Tear Proteomics in Infants at Risk of Retinopathy of Prematurity: A Feasibility Study.

Purpose: This feasibility study investigated the practicability of collecting and analyzing tear proteins from preterm infants at risk of retinopathy of prematurity (ROP). We sought to identify any tear proteins which might be implicated in the pathophysiology of ROP as well as prognostic markers. Methods: Schirmer's test was used to obtain tear samples from premature babies, scheduled for ROP screening, after parental informed consent. Mass spectrometry was used for proteomic analysis. Results: Samples were collected from 12 infants, which were all adequate for protein analysis. Gestational age ranged from 25 + 6 to 31 + 1 weeks. Postnatal age at sampling ranged from 19 to 66 days. One infant developed self-limiting ROP. Seven hundred one proteins were identified; 261 proteins identified in the majority of tear samples, including several common tear proteins, were used for analyses. Increased risk of ROP as determined by the postnatal growth ROP (G-ROP) criteria was associated with an increase in lactate dehydrogenase B chain in tears. Older infants demonstrated increased concentration of immunoglobulin complexes within their tear samples and two sets of twins in the cohort showed exceptionally similar proteomes, supporting validity of the analysis. Conclusions: Tear sampling by Schirmer test strips and subsequent proteomic analysis by mass spectrometry in preterm infants is feasible. A larger study is required to investigate the potential use of tear proteomics in identification of ROP. Translational Relevance: Tear sampling and subsequent mass spectrometry in preterm infants is feasible. Investigation of the premature tear proteome may increase our understanding of retinal development and provide noninvasive biomarkers for identification of treatment-warranted ROP.

Biodesulfurization of organosulfur compounds by a trehalose biosurfactant producing Gordonia sp. isolated from crude oil contaminated soil.

Certain factors hinder the commercialization of biodesulfurization process, including low substrate-specificity of the currently reported desulfurizing bacteria and restricted mass transfer of organic-sulfur compounds in biphasic systems. These obstacles must be addressed to clean organic-sulfur rich petro-fuels that pose serious environmental and health challenges. In current study, a dibenzothiophene desulfurizing strain, Gordonia rubripertincta W3S5 (source: oil contaminated soil) was systematically evaluated for its potential to remove sulfur from individual compounds and mixture of organic-sulfur compounds. Metabolic and genetic analyses confirmed that strain W3S5 desulfurized dibenzothiophene to 2-hydroxybiphenyl, suggesting that it follows the sulfur specific 4 S pathway. Furthermore, this strain demonstrated the ability to produce trehalose biosurfactants (with an EI24 of 53%) in the presence of dibenzothiophene, as confirmed by TLC and FTIR analyses. Various genome annotation tools, such as ClassicRAST, BlastKOALA, BV-BRC, and NCBI-PGAP, predicted the presence of otsA, otsB, treY, treZ, treP, and Trehalose-monomycolate lipid synthesis genes in the genomic pool of strain W3S5, confirming the existence of the OtsAB, TreYZ, and TreP pathways. Overall, these results underscore the potential of strain W3S5 as a valuable candidate for enhancing desulfurization efficiency and addressing the mass transfer challenges essential for achieving a scaled-up scenario.

Identification of a unique conserved region from a kinetoplastid genome of Leishmania orientalis (formerly named Leishmania siamensis) strain PCM2 in Thailand.

Mitochondrial DNAs (mtDNAs) appear in almost all eukaryotic species and are useful molecular markers for phylogenetic studies and species identification. Kinetoplast DNAs (kDNAs) are structurally complex circular mtDNA networks in kinetoplastids, divided into maxicircles and minicircles. Despite several kDNAs of many Leishmania species being examined, the kDNAs of the new species, Leishmania orientalis (formerly named Leishmania siamensis) strain PCM2, have not been explored. This study aimed to investigate the maxicircle and minicircle DNAs of L. orientalis strain PCM2 using hybrid genome sequencing technologies and bioinformatic analyses. The kDNA sequences were isolated and assembled using the SPAdes hybrid assembler from the Illumina short-read and PacBio long-read data. Circular contigs of the maxicircle and minicircle DNAs were reconstructed and confirmed by BLASTn and rKOMICs programs. The kDNA genome was annotated by BLASTn before the genome comparison and phylogenetic analysis by progressiveMauve, MAFFT, and MEGA programs. The maxicircle of L. orientalis strain PCM2 (18,215 bp) showed 99.92% similarity and gene arrangement to Leishmania enriettii strain LEM3045 maxicircle with variation in the 12s rRNA gene and divergent region. Phylogenetics of the whole sequence, coding regions, divergent regions, and 12s rRNA gene also confirmed this relationship and subgenera separation. The identified 105 classes of minicircles (402-1177 bp) were clustered monophyletically and related to the Leishmania donovani minicircles. The kinetoplast maxicircle and minicircle DNAs of L. orientalis strain PCM2 contained a unique conserved region potentially useful for specific diagnosis of L. orientalis and further exploration of this parasite population genetics in Thailand and related regions.

RAD51-mediated R-loop formation acts to repair transcription-associated DNA breaks driving antigenic variation in Trypanosoma brucei.

RNA-DNA hybrids are epigenetic features of all genomes that intersect with many processes, including transcription, telomere homeostasis, and centromere function. Increasing evidence suggests that RNA-DNA hybrids can provide two conflicting roles in the maintenance and transmission of genomes: They can be the triggers of DNA damage, leading to genome change, or can aid the DNA repair processes needed to respond to DNA lesions. Evasion of host immunity by African trypanosomes, such as Trypanosoma brucei, relies on targeted recombination of silent Variant Surface Glycoprotein (VSG) genes into a specialized telomeric locus that directs transcription of just one VSG from thousands. How such VSG recombination is targeted and initiated is unclear. Here, we show that a key enzyme of T. brucei homologous recombination, RAD51, interacts with RNA-DNA hybrids. In addition, we show that RNA-DNA hybrids display a genome-wide colocalization with DNA breaks and that this relationship is impaired by mutation of RAD51. Finally, we show that RAD51 acts to repair highly abundant, localised DNA breaks at the single transcribed VSG and that mutation of RAD51 alters RNA-DNA hybrid abundance at 70 bp repeats both around the transcribed VSG and across the silent VSG archive. This work reveals a widespread, generalised role for RNA-DNA hybrids in directing RAD51 activity during recombination and uncovers a specialised application of this interplay during targeted DNA break repair needed for the critical T. brucei immune evasion reaction of antigenic variation.

Metabolome Profiling in the Plasma of Dogs with Idiopathic Dilated Cardiomyopathy: A Multiplatform Mass-Spectrometry-Based Approach.

Dilated cardiomyopathy is one of the important diseases in dogs and humans. The second most common cause of heart failure in dogs is idiopathic dilated cardiomyopathy (iDCM), which results in heart failure or sudden cardiac death due to arrhythmia. This study aimed to determine changes in the plasma metabolome of dogs with iDCM compared to healthy dogs. For that purpose, a multiplatform mass-spectrometry-based approach was used. In this study, we included two groups of dogs: 12 dogs with iDCM and 8 healthy dogs. A total of 272 metabolites were detected in the plasma samples of dogs by combining three approaches but four MS-based platforms (GC-MS, LC-MS (untargeted), LC-MS (targeted), and FIA-MS (targeted) methods). Our findings demonstrated changes in the canine plasma metabolome involved in the development of iDCM, including the different concentrations of amino acids, biogenic amines, acylcarnitines, triglycerides and diglycerides, sphingomyelins, and organic acids. The results of this study will enable the detection and monitoring of pathophysiological mechanisms involved in the development of iDCM in the future.

Immunoprecipitation of RNA-DNA hybrid interacting proteins in Trypanosoma brucei reveals conserved and novel activities, including in the control of surface antigen expression needed for immune evasion by antigenic variation.

RNA-DNA hybrids are epigenetic features of genomes that provide a diverse and growing range of activities. Understanding of these functions has been informed by characterising the proteins that interact with the hybrids, but all such analyses have so far focused on mammals, meaning it is unclear if a similar spectrum of RNA-DNA hybrid interactors is found in other eukaryotes. The African trypanosome is a single-cell eukaryotic parasite of the Discoba grouping and displays substantial divergence in several aspects of core biology from its mammalian host. Here, we show that DNA-RNA hybrid immunoprecipitation coupled with mass spectrometry recovers 602 putative interactors in T. brucei mammal- and insect-infective cells, some providing activities also found in mammals and some lineage-specific. We demonstrate that loss of three factors, two putative helicases and a RAD51 paralogue, alters T. brucei nuclear RNA-DNA hybrid and DNA damage levels. Moreover, loss of each factor affects the operation of the parasite immune survival mechanism of antigenic variation. Thus, our work reveals the broad range of activities contributed by RNA-DNA hybrids to T. brucei biology, including new functions in host immune evasion as well as activities likely fundamental to eukaryotic genome function.

The catalytic subunit of DNA-PK regulates transcription and splicing of AR in advanced prostate cancer.

Aberrant androgen receptor (AR) signaling drives prostate cancer (PC), and it is a key therapeutic target. Although initially effective, the generation of alternatively spliced AR variants (AR-Vs) compromises efficacy of treatments. In contrast to full-length AR (AR-FL), AR-Vs constitutively activate androgenic signaling and are refractory to the current repertoire of AR-targeting therapies, which together drive disease progression. There is an unmet clinical need, therefore, to develop more durable PC therapies that can attenuate AR-V function. Exploiting the requirement of coregulatory proteins for AR-V function has the capacity to furnish tractable routes for attenuating persistent oncogenic AR signaling in advanced PC. DNA-PKcs regulates AR-FL transcriptional activity and is upregulated in both early and advanced PC. We hypothesized that DNA-PKcs is critical for AR-V function. Using a proximity biotinylation approach, we demonstrated that the DNA-PK holoenzyme is part of the AR-V7 interactome and is a key regulator of AR-V-mediated transcription and cell growth in models of advanced PC. Crucially, we provide evidence that DNA-PKcs controls global splicing and, via RBMX, regulates the maturation of AR-V and AR-FL transcripts. Ultimately, our data indicate that targeting DNA-PKcs attenuates AR-V signaling and provide evidence that DNA-PKcs blockade is an effective therapeutic option in advanced AR-V-positive patients with PC.

Serum proteome profiling of naturally acquired Babesia rossi infection in dogs.

Babesiosis is a disease of significant medically and veterinary importance with worldwide distribution. It is caused by intra-erythrocyte protozoal parasites, with Babesia rossi causing the most severe clinical signs of all the large Babesia parasites infecting dogs. The disease can be clinically classified into uncomplicated and complicated forms with a wide range of clinical presentations from a mild, subclinical illness to complicated forms and death. The aim of this study was to assess serum proteomic profiles from dogs with babesiosis and healthy dogs using a label-based proteomics approach. Altogether 32 dogs naturally infected with B. rossi (subdivided into 18 uncomplicated cases and 14 complicated cases of babesiosis) and 20 healthy dogs were included. There were 78 proteins with significantly different abundances between the three groups of dogs. Elucidation of proteins and pathways involved in canine babesiosis caused by B. rossi have revealed key differences associated with haemostasis, innate immune system, lipid metabolism and inflammation. Shotgun proteomic profiling allowed identification of potential serum biomarkers for differentiation of disease severity in canine babesiosis caused by B. rossi. These findings may be applicable to the study of host-parasite interactions and the development of novel therapeutic targets.

Cloning and Characterization of Trypanosoma congolense and T. vivax Nucleoside Transporters Reveal the Potential of P1-Type Carriers for the Discovery of Broad-Spectrum Nucleoside-Based Therapeutics against Animal African Trypanosomiasis.

African Animal Trypanosomiasis (AAT), caused predominantly by Trypanosoma brucei brucei, T. vivax and T. congolense, is a fatal livestock disease throughout Sub-Saharan Africa. Treatment options are very limited and threatened by resistance. Tubercidin (7-deazaadenosine) analogs have shown activity against individual parasites but viable chemotherapy must be active against all three species. Divergence in sensitivity to nucleoside antimetabolites could be caused by differences in nucleoside transporters. Having previously characterized the T. brucei nucleoside carriers, we here report the functional expression and characterization of the main adenosine transporters of T. vivax (TvxNT3) and T. congolense (TcoAT1/NT10), in a Leishmania mexicana cell line ('SUPKO') lacking adenosine uptake. Both carriers were similar to the T. brucei P1-type transporters and bind adenosine mostly through interactions with N3, N7 and 3'-OH. Expression of TvxNT3 and TcoAT1 sensitized SUPKO cells to various 7-substituted tubercidins and other nucleoside analogs although tubercidin itself is a poor substrate for P1-type transporters. Individual nucleoside EC50s were similar for T. b. brucei, T. congolense, T. evansi and T. equiperdum but correlated less well with T. vivax. However, multiple nucleosides including 7-halogentubercidines displayed pEC50>7 for all species and, based on transporter and anti-parasite SAR analyses, we conclude that nucleoside chemotherapy for AAT is viable.

Proteomic changes associated with maternal dietary low ω6:ω3 ratio in piglets supplemented with seaweed Part II: Ileum proteomes.

This study evaluates how long-term dietary low ω6:ω3 ratio in sows and offspring's seaweed (SW) intake affects piglet intestinal function and growth through modifying ileum proteome. Sows were assigned to either control diet (CR, ω6:ω3 ratio = 13:1) or treatment diet (LR, ω6:ω3 = 4:1) during gestation and lactation (n = 8 each). The male weaned offspring were received a basal diet with or without SW powder supplementation (4 g/kg) for 21 days, denoted as SW and CT groups, respectively. In total, four groups of weaned piglets were formed following maternal and offspring's diets combination, represented by CRCT, CRSW, LRCT, and LRSW (n = 10 each). Piglet ileum tissue was collected on day 22 post-weaning and analysed using TMT-based quantitative proteomics. The differentially abundant proteins (n = 300) showed the influence of maternal LR diet on protein synthesis, cell proliferation, and cell cycle regulation. In contrast, the SW diet lowered the inflammation severity and promoted ileal tissue development in CRSW piglets but reduced the fat absorption capacity in LRSW piglets. These results uncovered the mechanism behind the anti-inflammation and intestinal-boosting effects of maternal LR diet in piglets supplemented with SW.

Proteomic changes associated with maternal dietary low ω6:ω3 ratio in piglets supplemented with seaweed. Part I: Serum proteomes.

This study examines whether maternal low ω6:ω3 ratio diet and offspring SW supplementation can improve offspring immunity and performance by elucidating the effects on piglet serum proteome. A total of 16 sows were given either a standard (CR, 13:1) or low ω6:ω3 ratio diet (LR, 4:1) during pregnancy and lactation and their male weaned piglets were supplemented with SW powder (4 g/kg, SW) or not (CT) in a 21-day post-weaning (PW) diet. Four PW piglet groups were then identified based on dam and piglet treatment, namely CRCT, CRSW, LRCT, and LRSW (n = 10 each). Piglet serum collected at weaning and d21 PW were analysed (n = 5 each) using TMT-based quantitative proteomics and validated by appropriate assays. The differentially abundant proteins (n = 122) displayed positive effects of maternal LR diet on anti-inflammatory properties and innate immune stimulation. Progeny SW diet activated the innate immunity and enhance the host defence during inflammation. These data demonstrate the value of decreasing ω6:ω3 ratio in maternal diet and SW supplementation in PW piglet's diet to boost their immunity and anti-inflammation properties. SIGNIFICANCE: This novel proteomic study in post-weaned piglets addresses the interplay between maternal and offspring nutritional interventions in a context of rapid and dynamic alterations in piglet metabolic status around weaning. Decreasing ω6:ω3 ratio in maternal diet and SW supplementation in PW piglet's diet can boost their immunity and anti-inflammation properties. This study also provides new insights into piglet serum proteome regulation during post-weaning, a critical development period in swine.

Identification of a conserved maxicircle and unique minicircles as part of the mitochondrial genome of Leishmania martiniquensis strain PCM3 in Thailand.

BACKGROUND: The mitochondrial DNA of trypanosomatids, including Leishmania, is known as kinetoplast DNAs (kDNAs). The kDNAs form networks of hundreds of DNA circles that are evidently interlocked and require complex RNA editing. Previous studies showed that kDNA played a role in drug resistance, adaptation, and survival of Leishmania. Leishmania martiniquensis is one of the most frequently observed species in Thailand, and its kDNAs have not been illustrated. METHODS: This study aimed to extract the kDNA sequences from Illumina short-read and PacBio long-read whole-genome sequence data of L. martiniquensis strain PCM3 priorly isolated from the southern province of Thailand. A circular maxicircle DNA was reconstructed by de novo assembly using the SPAdes program, while the minicircle sequences were retrieved and assembled by the rKOMIC tool. The kDNA contigs were confirmed by blasting to the NCBI database, followed by comparative genomic and phylogenetic analysis. RESULTS: We successfully constructed the complete circular sequence of the maxicircle (19,008 bp) and 214 classes of the minicircles from L. martiniquensis strain PCM3. The genome comparison and annotation showed that the maxicircle structure of L. martiniquensis strain PCM3 was similar to those of L. enriettii strain LEM3045 (84.29%), L. arabica strain LEM1108 (82.79%), and L. tarentolae (79.2%). Phylogenetic analysis also showed unique evolution of the minicircles of L. martiniquensis strain PCM3 from other examined Leishmania species. CONCLUSIONS: This was the first report of the complete maxicircle and 214 minicircles of L. martiniquensis strain PCM3 using integrated whole-genome sequencing data. The information will be helpful for further improvement of diagnosis methods and monitoring genetic diversity changes of this parasite.

Metabolomic Profiling of End-Stage Heart Failure Secondary to Chronic Chagas Cardiomyopathy.

Chronic Chagas cardiomyopathy (CCC) is the most frequent and severe clinical form of chronic Chagas disease, representing one of the leading causes of morbidity and mortality in Latin America, and a growing global public health problem. There is currently no approved treatment for CCC; however, omics technologies have enabled significant progress to be made in the search for new therapeutic targets. The metabolic alterations associated with pathogenic mechanisms of CCC and their relationship to cellular and immunopathogenic processes in cardiac tissue remain largely unknown. This exploratory study aimed to evaluate the potential underlying pathogenic mechanisms in the failing myocardium of patients with end-stage heart failure (ESHF) secondary to CCC by applying an untargeted metabolomic profiling approach. Cardiac tissue samples from the left ventricle of patients with ESHF of CCC etiology (n = 7) and healthy donors (n = 7) were analyzed using liquid chromatography-mass spectrometry. Metabolite profiles showed altered branched-chain amino acid and acylcarnitine levels, decreased fatty acid uptake and oxidation, increased activity of the pentose phosphate pathway, dysregulation of the TCA cycle, and alterations in critical cellular antioxidant systems. These findings suggest processes of energy deficit, alterations in substrate availability, and enhanced production of reactive oxygen species in the affected myocardium. This profile potentially contributes to the development and maintenance of a chronic inflammatory state that leads to progression and severity of CCC. Further studies involving larger sample sizes and comparisons with heart failure patients without CCC are needed to validate these results, opening an avenue to investigate new therapeutic approaches for the treatment and prevention of progression of this unique and severe cardiomyopathy.

Amphotericin B resistance in Leishmania mexicana: Alterations to sterol metabolism and oxidative stress response.

Amphotericin B is increasingly used in treatment of leishmaniasis. Here, fourteen independent lines of Leishmania mexicana and one L. infantum line were selected for resistance to either amphotericin B or the related polyene antimicrobial, nystatin. Sterol profiling revealed that, in each resistant line, the predominant wild-type sterol, ergosta-5,7,24-trienol, was replaced by other sterol intermediates. Broadly, two different profiles emerged among the resistant lines. Whole genome sequencing then showed that these distinct profiles were due either to mutations in the sterol methyl transferase (C24SMT) gene locus or the sterol C5 desaturase (C5DS) gene. In three lines an additional deletion of the miltefosine transporter gene was found. Differences in sensitivity to amphotericin B were apparent, depending on whether cells were grown in HOMEM, supplemented with foetal bovine serum, or a serum free defined medium (DM). Metabolomic analysis after exposure to AmB showed that a large increase in glucose flux via the pentose phosphate pathway preceded cell death in cells sustained in HOMEM but not DM, indicating the oxidative stress was more significantly induced under HOMEM conditions. Several of the lines were tested for their ability to infect macrophages and replicate as amastigote forms, alongside their ability to establish infections in mice. While several AmB resistant lines showed reduced virulence, at least two lines displayed heightened virulence in mice whilst retaining their resistance phenotype, emphasising the risks of resistance emerging to this critical drug.

Making the Most of Its Short Reads: A Bioinformatics Workflow for Analysing the Short-Read-Only Data of Leishmania orientalis (Formerly Named Leishmania siamensis) Isolate PCM2 in Thailand.

BACKGROUND: Leishmania orientalis (formerly named Leishmania siamensis) has been neglected for years in Thailand. The genomic study of L. orientalis has gained much attention recently after the release of the first high-quality reference genome of the isolate LSCM4. The integrative approach of multiple sequencing platforms for whole-genome sequencing has proven effective at the expense of considerably expensive costs. This study presents a preliminary bioinformatic workflow including the use of multi-step de novo assembly coupled with the reference-based assembly method to produce high-quality genomic drafts from the short-read Illumina sequence data of L. orientalis isolate PCM2. RESULTS: The integrating multi-step de novo assembly by MEGAHIT and SPAdes with the reference-based method using the L. enriettii genome and salvaging the unmapped reads resulted in the 30.27 Mb genomic draft of L. orientalis isolate PCM2 with 3367 contigs and 8887 predicted genes. The results from the integrated approach showed the best integrity, coverage, and contig alignment when compared to the genome of L. orientalis isolate LSCM4 collected from the northern province of Thailand. Similar patterns of gene ratios and frequency were observed from the GO biological process annotation. Fifty GO terms were assigned to the assembled genomes, and 23 of these (accounting for 61.6% of the annotated genes) showed higher gene counts and ratios when results from our workflow were compared to those of the LSCM4 isolate. CONCLUSIONS: These results indicated that our proposed bioinformatic workflow produced an acceptable-quality genome of L. orientalis strain PCM2 for functional genomic analysis, maximising the usage of the short-read data. This workflow would give extensive information required for identifying strain-specific markers and virulence-associated genes useful for drug and vaccine development before a more exhaustive and expensive investigation.

Nucleoside Transport and Nucleobase Uptake Null Mutants in Leishmania mexicana for the Routine Expression and Characterization of Purine and Pyrimidine Transporters.

The study of transporters is highly challenging, as they cannot be isolated or studied in suspension, requiring a cellular or vesicular system, and, when mediated by more than one carrier, difficult to interpret. Nucleoside analogues are important drug candidates, and all protozoan pathogens express multiple equilibrative nucleoside transporter (ENT) genes. We have therefore developed a system for the routine expression of nucleoside transporters, using CRISPR/cas9 to delete both copies of all three nucleoside transporters from Leishmania mexicana (ΔNT1.1/1.2/2 (SUPKO)). SUPKO grew at the same rate as the parental strain and displayed no apparent deficiencies, owing to the cells' ability to synthesize pyrimidines, and the expression of the LmexNT3 purine nucleobase transporter. Nucleoside transport was barely measurable in SUPKO, but reintroduction of L. mexicana NT1.1, NT1.2, and NT2 restored uptake. Thus, SUPKO provides an ideal null background for the expression and characterization of single ENT transporter genes in isolation. Similarly, an LmexNT3-KO strain provides a null background for transport of purine nucleobases and was used for the functional characterization of T. cruzi NB2, which was determined to be adenine-specific. A 5-fluorouracil-resistant strain (Lmex5FURes) displayed null transport for uracil and 5FU, and was used to express the Aspergillus nidulans uracil transporter FurD.

The Identification and Characterization of Immunoreactive Fungal Proteins Recognized by Sera from Zimbabweans Sensitized to Fungi.

BACKGROUND: Exposure to fungal allergens poses a serious threat to human health, especially to mould-allergic individuals. The prevalence of fungal allergic disease is increasing globally but is poorly studied in Africa. Here, we aimed to identify and characterize fungal proteins that were immunoreactive against serum samples from fungal-sensitized Zimbabweans from Shamva district to inform the development of diagnostics and therapeutics. METHODS: Crude protein extracts of the Ascomycota Aspergillus fumigatus, Alternaria alternata, Cladosporium herbarum, Epicoccum nigrum, Penicillium chrysogenum, and Saccharomyces cerevisiae as well as mucoromycota Rhizopus nigricans were individually separated by one-dimensional gel electrophoresis for protein staining and immunoblotting. A pool of eight sera from fungi-sensitive Zimbabwean children aged 3-5 years was used to screen the crude extracts to determine their immunoreactivity. Protein bands recognized by the sera were subjected to mass spectrometry to identify the individual proteins reactive with the sera. RESULTS: The pooled serum sample reacted with 20 bands, which resolved to 34 distinct proteins, most of which were novel immunogens. The pool was most reactive to A. alternata. The proteins identified included peptidases (8/34), hydrolases (6/34), oxidoreductases (5/34), and glucosidases (4/34), while 11/34 were unknown. Eight of the proteins were predicted to be allergens using the Structural Database of Allergenic Proteins (SDAP). CONCLUSIONS: We identified novel immunogens from fungi expanding the number of known fungal allergens. These form a potential basis for diagnostics specific for the Zimbabwean population. Validation assays will now need to be carried out to further evaluate the cross-reactivity of the identified allergen candidates as well as investigate their potential recognition in a larger cohort of patients. Furthermore, there is now a need to conduct studies relating sensitization to these immunogens and clinical diseases in the population.

Comparative Draft Genomes of Leishmania orientalis Isolate PCM2 (Formerly Named Leishmania siamensis) and Leishmania martiniquensis Isolate PCM3 from the Southern Province of Thailand.

(1) Background: Autochthonous leishmaniasis, a sandfly-borne disease caused by the protozoan parasites Leishmania orientalis (formerly named Leishmania siamensis) and Leishmania martiniquensis, has been reported for immunocompromised and immunocompetent patients in the southern province of Thailand. Apart from the recent genomes of the northern isolates, limited information is known on the emergence and genetics of these parasites. (2) Methods: This study sequenced and compared the genomes of L. orientalis isolate PCM2 and L. martiniquensis isolate PCM3 with those of the northern isolates and other 14 Leishmania species using short-read whole-genome sequencing methods and comparative bioinformatic analyses. (3) Results: The genomes of the southern isolates of L. orientalis and L. martiniquensis were 30.01 Mbp and 32.39 Mbp, and the comparison with the genomes of the northern isolates revealed species-level similarity with a level of genome and proteome variation, suggesting the different strains. Comparative proteome analysis showed six protein groups with 53 unique proteins for the strain PCM2 and 97 for the strain PCM3. Certain proteins were related to virulence, drug resistance, and stress response. (4) Conclusion: Therefore, the findings could indicate the need for more genetic and population genomic investigation, and the close monitoring of L. orientalis and L. martiniquensis in Thailand and neighboring regions.

Technical report: In-gel sample preparation prior to proteomic analysis of bovine faeces increases protein identifications by removal of high molecular weight glycoproteins.

Bovine faecal composition is complex and a knowledge gap exists in the understanding of the bovine faecal proteome. In the present study, in-gel sample preparation (IGSP) of faecal samples prior to proteomics showed an increase in the number of proteins identified in faecal samples compared to those processed by filter-aided sample preparation (FASP). The optimised sample preparation method removed high molecular weight glycoproteins as part of the clean-up process of the faecal samples, and in combination with in-gel digestion before liquid chromatography with tandem mass spectrometry (LC-MS/MS). The use of IGSP led to enhanced protein identification with increases in the number of peptides identified and in the percent coverage of proteins in the bovine faecal samples. SIGNIFICANCE: Characterization of faecal proteins has the potential to increase our understanding of host responses to changes such as diet, disease and drug-treatment. In-gel sample preparation prior to proteomics can be used to remove high molecular weight glycoproteins and reduce protein/peptide loss in FASP. This method of sample preparation will have application not only in the investigation of bovine faecal extracts but also in studies where large molecules such as glycoproteins or oligosaccharides could have detrimental influences on sample preparation involving ultrafiltration.

Abundance of plasma proteins in response to divergent ratios of dietary ω6:ω3 fatty acids in gestating and lactating sows using a quantitative proteomics approach.

This study aimed to investigate the characteristic proteomic pattern of plasma from sows supplemented with low dietary ω6:ω3 fatty acids (FAs) ratio during gestation and lactation. Two dietary treatments (n = 8 each) comprised either a control ratio of ω6:ω3 FAs (CR, 13:1 during gestation and 10:1 during lactation) or a low ratio (LR, 4:1 during gestation and lactation) by adding soybean oil or linseed oil, respectively. High-resolution mass spectrometry-based quantitative proteomics was applied on plasma (n = 5 each) at day 108 of gestation (G108) and at the end of lactation (L-End), and a total of 379 proteins and 202 master proteins were identified. Out of these, four differentially abundant proteins between LR and CR samples at G108 may relate to serine-type endopeptidase inhibitor activity. Differentially abundant proteins in L-End versus G108 (12 up-regulated and 10 down-regulated) were positively correlated with the events that regulate plasma lipoproteins, stimulus- and defence-responses. These findings demonstrate the benefit of increased dietary ω3 FAs in modifying proteins involved in protective mechanisms against increased stresses in key life cycle phases in pigs. In addition, proteome changes from late gestation to late lactation disclosed the underlying mechanism of pigs in response to reproduction-related stimuli. SIGNIFICANCE: This study aimed to provide a proteomics insight into the beneficial effects of maternal diet supplementation with a low ω6:ω3 fatty acids ratio, based on previously reported performance and zootechnical data. The results suggest that a low dietary ω6:ω3 fatty acids ratio could enhance the cellular defence mechanisms against increased stresses and in particular to oxidative stress in sows during gestation and lactation, as reflected in proteomic changes of haptoglobin (HP), alpha-1-antitrypsin (SERPINA1) and serum amyloid P-component (APCS). Furthermore, significantly changed proteome profiles in sow plasma between late gestation and lactation phases have been revealed for the first time. This finding identified the adaptation mechanisms of sows to changing physiological events during reproduction.