease-related questions., Results: In a n=110 patient sample, lower education level showed a negative impact on the Psychological Distress dimension (p=0,013). In the Barriers to activity dimension, significant differences were found between different age groups (p=0,033) - a better health profile was observed in the 40-59 years group and also between gender (p=0,039) - greater impact in female subjects. Disinhibited Eating dimension wasn't associated with any of the studied variables. However, all the groups showed worse results in this domain. No associations were found between DHP and medication adherence. Worse medication adherence is more common when there is a prevailing notion of lack of control of diabetes., Discussion and Conclusion: Using DHP, a larger negative impact related to type 2 diabetes mellitus is found in those with lower educational level, female and younger (20-39 years) or older (60-79 years) subjects. Broader information strategies aimed at improving the quality of life of diabetic patients is necessary, particularly those related to nutrition., (Copyright © 2019 Primary Care Diabetes Europe. Published by Elsevier Ltd. All rights reserved.)

Osteoarthritis (OA) is one of the most frequent and incapacitating pathologies today, especially of the knee. Among the possible approaches for knee OA, the neurotomy of the genicular nerves by radiofrequency (RF) has been gaining prominence. However, as this is a relatively new procedure, indications for its implementation are still unclear. The objective of the present review is to identify the main indications of the use of RF for the treatment of knee OA in the medical literature. A review of the literature was performed in January 2018 through a search in the PubMed, ClinicalKey and Google Scholar databases. After reviewing the main articles on the subject, it was concluded that the main indications of the use of RF for the treatment of knee OA were: OA Kellgren-Lawrence grades 3 and 4, with moderate to severe pain and failure of conservative treatment, mainly in elderly people; persistence of pain even after total knee arthroplasty (TKA); patients with an indication for TKA who refuse to undergo surgical treatment.

Objective. To compare the quality indicators of public and private pharmacy schools in Brazil. Method. Descriptive data regarding undergraduate pharmacy schools recorded as of April 2016 was obtained from the website of Brazil's Ministry of Education. The quality indicators analyzed included scores on the National Student Performance Examination, preliminary classification of the school, and final classification of the school. The schools were graded on a scale of 1 to 5, with 5 being excellent. Results. Four hundred sixty-seven Brazilian pharmacy schools were analyzed. The number of schools and class sizes has been increasing since 1832. The number of private pharmacy schools has increased since 1996, with the majority located in non-capital cities. The results showed an isomorphic characteristic of the quality indicators and a central tendency of the scores (score of 3), which are indicators of satisfactory quality. Most public institutions received a score of excellent. Conclusion. The analysis found an increase in the number of pharmacy schools in the private sector in Brazil and that the quality of public institutions was better than that of private institutions. Qualitative research should be carried out to complement the quantitative findings reported here.

Objective: To evaluate the survival of wounds in lower limbs of diabetic and non-diabetic patients., Method: Retrospective cohort study of patients with lower limb ulcers treated at a specialized center between 2011 and 2013. Outcome: healing of lower limb injuries in days. The survival function of lower limb wounds and the differences between diabetic and non-diabetic were analyzed. The Log-rank test was used to compare the survival curves between the study groups., Results: In up to 600 days, 23% of the diabetic patients presented wound healing, while 63% of the non-diabetic patients had their wounds healed, with a statistically significant difference in survival curves in comparison between the groups. The Hazard Ratios (RH) of healing were lower for diabetic patients (HR = 0.13, 95% CI = 0.02-0.97)., Conclusion: The results show that there is a delay in wound healing in diabetic patients.

As the number of immunotherapies increases, so does the need for biomarkers that can aid in identifying an optimal therapy or combination therapy for patients. These predictive biomarkers are of enormous value to patients but present unique challenges to researchers due to the complexity of the immune system and the variability of individual patient molecular profiles. This chapter draws on recent examples of the use of biomarkers to explore the range of phenotypes encountered in immunotherapy trials for the treatment of neoplastic disease. These examples are discussed in the context of immunoproteomic analysis with a particular focus on the unique challenges that are presented when a high dimensionality technique such as immunoproteomics is applied to study a complex system, the immune system in this case. In order to overcome these challenges, immunoproteomic researchers must pay close attention to study design in order to ensure that the results are not only statistically valid but also that the biomarker strategy as a whole is compatible with the standard of care. We propose that, in spite of its limitations, the use of immunoproteomic analysis of liquid biopsies may present a unique opportunity for translation of immunoproteomic biomarkers to the clinic.

Advanced fluorescence imaging methods require careful matching of excitation sources, dichroics, emission filters, detectors, and dyes to operate at their best. This complex task is often left to guesswork, preventing optimal dye:filter combinations, particularly for multicolour applications. To overcome this challenge we developed SPEKcheck, a web application to visualise the efficiency of the light path in a fluorescence microscope. The software reports values for the excitation efficiency of a dye, the collection efficiency of the emitted fluorescence, and a "brightness" score, allowing easy comparison between different fluorescent labels. It also displays a spectral plot of various elements in the configuration, enabling users to readily spot potential problems such as low efficiency excitation, emission, or high bleedthrough. It serves as an aid to exploring the performance of different dyes and filter sets., Competing Interests: No competing interests were disclosed.

Objectives: To estimate productivity losses due to absenteeism and presenteeism and their determinants in patients with depression from five Colombian cities., Methods: We used data from a multicenter, mixed-methods study of adult patients diagnosed with major depressive disorder or double depression (major depressive disorder plus dysthymia) during 2010. The World Health Organization's Health and Work Performance Questionnaire was used to assess absenteeism and presenteeism. We explored the determinants of productivity losses using a two-part model. We also used a costing model to calculate the corresponding monetary losses., Results: We analyzed data from 107 patients employed in the last 4 weeks. Absenteeism was reported by 70% of patients; presenteeism was reported by all but one. Half of the patients reported a level of performance at work at least 50% below usual. Average number of hours per month lost to absenteeism and presenteeism was 43 and 51, respectively. The probability of any absenteeism was 17 percentage points lower in patients rating their mental health favorably compared with those rating it poorly (standard error [SE] 0.09; P < 0.10) and 19 percentage points higher in patients with at least one comorbidity compared with patients with none (SE 0.10; P < 0.10). All other covariates showed no significant associations on hours lost to absenteeism. Patients with favorable mental health self-ratings had 16.4 fewer hours per month of presenteeism compared with those with poor self-ratings (SE 4.52; P < 0.01). The 2015 monetary value of productivity losses amounted to US $840 million., Conclusions: This study in a middle-income country confirms the high economic burden of depression. Health policies and workplace interventions ensuring adequate diagnosis and treatment of depression are recommended., (Copyright © 2017. Published by Elsevier Inc.)

Centrioles organise centrosomes and cilia, and these organelles have an important role in many cell processes. In flies, the centriole protein Ana1 is required for the assembly of functional centrosomes and cilia. It has recently been shown that Cep135 (also known as Bld10) initially recruits Ana1 to newly formed centrioles, and that Ana1 then recruits Asl (known as Cep152 in mammals) to promote the conversion of these centrioles into centrosomes. Here, we show that ana1 mutants lack detectable centrosomes in vivo, that Ana1 is irreversibly incorporated into centrioles during their assembly and appears to play a more important role in maintaining Asl at centrioles than in initially recruiting Asl to centrioles. Unexpectedly, we also find that Ana1 promotes centriole elongation in a dose-dependent manner: centrioles are shorter when Ana1 dosage is reduced and are longer when Ana1 is overexpressed. This latter function of Ana1 appears to be distinct from its role in centrosome and cilium function, as a GFP-Ana1 fusion lacking the N-terminal 639 amino acids of the protein can support centrosome assembly and cilium function but cannot promote centriole over-elongation when overexpressed., (© 2016. Published by The Company of Biologists Ltd.)

Cationic antimicrobial peptides (CAPs) defend against pathogens and, in some cases, exhibit potent anticancer activities. We previously reported that the pleurocidin NRC-03 causes lysis of breast cancer and multiple myeloma cells. NRC-03 also reduces the EC50 of other cytotoxic compounds and prevents tumor growth in vivo. However, the therapeutic utility of NRC-03 may be limited by its susceptibility to degradation by proteases. The goal of this study was to characterize the anticancer activities of a d-amino acid analog of NRC-03 ([D]-NRC-03) that was predicted to be resistant to proteolytic degradation. Unlike NRC-03, [D]-NRC-03 was not degraded by human serum or trypsin and, in comparison to NRC-03, showed increased killing of breast cancer cells, including multidrug-resistant cells; however, [D]-NRC-03 was somewhat more cytotoxic than NRC-03 for several types of normal cells. Importantly, [D]-NRC-03 was more effective than NRC-03 in vivo since 4-fold less peptide was required for an equivalent inhibitory effect on the growth of breast cancer cell xenografts in immune-deficient mice. These findings demonstrate that a d-amino acid analog of NRC-03 overcomes a major limitation to the therapeutic use of NRC-03, namely peptide stability. Further modification of [D]-NRC-03 is required to improve its selectivity for cancer cells., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Objective: To analyze the care implemented by the nursing team to promote the safety of adult patients and prevention of skin and mucosal lesions associated with the presence of lower airways invasive devices., Method: Study with qualitative and quantitative approach, descriptive and exploratory type, whose investigative scenarios were adult inpatient units of a hospital in the West Frontier of Rio Grande do Sul. The study subjects consisted of nurses, nursing technicians and nursing assistants., Results: A total of 118 professionals were interviewed. We highlight the observed specific care with endotracheal tube and tracheostomy, management and assessment of the cuff and the criteria used to secretion aspiration., Conclusion: There is a superficial nursing work in the patient direct care and a differentiation in relation to the perception of nurse technicians, especially those working in the intensive care unit, who presented major property and view of the patient's clinical status.

Centrosomes comprise a pair of centrioles surrounded by a matrix of pericentriolar material (PCM). In vertebrate cells, Pericentrin plays an important part in mitotic PCM assembly, but the Drosophila Pericentrin-like protein (PLP) appears to have a more minor role in mitotic fly cells. Here we investigate the function of PLP during the rapid mitotic cycles of the early Drosophila embryo. Unexpectedly, we find that PLP is specifically enriched in the outer-most regions of the PCM, where it largely co-localizes with the PCM scaffold protein Cnn. In the absence of PLP the outer PCM appears to be structurally weakened, and it rapidly disperses along the centrosomal microtubules (MTs). As a result, centrosomal MTs are subtly disorganized in embryos lacking PLP, although mitosis is largely unperturbed and these embryos develop and hatch at near-normal rates. Y2H analysis reveals that PLP can potentially form multiple interactions with itself and with the PCM recruiting proteins Asl, Spd-2 and Cnn. A deletion analysis suggests that PLP participates in a complex network of interactions that ultimately help to strengthen the PCM., (© 2015. Published by The Company of Biologists Ltd.)

Objectives: To evaluate the prevalence of a vascular network adjacent to the insertion of the pes anserinus, so that it could be used as an anatomical reference point to facilitate harvesting flexor grafts for arthroscopic reconstruction of the anterior cruciate ligament (ACL)., Methods: Thirty patients with ACL tears who were going to undergo ACL reconstruction using the tendons of the semitendinosus and gracilis muscles as grafts were selected randomly. During the harvesting of these tendons, the presence or absence of this anatomical reference point was noted., Results: All the patients presented a vascular network of greater or lesser diameter., Conclusion: The vascular network seems to be a good reference point during harvesting of the tendons of the semitendinosus and gracilis muscles, for facilitating graft harvesting.

Induced pluripotent cell-derived motoneurons (iPSCMNs) are sought for use in cell replacement therapies and treatment strategies for motoneuron diseases such as amyotrophic lateral sclerosis (ALS). However, much remains unknown about the physiological properties of iPSCMNs and how they compare with endogenous spinal motoneurons or embryonic stem cell-derived motoneurons (ESCMNs). In the present study, we first used a proteomic approach and compared protein expression profiles between iPSCMNs and ESCMNs to show that <4% of the proteins identified were differentially regulated. Like ESCs, we found that mouse iPSCs treated with retinoic acid and a smoothened agonist differentiated into motoneurons expressing the LIM homeodomain protein Lhx3. When transplanted into the neural tube of developing chick embryos, iPSCMNs selectively targeted muscles normally innervated by Lhx3 motoneurons. In vitro studies showed that iPSCMNs form anatomically mature and functional neuromuscular junctions (NMJs) when cocultured with chick myofibers for several weeks. Electrophysiologically, iPSCMNs developed passive membrane and firing characteristic typical of postnatal motoneurons after several weeks in culture. Finally, iPSCMNs grafted into transected mouse tibial nerve projected axons to denervated gastrocnemius muscle fibers, where they formed functional NMJs, restored contractile force. and attenuated denervation atrophy. Together, iPSCMNs possess many of the same cellular and physiological characteristics as ESCMNs and endogenous spinal motoneurons. These results further justify using iPSCMNs as a source of motoneurons for cell replacement therapies and to study motoneuron diseases such as ALS., (Copyright © 2015 the authors 0270-6474/15/351291-16$15.00/0.)

Dysregulation of Ras signaling is the major cause of various cancers. Aberrant Ras signaling, however, provides a favorable environment for many viruses, making them suitable candidates as cancer-killing therapeutic agents. Susceptibility of cancer cells to such viruses is mainly due to impaired type I interferon (IFN) response, often as a result of activated Ras/ERK signaling in these cells. In this study, we searched for cellular factors modulated by Ras signaling and their potential involvement in promoting viral oncolysis. We found that upon Ras transformation of NIH-3T3 cells, the N-terminus of Nogo-B (reticulon 4) was proteolytically cleaved. Interestingly, Nogo knockdown (KD) in non-transformed and Ras-transformed cells both enhanced virus-induced IFN response, suggesting that both cleaved and uncleaved Nogo can suppress IFN response. However, pharmacological blockade of Nogo cleavage in Ras-transformed cells significantly enhanced virus-induced IFN response, suggesting that cleaved Nogo contributes to enhanced IFN suppression in these cells. We further showed that IFN suppression associated with Ras-induced Nogo-B cleavage was distinct from but synergistic with that associated with an activated Ras/ERK pathway. Our study therefore reveals an important and novel role of Nogo-B and its cleavage in the suppression of anti-viral immune responses by oncogenic Ras transformation.

We present a combined proteomic and bioinformatic investigation of mitochondrial proteins from the amoeboid protist Acanthamoeba castellanii, the first such comprehensive investigation in a free-living member of the supergroup Amoebozoa. This protist was chosen both for its phylogenetic position (as a sister to animals and fungi) and its ecological ubiquity and physiological flexibility. We report 1033 A. castellanii mitochondrial protein sequences, 709 supported by mass spectrometry data (676 nucleus-encoded and 33 mitochondrion-encoded), including two previously unannotated mtDNA-encoded proteins, which we identify as highly divergent mitochondrial ribosomal proteins. Other notable findings include duplicate proteins for all of the enzymes of the tricarboxylic acid (TCA) cycle-which, along with the identification of a mitochondrial malate synthase-isocitrate lyase fusion protein, suggests the interesting possibility that the glyoxylate cycle operates in A. castellanii mitochondria. Additionally, the A. castellanii genome encodes an unusually high number (at least 29) of mitochondrion-targeted pentatricopeptide repeat (PPR) proteins, organellar RNA metabolism factors in other organisms. We discuss several key mitochondrial pathways, including DNA replication, transcription and translation, protein degradation, protein import and Fe-S cluster biosynthesis, highlighting similarities and differences in these pathways in other eukaryotes. In compositional and functional complexity, the mitochondrial proteome of A. castellanii rivals that of multicellular eukaryotes., Biological Significance: Comprehensive proteomic surveys of mitochondria have been undertaken in a limited number of predominantly multicellular eukaryotes. This phylogenetically narrow perspective constrains and biases our insights into mitochondrial function and evolution, as it neglects protists, which account for most of the evolutionary and functional diversity within eukaryotes. We report here the first comprehensive investigation of the mitochondrial proteome in a member (A. castellanii) of the eukaryotic supergroup Amoebozoa. Through a combination of tandem mass spectrometry (MS/MS) and in silico data mining, we have retrieved 1033 candidate mitochondrial protein sequences, 709 having MS support. These data were used to reconstruct the metabolic pathways and protein complexes of A. castellanii mitochondria, and were integrated with data from other characterized mitochondrial proteomes to augment our understanding of mitochondrial proteome evolution. Our results demonstrate the power of combining direct proteomic and bioinformatic approaches in the discovery of novel mitochondrial proteins, both nucleus-encoded and mitochondrion-encoded, and highlight the compositional complexity of the A. castellanii mitochondrial proteome, which rivals that of animals, fungi and plants., (Copyright © 2014 Elsevier B.V. All rights reserved.)

This article describes and directly links to 1033 Acanthamoeba castellanii mitochondrial protein sequences. Of these, 709 are supported by Mass Spectrometry (MS) data (676 nucleus-encoded and 33 mitochondrion-encoded). Two of these entries are previously unannotated mtDNA-encoded proteins, which we identify as highly divergent mitochondrial ribosomal proteins. Our analysis corrects many A. castellanii protein sequences that were incorrectly inferred previously from genomic data deposited in NCBI.

Phosphite (salts of phosphorous acid; Phi)-based fungicides are increasingly used in controlling oomycete pathogens, such as the late blight agent Phytophthora infestans. In plants, low amounts of Phi induce pathogen resistance through an indirect mode of action. We used iTRAQ-based quantitative proteomics to investigate the effects of phosphite on potato plants before and after infection with P. infestans. Ninety-three (62 up-regulated and 31 down-regulated) differentially regulated proteins, from a total of 1172 reproducibly identified proteins, were identified in the leaf proteome of Phi-treated potato plants. Four days post-inoculation with P. infestans, 16 of the 31 down-regulated proteins remained down-regulated and 42 of the 62 up-regulated proteins remained up-regulated, including 90% of the defense proteins. This group includes pathogenesis-related, stress-responsive, and detoxification-related proteins. Callose deposition and ultrastructural analyses of leaf tissues after infection were used to complement the proteomics approach. This study represents the first comprehensive proteomics analysis of the indirect mode of action of Phi, demonstrating broad effects on plant defense and plant metabolism. The proteomics data and the microscopy study suggest that Phi triggers a hypersensitive response that is responsible for induced resistance of potato leaves against P. infestans., Biological Significance: Phosphie triggers complex functional changes in potato leaves that are responsible for the induced resistance against Phytophthora infestans. This article is part of a Special Issue entitled: Translational Plant Proteomics., (Copyright © 2013 Elsevier B.V. All rights reserved.)