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Cutaneous leishmaniasis is atypical in Sri Lanka because Leishmania donovani, which typically causes visceral disease, is the causative agent. The origins of recently described hybrids between L. donovani and other Leishmania spp. usually responsible for cutaneous leishmaniasis remain unknown. Other endemic dermotropic Leishmania spp. have not been reported in Sri Lanka. Genome analysis of 27 clinical isolates from Sri Lanka and 32 Old World Leishmania spp. strains found 8 patient isolates clustered with L. tropica and 19 with L. donovani. The L. tropica isolates from Sri Lanka shared markers with strain LtK26 reported decades ago in India, indicating they were not products of recent interspecies hybridization. Because L. tropica was isolated from patients with leishmaniasis in Sri Lanka, our findings indicate L. donovani is not the only cause of cutaneous leishmaniasis in Sri Lanka and potentially explains a haplotype that led to interspecies dermotropic L. donovani hybrids.

Abstract Tissue-resident macrophages are critical for tissue homeostasis and repair. We previously showed that dermis-resident macrophages produce CCL24 which mediates their interaction with IL-4+ eosinophils, required to maintain their M2-like properties in the TH1 environment of the Leishmania major infected skin. Here, we show that thymic stromal lymphopoietin (TSLP) and IL-5+ type 2 innate lymphoid cells are also required to maintain dermis-resident macrophages and promote infection. Single cell RNA sequencing reveals the dermis-resident macrophages as the sole source of TSLP and CCL24. Generation of Ccl24-cre mice permits specific labeling of dermis-resident macrophages and interstitial macrophages from other organs. Selective ablation of TSLP in dermis-resident macrophages reduces the numbers of IL-5+ type 2 innate lymphoid cells, eosinophils and dermis-resident macrophages, and ameliorates infection. Our findings demonstrate that dermis-resident macrophages are self-maintained as a replicative niche for L. major by orchestrating localized type 2 circuitries with type 2 innate lymphoid cells and eosinophils.

While adverse local tissue reactions are well described in the total hip arthroplasty literature, there have only been case reports and case series in the total knee arthroplasty literature. There have been no cases described in the setting of a distal femoral replacement. In this case, we describe a 69-year-old female with a complex history of left knee revision arthroplasty with a distal femoral and proximal tibial replacement who presented with left knee pain and was found to have extensive adverse local tissue reaction with corrosion at the femoral stem-extension piece junction and the extension piece-distal femoral component junction. The femoral taper was then manually cleaned and modular components replaced. Corrosion at the stem-distal femoral component junction can result in adverse local tissue reaction in patients with distal femoral replacements. It is important to consider this diagnosis when evaluating patients with knee pain following distal femoral replacement.

Acidity and tannins are among the grape berry quality traits that influence wine quality. Despite advantageous environmental tolerances of Vitis aestivalis-derived 'Norton', its acidity and tannin concentrations often deviate from expectations set for V. vinifera. Identification of the genetic determinants of malic acid, tartaric acid, pH, and tannin can assist in the improvement of new hybrid cultivars. For this purpose, a 'Norton' and V. vinifera 'Cabernet Sauvignon' hybrid population containing 223 individuals was used to construct a linkage map containing 384 simple sequence repeat (SSR) and 2,084 genotyping-by-sequencing (GBS)-derived single nucleotide polymorphism (SNP) markers. The resulting map was 1,441.9 cM in length with an average inter-marker distance of 0.75 cM and spanned 19 linkage groups (LGs). Quantitative trait loci (QTLs) were detected for malic acid, tartaric acid, pH, and tannin. QTLs for malic acid (LG 8) and pH (LG 6) were observed across multiple years and explained approximately 17.7% and 18.5% of the phenotypic variation, respectively. Additionally, QTLs for tartaric acid were identified on linkage groups 1, 6, 7, 9, and 17 and tannin on LG 2 in single-year data. The QTLs for tartaric acid explained between 8.8−14.3% and tannin explained 24.7% of the phenotypic variation. The markers linked to these QTLs can be used to improve hybrid cultivar breeding through marker-assisted selection.

Parallel extraction of headspace volatiles from multiwell plates using sorbent sheets (HS-SPMESH) followed by direct analysis in real-time high-resolution mass spectrometry (DART-HRMS) can be used as a rapid alternative to solid-phase micro-extraction (SPME) gas-chromatography mass-spectrometry (GC-MS) for trace level volatile analyses. However, an earlier validation study of SPMESH-DART-MS using 3-isobutyl-2-methoxypyrazine (IBMP) in grape juice showed poor correlation between SPMESH-DART-MS and a gold standard SPME-GC-MS around the compound’s odor detection threshold (n = 302) and achieved good correlation and agreement with SPME-GC-MS (R2 = 0.84) over the native range of IBMP (

Leishmania major is the main causative agent of cutaneous leishmaniasis in the Old World. In Leishmania parasites, the lack of transcriptional control is mostly compensated by post-transcriptional mechanisms. Methylation of arginine is a conserved post-translational modification executed by Protein Arginine Methyltransferase (PRMTs). The genome from L. major encodes five PRMT homologs, including the cytosolic protein associated with several RNA-binding proteins, LmjPRMT7. It has been previously reported that LmjPRMT7 could impact parasite infectivity. In addition, a more recent work has clearly shown the importance of LmjPRMT7 in RNA-binding capacity and protein stability of methylation targets, demonstrating the role of this enzyme as an important epigenetic regulator of mRNA metabolism. In this study, we unveil the impact of PRMT7-mediated methylation on parasite development and virulence. Our data reveals that higher levels of LmjPRMT7 can impair parasite pathogenicity, and that deletion of this enzyme rescues the pathogenic phenotype of an attenuated strain of L. major. Interestingly, lesion formation caused by LmjPRMT7 knockout parasites is associated with an exacerbated inflammatory reaction in the tissue correlated with an excessive neutrophil recruitment. Moreover, the absence of LmjPRMT7 also impairs parasite development within the sand fly vector Phlebotomus duboscqi. Finally, a transcriptome analysis shed light onto possible genes affected by depletion of this enzyme. Taken together, this study highlights how post-transcriptional regulation can affect different aspects of the parasite biology.

Despite major advances over the last decade in our understanding of Leishmania reproductive strategies, the sexual cycle in Leishmania has defied direct observation and remains poorly investigated due to experimental constraints. Here, we summarize the findings and conclusions drawn from genetic analysis of experimental hybrids generated in sand flies and highlight the recent advances in generating hybrids in vitro. The ability to hybridize between culture forms of different species and strains of Leishmania should invite more intensive investigation of the mechanisms underlying genetic exchange and provide a rich source of recombinant parasites for future genetic analyses.

Leishmania donovani causes visceral leishmaniasis (VL), which is typically fatal without treatment. There is substantial variation between individuals in rates of disease progression, response to treatment and incidence of post-treatment sequelae, specifically post-kala-azar dermal leishmaniasis (PKDL). Nevertheless, the majority of infected people are asymptomatic carriers. Hamsters and mice are commonly used as models of fatal and non-fatal VL, respectively. Host and parasite genetics are likely to be important factors, but in general the reasons for heterogeneous disease presentation in humans and animal models are poorly understood. Host microbiota has become established as a factor in cutaneous forms of leishmaniasis but this has not been studied in VL. We induced intestinal dysbiosis in mice and hamsters by long-term treatment with broad-spectrum antibiotics in their drinking water. There were no significant differences in disease presentation in dysbiotic mice. In contrast, dysbiotic hamsters infected with L. donovani had delayed onset and progression of weight loss. Half of control hamsters had a rapid progression phenotype compared with none of the ABX-treated animals and the nine-month survival rate was significantly improved compared to untreated controls (40% vs. 10%). Antibiotic-treated hamsters also had significantly less severe hepatosplenomegaly, which was accompanied by a distinct cytokine gene expression profile. The protective effect was not explained by differences in parasite loads or haematological profiles. We further found evidence that the gut-liver axis is a key aspect of fatal VL progression in hamsters, including intestinal parasitism, bacterial translocation to the liver, malakoplakia and iron sequestration, none of which occurred in non-progressing murine VL. Diverse bacterial genera were cultured from VL affected livers, of which Rodentibacter was specifically absent from ABX-treated hamsters, indicating this pathobiont may play a role in promoting disease progression. The results provide experimental support for antibiotic prophylaxis against secondary bacterial infections as an adjunct therapy in human VL patients.

ABSTRACT Sand flies are the natural vectors for the Leishmania species that produce a spectrum of diseases in their mammalian hosts, including humans. Studies of sand fly/Leishmania interactions have been limited by the absence of genome editing techniques applicable to these insects. In this report, we adapted CRISPR (clustered regularly interspaced palindromic repeat)/Cas9 (CRISPR-associated protein 9) technology to the Phlebotomus papatasi sand fly, a natural vector for Leishmania major, targeting the sand fly immune deficiency (IMD) pathway in order to decipher its contribution to vector competence. We established a protocol for transformation in P. papatasi and were able to generate transmissible null mutant alleles for Relish (Rel), the only transcription factor of the IMD pathway. While the maintenance of a homozygous mutant stock was severely compromised, we were able to establish in an early generation their greater susceptibility to infection with L. major. Flies carrying different heterozygous mutant alleles variably displayed a more permissive phenotype, presenting higher loads of parasites or greater numbers of infective-stage promastigotes. Together, our data show (i) the successful adaptation of the CRISPR/Cas9 technology to sand flies and (ii) the impact of the sand fly immune response on vector competence for Leishmania parasites. IMPORTANCE Sand flies are the natural vectors of Leishmania parasites. Studies of sand fly/Leishmania interactions have been limited by the lack of successful genomic manipulation of these insects. This paper shows the first example of successful targeted mutagenesis in sand flies via adaptation of the CRISPR/Cas9 editing technique. We generated transmissible null mutant alleles of relish, a gene known to be essential for the control of immune response in other insects. In addition to the expected higher level of susceptibility to bacteria, the mutant flies presented higher loads of parasites when infected with L. major, showing that the sand fly immune response impacts its vector competence for this pathogen.

Etched polymeric sorbent sheets (solid-phase mesh-enhanced sorption from headspace (SPMESH) sheets) were recently described as an alternative to solid-phase microextraction (SPME) for rapid, parallel, multi-sample extraction and pre-concentration of headspace volatiles. In this report, a workflow was evaluated based on SPMESH sheet extraction followed by direct analysis in real time-mass spectrometry (DART-MS) using grape samples harvested from multiple commercial vineyards at different maturities. SPMESH sheet-DART-MS(-MS) was performed on two grape-derived odorants related to wine quality: 3-isobutyl-2-methoxypyrazine (IBMP) in Cabernet Sauvignon and Merlot grape homogenate (n = 86 samples) and linalool in Muscat-type grape juice samples (n = 18 samples). As part of the optimization process, an MS-MS method was developed for IBMP and an equilibration procedure prior to extraction was established for homogenate samples. Following optimization, we achieved good correlation between SPMESH sheet-DART-MS and SPME-GC-MS for both IBMP (range by GC-MS = < 2 ng/L to 28 ng/L, R2 = 0.70) and linalool (range by GC-MS = 135 to 415 μg/L, R2 = 0.66). The results indicate SPMESH sheet-DART-MS is suitable for rapid measurements of trace level volatiles in grapes.

Analyses of key odorous polyfunctional volatile thiols in wines (3-mercaptohexanol (3-MH), 3-mercaptohexylacetate (3-MHA), and 4-mercapto-4-methyl-2-pentanone (4-MMP)) are challenging due to their high reactivity and ultra-trace concentrations, especially when using conventional gas-chromatography electron impact mass spectrometry (GC-EI-MS). We describe a method in which thiols are converted to pentafluorobenzyl (PFB) derivatives by extractive alkylation and the organic layer is evaporated prior to headspace solid phase microextraction (HS-SPME) and GC-EI-MS analysis. Optimal parameters were determined by response surface area modeling. The addition of NaCl solution to the dried SPME vials prior to extraction resulted in up to less than fivefold improvement in detection limits. Using 40 mL wine samples, limits of detection for 4-MMP, 3-MH, and 3-MHA were 0.9 ng/L, 1 ng/L, and 17 ng/L, respectively. Good recovery (90%–109%) and precision (5%–11% RSD) were achieved in wine matrices. The new method was used to survey polyfunctional thiol concentrations in 61 commercial California and New York State wines produced from V. vinifera (Riesling, Gewürztraminer, Cabernet Sauvignon, Sauvignon blanc and non-varietal rosé wines), V. labruscana (Niagara), and Vitis spp. (Cayuga White). Mean 4-MMP concentrations in New York Niagara (17 ng/L) were not significantly different from concentrations in Sauvignon blanc, but were significantly higher than 4-MMP in other varietal wines.

Anthocyanin pigments in grape juice can coprecipitate with potassium bitartrate (KHT) crystals during cold stabilization, but factors that reduce these adsorptive losses are not well understood. We hypothesized that coprecipitation on a % w/w basis should be decreased at lower pH. In initial experiments, model juice solutions containing an anthocyanin monoglucoside extract and varying pH values were subjected to cold-storage to induce KHT crystallization, and anthocyanins in the resulting precipitant were characterized by HPLC. The pH of the model juice was directly correlated with the % w/w concentration of anthocyanins in the KHT crystals, with a maximum observed at pH 3.40 (0.20% w/w) and a minimum at pH 2.35 (0.01% w/w). A pH dependency was also observed for anthocyanin-KHT coprecipitation in purple Concord grape juice, although the effect was smaller. Coprecipitation was significantly greater for anthocyanin monoglucosides and acylated anthocyanins as compared to anthocyanin diglucosides at pH > 3.05, but coprecipitation of mono- and acylated forms declined more sharply at lower pH values.

ABSTRACT Leishmania tropica, a unicellular eukaryotic parasite present in North and East Africa, the Middle East, and the Indian subcontinent, has been linked to large outbreaks of cutaneous leishmaniasis in displaced populations in Iraq, Jordan, and Syria. Here, we report the genome sequence of this pathogen and 7,863 identified protein-coding genes, and we show that the majority of clinical isolates possess high levels of allelic diversity, genetic admixture, heterozygosity, and extensive aneuploidy. By utilizing paired genome-wide high-throughput DNA sequencing (DNA-seq) with RNA-seq, we found that gene dosage, at the level of individual genes or chromosomal “somy” (a general term covering disomy, trisomy, tetrasomy, etc.), accounted for greater than 85% of total gene expression variation in genes with a 2-fold or greater change in expression. High gene copy number variation (CNV) among membrane-bound transporters, a class of proteins previously implicated in drug resistance, was found for the most highly differentially expressed genes. Our results suggest that gene dosage is an adaptive trait that confers phenotypic plasticity among natural Leishmania populations by rapid down- or upregulation of transporter proteins to limit the effects of environmental stresses, such as drug selection. IMPORTANCE Leishmania is a genus of unicellular eukaryotic parasites that is responsible for a spectrum of human diseases that range from cutaneous leishmaniasis (CL) and mucocutaneous leishmaniasis (MCL) to life-threatening visceral leishmaniasis (VL). Developmental and strain-specific gene expression is largely thought to be due to mRNA message stability or posttranscriptional regulatory networks for this species, whose genome is organized into polycistronic gene clusters in the absence of promoter-mediated regulation of transcription initiation of nuclear genes. Genetic hybridization has been demonstrated to yield dramatic structural genomic variation, but whether such changes in gene dosage impact gene expression has not been formally investigated. Here we show that the predominant mechanism determining transcript abundance differences (>85%) in Leishmania tropica is that of gene dosage at the level of individual genes or chromosomal somy.

Inflammatory monocytes can be manipulated by environmental cues to perform multiple functions. To define the role of monocytes during primary or secondary infection with an intra-phagosomal pathogen we employed Leishmania major-red fluorescent protein (RFP) parasites and multi-color flow cytometry to define and enumerate infected and uninfected inflammatory cells in the skin. During primary infection, infected monocytes had altered maturation and were the initial mononuclear host cell for parasite replication. In contrast, at a distal site of secondary infection in mice with a healed but persistent primary infection, this same population rapidly produced inducible nitric oxide synthase (iNOS) in an IFN-γ dependent manner and was critical for parasite killing. Maturation to a dendritic cell-like phenotype was not required for monocyte iNOS-production, and enhanced monocyte recruitment correlated with IFN-γ dependent cxcl10 expression. In contrast, neutrophils appeared to be a safe haven for parasites in both primary and secondary sites. Thus, inflammatory monocytes play divergent roles during primary versus secondary infection with an intra-phagosomal pathogen.

ABSTRACT The life cycle of the Leishmania parasite in the sand fly vector involves differentiation into several distinctive forms, each thought to represent an adaptation to specific microenvironments in the midgut of the fly. Based on transcriptome sequencing (RNA-Seq) results, we describe the first high-resolution analysis of the transcriptome dynamics of four distinct stages of Leishmania major as they develop in a natural vector, Phlebotomus duboscqi. The early transformation from tissue amastigotes to procyclic promastigotes in the blood-fed midgut was accompanied by the greatest number of differentially expressed genes, including the downregulation of amastins, and upregulation of multiple cell surface proteins, sugar and amino acid transporters, and genes related to glucose metabolism and cell cycle progression. The global changes accompanying post-blood meal differentiation of procyclic promastigotes to the nectomonad and metacyclic stages were less extensive, though each displayed a unique signature. The transcriptome of nectomonads, which has not been studied previously, revealed changes consistent with cell cycle arrest and the upregulation of genes associated with starvation and stress, including autophagic pathways of protein recycling. Maturation to the infective, metacyclic stage was accompanied by changes suggesting preadaptation to the intracellular environment of the mammalian host, demonstrated by the amastigote-like profiles of surface proteins and metabolism-related genes. Finally, a direct comparison between sand fly-derived and culture-derived metacyclics revealed a reassuring similarity between the two forms, with the in vivo forms distinguished mainly by a stronger upregulation of transcripts associated with nutrient stress. IMPORTANCE The life cycle of Leishmania parasites in the sand fly vector includes their growth and development as morphologically distinct forms of extracellular promastigotes found within the different microenvironments of the gut. Based on RNA-Seq, we provide here the first high-resolution, transcriptomic analysis of Leishmania insect stages during their cyclical development in vivo, from tissue amastigotes ingested with the blood meal to infective, metacyclic promastigotes that initiate infection in the mammalian host. The most extensive genetic reprogramming occurred during the early transformation of amastigotes to rapidly dividing procyclic promastigotes in the blood-fed midgut, with major changes in the abundance of mRNAs for surface proteins and metabolism. The post-blood meal-adapted nectomonad stage was characterized by the downregulation of cell cycle-related genes and the upregulation of stress- and starvation-related genes. Finally, the transcriptome of metacyclic promastigotes shifted to a more amastigote-like profile, suggesting their preadaptation to the intracellular host environment.

ABSTRACT: Objective: The malignant spread of histologically benign struma ovarii was historically referred to as peritoneal strumosis. Recently, highly differentiated follicular carcinoma of ovarian origin (HDFCO) was proposed as a more accurate diagnostic term for this rare disease. We report the unusual case presentation of a woman with HDFCO and the subsequent management of her cancer.Methods: Her interventions included abdominal surgery, postoperative laboratory tests, imaging, thyroidectomy, radioactive iodine-123 (RAI-123) diagnostic screening, and thyroid function studies.Results: A 72-year-old Chinese female was diagnosed with metastatic HDFCO approximately 30 years after hysterectomy and bilateral oophorectomy. While undergoing evaluation for an upper gastrointestinal bleed, a computed tomography scan revealed a 10 cm pelvic mass and multiple other peritoneal tumors. She underwent tumor debulking surgery, and the pathology was interpreted at two institutions; both made the diagnosis of a very well-differentiated follicular carcinoma arising in struma ovarii. Lab tests revealed hyperthyroidism and RAI-123 uptake confirmed tumor production of thyroid hormone. A total thyroidectomy was performed in anticipation of treatment with radioactive iodine-131 (RAI-131). However, the patient remained clinically asymptomatic and, therefore, neither additional tumor debulking nor RAI-131 treatment were performed. No disease progression has been noted for over 3 years.Conclusion: This case highlights the potentially slow-growing nature of HDFCO which, in such cases, may be suited to less aggressive disease management.Abbreviations: CT computed tomography; Hb hemoglobin; HDFCO highly differentiated follicular carcinoma of ovarian origin; RAI-123 radioactive iodine-123; RAI-131 radioactive iodine-131; T3 triiodothyronine; T4 thyroxine; TSH thyroid-stimulating hormone;

First paragraphs: Twenty-five years ago, my in-laws came to visit us in central Ohio. They were city folks from Philadelphia who couldn’t understand why my wife, Judy, and I had moved to the country. We timed their visit to coincide with Knox County’s Heart of Ohio tour. Each fall, this selfguided driving tour along the area’s scenic back roads features stops at farms, grange halls, and other sites that offer a glimpse into local rural life. This particular tour included a local turkey farm outside the town of Danville. Danville was well known for its many turkey operations; we were always thankful that it was easy to get a fresh bird for the Thanksgiving table. It remains a family story to this day of how Grandpop Irv felt compelled to let out a gobble during our visit, only to generate a vocal response from what seemed like thousands of birds in the adjacent field. A quarter century later there’s barely a gobble to be heard around Danville, and the only talk of turkeys is at the annual Danville Turkey Festival. Why aren’t there any turkeys at the Danville Turkey Festival? The answer is simple enough. The local processing plant moved 200 miles north into Michigan, in keeping with the trend toward centralization so characteristic of the current global food system. For local farmers, 200 extra miles was the difference between profit and loss, and so turkey farming disappeared....

In winegrapes (Vitis spp.), fruit quality traits such as berry color, total soluble solids content (SS), malic acid content (MA), and yeast assimilable nitrogen (YAN) affect fermentation or wine quality, and are important traits in selecting new hybrid winegrape cultivars. Given the high genetic diversity and heterozygosity of Vitis species and their tendency to exhibit inbreeding depression, linkage map construction and quantitative trait locus (QTL) mapping has relied on F1 families with the use of simple sequence repeat (SSR) and other markers. This study presents the construction of a genetic map by single nucleotide polymorphisms identified through genotyping-by-sequencing (GBS) technology in an F2 mapping family of 424 progeny derived from a cross between the wild species V. riparia Michx. and the interspecific hybrid winegrape cultivar, 'Seyval'. The resulting map has 1449 markers spanning 2424 cM in genetic length across 19 linkage groups, covering 95% of the genome with an average distance between markers of 1.67 cM. Compared to an SSR map previously developed for this F2 family, these results represent an improved map covering a greater portion of the genome with higher marker density. The accuracy of the map was validated using the well-studied trait berry color. QTL affecting YAN, MA and SS related traits were detected. A joint MA and SS QTL spans a region with candidate genes involved in the malate metabolism pathway. We present an analytical pipeline for calling intercross GBS markers and a high-density linkage map for a large F2 family of the highly heterozygous Vitis genus. This study serves as a model for further genetic investigations of the molecular basis of additional unique characters of North American hybrid wine cultivars and to enhance the breeding process by marker-assisted selection. The GBS protocols for identifying intercross markers developed in this study can be adapted for other heterozygous species.