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Abstract Diet is a potentially modifiable neurodegenerative disease risk factor. We studied the effects of a typical Western diet (WD; high in refined carbohydrates, cholesterol and saturated fat), relative to a heart-healthy diet (HHD; high in unrefined carbohydrates, polyunsaturated fat and fiber, and low in cholesterol) on brain microvessel transcriptomics and brain metabolomics of the temporal region in Ossabaw minipigs. Thirty-two pigs (16 male and 16 females) were fed a WD or HHD starting at the age of 4 months for a period of 6 months. The WD and HHD were isocaloric and had a similar macronutrient content but differed in macronutrient quality. Within each dietary group, half of the pigs also received atorvastatin. Relative to HHD-fed pigs, WD-fed pigs had 175 genes differentially expressed (fold change > 1.3, FDR

Primary ciliary dyskinesia (PCD) is a rare genetic condition with a variable clinical presentation, making its diagnosis a challenge. The authors describe two unrelated sibling pairs with PCD: adult siblings in the first and perinatal/neonatal in the second. Both families highlight the commoner and less common clinical manifestations of PCD. The authors use these cases to highlight (1) the current understanding of the underlying genetic and pathophysiological mechanisms of PCD, (2) the range of cardiac and respiratory features of PCD across a wide age-range (3), aspects of history and clinical exam that should raise suspicion of PCD, and (4) the role of NGS gene panel testing in confirmation of diagnosis.

Abstract Pleural empyema is a serious condition leading to a significant burden on health care systems due to protracted hospitalisations. Treatment ranges from non-surgical interventions such as antibiotic therapy and chest tube placement to thoracoscopic or open surgery. Various risk factors which impact outcomes have been investigated. The RAPID (renal, age, purulence, infection source, and dietary factors) score is a clinical risk score which identifies patients at risk of death and may be used to formulate individual treatment strategies accordingly. All patients undergoing surgical interventions for empyema at a major tertiary medical centre in Germany from 2017 to 2020 were analysed. The aim was to identify perioperative risk factors which significantly impact treatment outcomes but are currently not included in the RAPID score. 245 patients with pleural empyema surgically treated at the Department of General, Visceral and Thoracic Surgery at the University Medical Centre, Hamburg, Germany (admitted from January 2017 to April 2020) were retrospectively analysed. All patients which received either minimally invasive or open thoracic surgery were included. Epidemiological as well as perioperative data was analysed to identify risk factors which impact long-term overall outcomes. 90-day mortality rate was the primary endpoint. The mean age was 59.4 years with a bimodal distribution. There was a male predominance across the cohort (71.4% compared to 28.6%), with no significant differences across ages below or above 60 years. 53 (21.6%) patients died within the first 90 days. Diabetes type 1 and 2, renal replacement therapy, immunosuppression, postoperative bleeding, intraoperative transfusion as well as microbiologically confirmed bacterial invasion of the pleura all led to higher mortality rates. Higher RAPID scores accurately predicted higher 90-day mortality rates. Modifying the RAPID score by adding the comorbidities diabetes and renal replacement therapy significantly increased the predictive value of the score. We demonstrated various perioperative and patient related risk-factors not included in the RAPID score which negatively impact postoperative outcome in patients receiving surgical treatment for pleural empyema. These should be taken into consideration when deciding on the best course of treatment. If confirmed in a prospective study including non-surgical patients with a significantly larger cohort, it may be worth considering expanding the RAPID score to include these.

Abstract In this paper, we present the first high‐speed video observation of a cloud‐to‐ground lightning flash and its associated downward‐directed Terrestrial Gamma‐ray Flash (TGF). The optical emission of the event was observed by a high‐speed video camera running at 40,000 frames per second in conjunction with the Telescope Array Surface Detector, Lightning Mapping Array, interferometer, electric‐field fast antenna, and the National Lightning Detection Network. The cloud‐to‐ground flash associated with the observed TGF was formed by a fast downward leader followed by a very intense return stroke peak current of −154 kA. The TGF occurred while the downward leader was below cloud base, and even when it was halfway in its propagation to ground. The suite of gamma‐ray and lightning instruments, timing resolution, and source proximity offer us detailed information and therefore a unique look at the TGF phenomena.

Abstract Rare diseases often result in delays in diagnosis. It is important to recognize conditions that have features of both inborn errors of immunity and predispose to myeloid neoplasia. Here we report a patient with GATA2 deficiency that presented with disseminated non‐tuberculous mycobacterial infection and pancytopenia secondary to myelodysplastic syndrome.

Summary: Type 2 T helper (Th2) cells and group 2 innate lymphoid cells (ILC2s) provide protection against helminth infection and are involved in allergic responses. However, their relative importance and crosstalk during type 2 immune responses are still controversial. By generating and utilizing mouse strains that are deficient in either ILC2s or Th2 cells, we report that interleukin (IL)-33-mediated ILC2 activation promotes the Th2 cell response to papain; however, the Th2 cell response to ovalbumin (OVA)/alum immunization is thymic stromal lymphopoietin (TSLP) dependent but independent of ILC2s. During helminth infection, ILC2s and Th2 cells collaborate at different phases of the immune responses. Th2 cells, mainly through IL-4 production, induce the expression of IL-25, IL-33, and TSLP, among which IL-25 and IL-33 redundantly promote ILC2 expansion. Thus, while Th2 cell differentiation can occur independently of ILC2s, activation of ILC2s may promote Th2 responses, and Th2 cells can expand ILC2s by inducing type 2 alarmins.

ABSTRACT Bacillus thuringiensis (Bt) is a Gram-positive soil bacterium that is widely and safely applied in the environment as an insecticide for combatting insect pests that damage crops or are disease vectors. Dominant active ingredients made by Bt are insect-killing crystal (Cry) proteins released as crystalline inclusions upon bacterial sporulation. Some Bt Cry proteins, e.g., Cry5B (formally Cry5Ba1), target nematodes (roundworms) and show exceptional promise as anthelmintics (cures for parasitic nematode diseases). We have recently described inactivated bacteria with cytosolic crystal(s) (IBaCC) in which bioactive Bt Cry crystals (containing Cry5B) are fully contained within the cytosol of dead bacterial ghosts. Here, we demonstrate that these IBaCC-trapped Cry5B crystals can be liberated and purified away from cellular constituents, yielding purified cytosolic crystals (PCC). Cry5B PCC contains ~95% Cry5B protein out of the total protein content. Cry5B PCC is highly bioactive against parasitic nematode larvae and adults in vitro. Cry5B PCC is also highly active in vivo against experimental human hookworm and Ascaris infections in rodents. The process was scaled up to the 100-liter scale to produce PCC for a pilot study to treat two foals infected with the ascarid Parascaris spp. Single-dose Cry5B PCC brought the fecal egg counts of both foals to zero. These studies describe the process for the scalable production of purified Bt crystals and define a new and attractive pharmaceutical ingredient form of Bt Cry proteins. IMPORTANCE Bacillus thuringiensis crystal proteins are widely and safely used as insecticides. Recent studies have shown they also can cure gastrointestinal parasitic worm (nematode) infections when ingested. However, reproducible, scalable, and practical techniques for purifying these proteins have been lacking. Here, we address this severe limitation and present scalable and practical methods for large-scale purification of potently bioactive B. thuringiensis crystals and crystal proteins. The resultant product, called purified cytosolic crystals (PCC), is highly compatible with ingestible drug delivery and formulation. Furthermore, there are growing applications in agriculture and insect control where access to large quantities of purified crystal proteins is desirable and where these methods will find great utility.

Haemonchus contortus is a critical parasite of goats and sheep. Infection by this blood-feeding gastrointestinal nematode (GIN) parasite has significant health consequences, especially in lambs and kids. The parasite has developed resistance to virtually all known classes of small molecule anthelmintics used to treat it, giving rise in some areas to multidrug resistant parasites that are very difficult to control. Thus, new anthelmintics are urgently needed. Bacillus thuringiensis (Bt) crystal protein 5B (Cry5B), a naturally occurring protein made by a bacterium widely and safely used around the world as a bioinsecticide, represents a new non-small molecule modality for treating GINs. Cry5B has demonstrated anthelmintic activities against parasites of monogastric animals, including some related to those that infect humans, but has not yet been studied in a ruminant. Here we show that H. contortus adults are susceptible to Cry5B protein in vitro. Cry5B produced in its natural form as a spore-crystal lysate against H. contortus infections in goats had no significant efficacy. However, a new Active Pharmaceutical Ingredient (API) paraprobiotic form of Cry5B called IBaCC (Inactivated Bacterium with Cytosolic Crystals), in which Cry5B crystals are encapsulated in dead Bt cell wall ghosts, showed excellent efficacy in vitro against larval stages of H. contortus and relative protein stability in bovine rumen fluid. When given to sheep experimentally infected with H. contortus as three 60 mg/kg doses, Cry5B IBaCC resulted in significant reductions in fecal egg counts (90%) and parasite burdens (72%), with a very high impact on female parasites (96% reduction). These data indicate that Cry5B IBaCC is a potent new treatment tool for small ruminants in the battle against H. contortus.

Abstract Background The anti-inflammatory property of ω-3 polyunsaturated fatty acids (PUFA) has been exploited in the management of inflammatory bowel disease (IBD) with promising results. However, it remains unclear if PUFA play a significant role in the resolution of inflammation and promotion of mucosal healing. Krill oil (KO) is a natural product rich in PUFA and the potent antioxidant, astaxanthin. In this study, we attempted to understand the mechanisms through which KO modulates the gut microbiome and metabolome using in vitro and in vivo colitis models and a multi-omics based approach. Results KO significantly decreased LPS-induced IL1β and TNFα expression in human macrophages in vitro in a dose-dependent manner by regulating a broad spectrum of signaling pathways, including NF-κB and NOD-like receptor signaling, and displayed a synergistic effect with COX2 and IKK2 inhibitors in attenuating inflammatory pathways. Moreover, KO was involved in the resolution of inflammation by promoting M2 polarization and enhancing macrophage-mediated intracellular bacterial killing. Parasite-dependent intestinal mucosal damage and microbial dysbiosis induced by Trichuris suis infection in pigs were partially restored by feeding KO. KO supplementation reduced the abundance of Rickettsiales and several species of Lactobacillus, which were among the important features identified by random forests analysis contributing to classification accuracy for KO supplementation. Several microbial signatures with strong predictive power for the status of both infection and supplementation were identified. The inhibitory effect of KO on histidine metabolism was identified using untargeted metabolomics. KO supplementation reduced several key metabolites related to histamine metabolism by suppressing the expression of a gene encoding l-histidine decarboxylase in the colon mucosa and reducing histamine biosynthesis of microbial origin. Moreover, the pro-resolving properties of KO were validated using a Citrobacter rodentium-induced Th1-dependent colitis murine model. Further, microbial signatures with high prediction accuracy for colitis-related pathophysiological traits were identified in mice. Conclusion The findings from this study provided a mechanistic basis for optimizing microbiome-inspired alternative therapeutics in the management of IBD. The microbial signatures identified, particularly those with strong predictive accuracy for colitis phenotypes, will facilitate the development of biomarkers associated with appropriate dietary intervention to manage intestinal inflammation. Video abstract.

Summary: The thymus has a high capacity to support the differentiation of ILCs, especially when E protein transcription factors are ablated. Whether it contributes to the homeostasis of ILC pools in tissues is not clear. Single-cell RNA sequencing analysis shows a substantial amount of ILC precursors in wild type but not athymic nude blood. The precursors express CD3 intracellularly (ic) but not on the surface. The abundance of Lin−CD127+CD62L+icCD3ε+ precursors varies with age, peaking at 2–3 months. These cells can differentiate into various ILC subsets on OP9-DL1 stroma in vitro. In the lung, small intestine, and epidermis, icCD3ε+ cells differentiate into diverse ILC subsets in different tissue environments in steady state. Helminth infection promotes their differentiation toward functional ILC2s. Thus, the thymus appears to play a role in replenishing ILC pools in different peripheral tissues. Because thymic activity is age-dependent, this finding may help explain age-related differences in immune responses.

Over 33% of Americans are labeled as obese, leading the World Health Organization to designate obesity as a major public health problem. One consequence of obesity is the development of metabolic syndrome, a condition which has been correlated to an increased risk for developing cardiovascular disease and Type 2 diabetes. Prolonged ingestion of a higher-fat diet, one cause of obesity, results in alterations to the gut microbiome. These alterations are implicated to have a profound role in the evolution and progression of obesity-linked diseases. Probiotics are associated with positive health effects such as limiting pathogen colonization, aiding in digestion, and vitamin synthesis. Using Ossabaw pigs as a model for obesity, and in conjunction with our previous research, we performed an in-depth, nontargeted, metabolomic analysis on select organs to elucidate the effects of dietary supplementation with the probiotic Lacticaseibacillus paracasei. We focused our analysis on the effects of probiotic supplementation on a higher-fat (obesogenic) diet and a nutritionally balanced diet. Notably, our findings reveal that the brain cortex is highly sensitive to dietary influencers, and with probiotic supplementation, several aberrant metabolites associated with a higher-fat diet revert to healthy levels, thus demonstrating the potential for a probiotic intervention for obesity-linked disease.

Abstract The Phagocytosis of fungal structures by neutrophils is a well-documented function of these immune cells. However, neutrophil phagocytosis of hyphal structures in the urine sediment is not usually observed during routine sample evaluation. This is a case of hyphal phagocytosis by neutrophils in the urine of a kidney allograft recipient patient.

Ascaris and Parascaris are important parasites in the family Ascarididae, large, ubiquitous intestinal-dwelling nematodes infecting all classes of vertebrates. Parasitic nematode drug resistance in veterinary medicine and drug recalcitrance in human medicine are increasing worldwide, with few if any new therapeutic classes on the horizon. Some of these parasites are zoonotic, e.g., Ascaris is passed from humans to pigs and vice versa. The development of new therapies against this family of parasites would have major implications for both human and livestock health. Here we tested the therapeutic ability of a paraprobiotic or dead probiotic that expresses the Bacillus thuringiensis Cry5B protein with known anthelmintic properties, against zoonotic Ascaris suum and Parascaris spp. This paraprobiotic, known as IBaCC, intoxicated A. suum larvae in vitro and was highly effective in vivo against intestinal A. suum infections in a new mouse model for this parasite. Fermentation was scaled up to 350 l to treat pigs and horses. Single dose Cry5B IBaCC nearly completely cleared A. suum infections in pigs. Furthermore, single dose Cry5B IBaCC drove fecal egg counts in Parascaris-infected foals to zero, showing at least parity with, and potential superiority to, current efficacy of anthelmintics used against this parasite. Cry5B IBaCC therefore represents a new, paraprobiotic One Health approach towards targeting Ascarididae that is safe, effective, massively scalable, stable, and useful in human and veterinary medicine in both the developed and developing regions of the world.

Interaction between microorganisms and their surroundings are generally mediated via the cell wall or cell envelope. An understanding of the overall chemical composition of these surface layers may give clues on how these interactions occur and suggest mechanisms to manipulate them. This knowledge is key, for instance, in research aiming to reduce colonization of medical devices and device-related infections from different types of microorganisms. In this context, X-ray photoelectron spectroscopy (XPS) is a powerful technique as its analysis depth below 10 nm enables studies of the outermost surface structures of microorganism. Of specific interest for the study of biological systems is cryogenic XPS (cryo-XPS). This technique allows studies of intact fast-frozen hydrated samples without the need for pre-treatment procedures that may cause the cell structure to collapse or change due to the loss of water. Previously, cryo-XPS has been applied to study bacterial and algal surfaces with respect to their composition of lipids, polysaccharides and peptide (protein and/or peptidoglycan). This contribution focuses onto two other groups of microorganisms with widely different architecture and modes of life, namely fungi and viruses. It evaluates to what extent existing models for data treatment of XPS spectra can be applied to understand the chemical composition of their very different surface layers. XPS data from model organisms as well as reference substances representing specific building blocks of their surface were collected and are presented. These results aims to guide future analysis of the surface chemical composition of biological systems.

The human whipworm Trichuris trichiura infects 289 million people worldwide, resulting in substantial morbidity. Whipworm infections are difficult to treat due to low cure rates and high reinfection rates. Interactions between whipworm and its host’s intestinal microbiome present a potential novel target for infection control or prevention but are very complicated and are identified using inconsistent methodology and sample types across the literature, limiting their potential usefulness. Here, we used a combined 16S rRNA gene OTU analysis approach (QIIME2) for samples from humans and mice infected with whipworm (T. trichiura and T. muris, respectively) to identify for the first time, bacterial taxa that were consistently associated with whipworm infection spanning host species and infection status using four independent comparisons (baseline infected vs uninfected and before vs after deworming for both humans and mice). Using these four comparisons, we identified significant positive associations for seven taxa including Escherichia, which has been identified to induce whipworm egg hatching, and Bacteroides, which has previously been identified as a major component of the whipworm internal microbiome. We additionally identified significant negative associations for five taxa including four members of the order Clostridiales, two from the family Lachnospiraceae, including Blautia which was previously identified as positively associated with whipworm in independent human and mouse studies. Using this approach, bacterial taxa of interest for future association and mechanistic studies were identified, and several were validated by RT-qPCR. We demonstrate the applicability of a mouse animal model for comparison to human whipworm infections with respect to whipworm-induced intestinal microbiome disruption and subsequent restoration following deworming. Overall, the novel cross-species analysis approach utilized here provides a valuable research tool for studies of the interaction between whipworm infection and the host intestinal microbiome.

Intra-abdominal infection (peritonitis) is a leading cause of severe disease in surgical intensive care units, as over 70% of patients diagnosed with peritonitis develop septic shock. A critical role of the immune system is to return to homeostasis after combating infection. S100A8/A9 (calprotectin) is an antimicrobial and pro-inflammatory protein complex used as a biomarker for diagnosis of numerous inflammatory disorders. Here we describe the role of S100A8/A9 in inflammatory collateral tissue damage (ICTD). Using a mouse model of disseminated intra-abdominal candidiasis (IAC) in wild-type and S100A8/A9-deficient mice in the presence or absence of S100A9 inhibitor paquinimod, the role of S100A8/A9 during ICTD and fungal clearance were investigated. S100A8/A9-deficient mice developed less ICTD than wild-type mice. Restoration of S100A8/A9 in knockout mice by injection of recombinant protein resulted in increased ICTD and fungal clearance comparable to wild-type levels. Treatment with paquinimod abolished ICTD and S100A9-deficient mice showed increased survival compared to wild-type littermates. The data indicates that S100A8/A9 controls ICTD levels and antimicrobial activity during IAC and that targeting of S100A8/A9 could serve as promising adjunct therapy against this challenging disease.

Efforts to identify new drugs for therapeutic and preventive treatments against parasitic nematodes have gained increasing interest with expanding pathogen omics databases and drug databases from which new anthelmintic compounds might be identified. Here, a novel approach focused on integrating a pan-Nematoda multi-omics data targeted to a specific nematode organ system (the intestinal tract) with evidence-based filtering and chemogenomic screening was undertaken. Based on de novo computational target prioritization of the 3,564 conserved intestine genes in A. suum, exocytosis was identified as a high priority pathway, and predicted inhibitors of exocytosis were tested using the large roundworm (Ascaris suum larval stages), a filarial worm (Brugia pahangi adult and L3), a whipworm (Trichuris muris adult), and the non-parasitic nematode Caenorhabditis elegans. 10 of 13 inhibitors were found to cause rapid immotility in A. suum L3 larvae, and five inhibitors were effective against the three phylogenetically diverse parasitic nematode species, indicating potential for a broad spectrum anthelmintics. Several distinct pathologic phenotypes were resolved related to molting, motility, or intestinal cell and tissue damage using conventional and novel histologic methods. Pathologic profiles characteristic for each inhibitor will guide future research to uncover mechanisms of the anthelmintic effects and improve on drug designs. This progress firmly validates the focus on intestinal cell biology as a useful resource to develop novel anthelmintic strategies.

We previously demonstrated that IL4, IL13, CLCA1, and CCL26 mRNA were significantly upregulated in the lungs of pigs given a low dose of all trans-retinoic acid (ATRA) and infected with Ascaris suum. We also demonstrated that in vitro ATRA induced a state of partial alternative activation in porcine macrophages (Mφs) and amplified certain aspects of M2a activation induced by IL-4. Given these results, we tested the effect of ATRA on IL-4 responses in two porcine intestinal epithelial cell lines, IPEC1 and IPEC-J2 and observed that ATRA increased mRNA for the IL-4 receptor alpha chain. ATRA also increased IL-4 induced phosphorylation of signal transducer and activator of transcription 6 (STAT6) and mRNA expression of the chloride channel, calcium activated, family member 1 (CLCA1), important for mucus formation, and chemokine (C-C motif) ligand 26 (CCL26), a potent eosinophil chemoattractant. We extended these findings to human Mφ THP-1 cells and showed that ATRA synergistically increased IL-4–induced CCL2, CCL13, and CCL26 mRNA and protein levels. Transglutaminase 2 mRNA, protein, and enzyme activity were synergistically induced in THP-1 cells pretreated with ATRA and then treated with IL-4, thus, ATRA increased signaling in response to IL-4 in porcine epithelial cells and porcine and human Mφs. Given the prevalence of allergic and parasitic diseases worldwide and the close similarities in the porcine and human immune responses, these findings have important implications for the nutritional regulation of allergic inflammation at mucosal surfaces.

Recent observations of near-surface soil temperatures over the circumpolar Arctic show accelerated warming of permafrost-affected soils. The availability of a comprehensive near-surface permafrost and active layer dataset is critical to better understanding climate impacts and to constraining permafrost thermal conditions and its spatial distribution in land system models. We compiled a soil temperature dataset from 72 monitoring stations in Alaska using data collected by the U.S. Geological Survey, the National Park Service, and the University of Alaska Fairbanks permafrost monitoring networks. The array of monitoring stations spans a large range of latitudes from 60.9 to 71.3° N and elevations from near sea level to ∼ 1300 m, comprising tundra and boreal forest regions. This dataset consists of monthly ground temperatures at depths up to 1 m, volumetric soil water content, snow depth, and air temperature during 1997–2016. These data have been quality controlled in collection and processing. Meanwhile, we implemented data harmonization evaluation for the processed dataset. The final product (PF-AK, v0.1) is available at the Arctic Data Center (https://doi.org/10.18739/A2KG55).

Hookworms are intestinal nematode parasites that infect nearly half a billion people and are globally one of the most important contributors to iron-deficiency anemia. These parasites have significant impacts in developing children, pregnant women and working adults. Of all the soil-transmitted helminths or nematodes (STNs), hookworms are by far the most important, with disease burdens conservatively estimated at four million DALYs (Disability-Adjusted Life Years) and with productivity losses of up to US$139 billion annually. To date, mainly one drug, albendazole is used for hookworm therapy in mass drug administration, which has on average ∼80% cure rate that is lower (

Th2 cells are critical in the resolution of helminth infection and allergy. Here the authors show that Bcl11b is required to license the Th2 program of helper T cell differentiation and restrict alternate lineage gene expression using in vivo models of helminth infection and asthma.

Sterols, bile acids, and acylcarnitines are key players in human metabolism. Precise annotations of these metabolites with mass spectrometry analytics are challenging because of the presence of several isomers and stereoisomers, variability in ionization, and their relatively low concentrations in biological samples. Herein, we present a sensitive and simple qualitative LC–MS/MS (liquid chromatography with tandem mass spectrometry) method by utilizing a set of pure chemical standards to facilitate the identification and distribution of sterols, bile acids, and acylcarnitines in biological samples including human stool and plasma; mouse ileum, cecum, jejunum content, duodenum content, and liver; and pig bile, proximal colon, cecum, heart, stool, and liver. With this method, we detected 24 sterol, 32 bile acid, and 27 acylcarnitine standards in one analysis that were separated within 13 min by reversed-phase chromatography. Further, we observed different sterol, bile acid, and acylcarnitine profiles for the different biological samples across the different species. The simultaneous detection and annotation of sterols, bile acids, and acylcarnitines from reference standards and biological samples with high precision represents a valuable tool for screening these metabolites in routine scientific research.

Summary: Helminth infection is known for generating large amounts of poly-specific IgE. Here we demonstrate that innate-like B1 cells are responsible for this IgE production during infection with the nematode parasites Nippostrongylus brasiliensis and Heligmosomoides polygyrus bakeri. In vitro analysis of B1 cell immunoglobulin class switch recombination to IgE demonstrated a requirement for anti-CD40 and IL-4 that was further enhanced when IL-5 was added or when the B1 source was helminth infected mice. An IL-25-induced upregulation of IgE in B1 cells was also demonstrated. In T cell-reconstituted RAG1−/− mice, N. brasiliensis clearance was enhanced with the addition of B2 cells in an IgE-dependent manner. This enhanced clearance was impeded by reconstitution with IgE sufficient B1 cells. Mucosal mast cells mediated the B2 cell enhancement of clearance in the absence of B1 cells. The data support B1 cell IgE secretion as a regulatory response exploited by the helminth.

A targeted metabolomic analysis was performed on tissues derived from pigs fed diets supplemented with white button mushrooms (WBM) to determine the effect on the liver and brain metabolome. Thirty-one pigs were fed a grower diet alone or supplemented with either three or six servings of freeze-dried WBM for six weeks. Tissue metabolomes were analyzed using targeted liquid chromatography-mass spectrometry (LC-MS) combined with chemical similarity enrichment analysis (ChemRICH) and correlated to WBM-induced changes in fecal microbiome composition. Results indicated that WBM can differentially modulate metabolites in liver, brain cortex and hippocampus of healthy pigs. Within the glycero-phospholipids, there was an increase in alkyl-acyl-phosphatidyl-cholines (PC-O 40:3) in the hippocampus of pigs fed six servings of WBM. A broader change in glycerophospholipids and sphingolipids was detected in the liver with a reduction in several lipid species in pigs fed both WBM diets but with an increase in amino acids known as precursors of neurotransmitters in the cortex of pigs fed six servings of WBM. Metabolomic changes were positively correlated with increased abundance of Cryomorphaceae, Lachnospiraceae, Flammeovirgaceae and Ruminococcaceae in the microbiome suggesting that WBM can also positively impact tissue metabolite composition.

Abstract Background Neutrophils are traditionally considered transcriptionally inactive. Compared to other immune cells, little is known about their transcriptional profile during interaction with pathogens. Methods We analyzed the meta-transcriptome of the neutrophil-Candida albicans interplay and the transcriptome of C. albicans challenged with neutrophil extracellular traps (NETs) by RNA-Seq, considering yeast and hypha individually in each approach. Results The neutrophil response to C. albicans yeast and hyphae was dominated by a morphotype-independent core response. However, 11 % of all differentially expressed genes were regulated in a specific manner when neutrophils encountered the hyphal form of C. albicans. While involving genes for transcriptional regulators, receptors, and cytokines, the neutrophil core response lacked typical antimicrobial effectors genes. Genes of the NOD-like receptor pathway, including NLRP3, were enriched. Neutrophil- and NET-provoked responses in C. albicans differed. At the same time, the Candida transcriptome upon neutrophil encounter and upon NET challenge included genes from various metabolic processes and indicate a mutual role of the regulators Tup1p, Efg1p, Hap43p, and Cap1p. Upon challenge with neutrophils and NETs, the overall Candida response was partially morphotype-specific. Yet again, actual oppositional regulation in yeasts and hyphae was only detected for the arginine metabolism in neutrophil-infecting C. albicans. Conclusions Taken together, our study provides a comprehensive and quantitative transcript profile of the neutrophil–C. albicans interaction. By considering the two major appearances of both, neutrophils and C. albicans, our study reveals yet undescribed insights into this medically relevant encounter. Hence, our findings will facilitate future research and potentially inspire novel therapy developments.

Abstract Background Hookworms belonging to the genus Ancylostoma (Dubini, 1843) cause ancylostomiasis, a disease of considerable concern in humans and domestic and wild animals. Molecular and epidemiological data support evidence for the zoonotic potential among species of Ancylostoma where transmission to humans is facilitated by rapid urbanization and increased human-wildlife interactions. It is important to assess and describe these potential zoonotic parasite species in wildlife, especially in hosts that have physiological similarities to humans and share their habitat. Moreover, defining species diversity within parasite groups that can circulate among free-ranging host species and humans also provides a pathway to understanding the distribution of infection and disease. In this study, we describe a previously unrecognized species of hookworm in the genus Ancylostoma in the giant panda, including criteria for morphological and molecular characterization. Methods The hookworm specimens were obtained from a wild giant panda that died in the Fengtongzai Natural Reserve in Sichuan Province of China in November 2013. They were microscopically examined and then genetically analyzed by sequencing the nuclear internal transcribed spacer (ITS, ITS1-5.8S-ITS2) and mitochondrial cytochrome c oxidase subunit 1 (cox1) genes in two representative specimens (one female and one male, FTZ1 and FTZ2, respectively). Results Ancylostoma ailuropodae n. sp. is proposed for these hookworms. Morphologically the hookworm specimens differ from other congeneric species primarily based on the structure of the buccal capsule in males and females, characterized by 2 pairs of ventrolateral and 2 pairs of dorsolateral teeth; males differ in the structure and shape of the copulatory bursa, where the dorsal ray possesses 2 digitations. Pairwise nuclear and mitochondrial DNA comparisons, genetic distance analysis, and phylogenetic data strongly indicate that A. ailuropodae from giant pandas is a separate species which shared a most recent common ancestor with A. ceylanicum Looss, 1911 in the genus Ancylostoma (family Ancylostomatidae). Conclusion Ancylostoma ailuropodae n. sp. is the fourth species of hookworm described from the Ursidae and the fifteenth species assigned to the genus Ancylostoma. A sister-species association with A. ceylanicum and phylogenetic distinctiveness from the monophyletic Uncinaria Frölich, 1789 among ursids and other carnivorans indicate a history of host colonization in the evolutionary radiation among ancylostomatid hookworms. Further, phylogenetic relationships among bears and a history of ecological and geographical isolation for giant pandas may be consistent with two independent events of host colonization in the diversification of Ancylostoma among ursid hosts. A history for host colonization within this assemblage and the relationship for A. ailuropodae n. sp. demonstrate the potential of this species as a zoonotic parasite and as a possible threat to human health. The cumulative morphological, molecular and phylogenetic data presented for A. ailuropodae n. sp. provides a better understanding of the taxonomy, diagnostics and evolutionary biology of the hookworms.

Background: In 2013, a pregnancy exposure registry and birth defects surveillance (PER/BDS) system was initiated in eThekwini District, KwaZulu-Natal (KZN), to assess the impact of antiretroviral treatment (ART) on birth outcomes. Objectives: At the end of the first year, we assessed the risk of major congenital malformations (CM) and other adverse birth outcomes (ABOs) detected at birth, in children born to women exposed to ART during pregnancy. Method: Data were collected from women who delivered at Prince Mshiyeni Memorial Hospital, Durban, from 07 October 2013 to 06 October 2014, using medicine exposure histories and birth outcomes from maternal interviews, clinical records and neonatal surface examination. Singleton births exposed to only one ART regimen were included in bivariable analysis for CM risk and multivariate risk analysis for ABO risk. Results: Data were collected from 10 417 women with 10 517 birth outcomes (4013 [38.5%] HIV-infected). Congenital malformations rates in births exposed to Efavirenz during the first trimester (T1) (RR 0.87 [95% CI 0.12–6.4; p = 0.895]) were similar to births not exposed to ART during T1. However, T1 exposure to Nevirapine was associated with the increased risk of CM (RR 9.28 [95% CI 2.3–37.9; p = 0.002]) when compared to the same group. Other ABOs were more frequent in the combination of HIV/ART-exposed births compared to HIV-unexposed births (29.9% vs. 26.0%, adjusted RR 1.23 [1.14–1.31; p 0.001]). Conclusion: No association between T1 use of EFV-based ART regimens and CM was observed. Associations between T1 NVP-based ART regimen and CM need further investigation. HIV- and ART-exposed infants had more ABOs compared to HIV-unexposed infants.

Invasive mycoses remain underdiagnosed and difficult to treat. Hospitalized individuals with compromised immunity increase in number and constitute the main risk group for severe fungal infections. Current antifungal therapy is hampered by slow and insensitive diagnostics and frequent toxic side effects of standard antifungal drugs. Identification of new antifungal compounds with high efficacy and low toxicity is therefore urgently required. We investigated the antifungal activity of tempol, a cell-permeable nitroxide. To narrow down possible mode of action we used RNA-seq technology and metabolomics to probe for pathways specifically disrupted in the human fungal pathogen Candida albicans due to tempol administration. We found genes upregulated which are involved in iron homeostasis, mitochondrial stress, steroid synthesis, and amino acid metabolism. In an ex vivo whole blood infection, tempol treatment reduced C. albicans colony forming units and at the same time increased the release of pro-inflammatory cytokines, such as interleukin 8 (IL-8, monocyte chemoattractant protein-1, and macrophage migration inhibitory factor). In a systemic mouse model, tempol was partially protective with a significant reduction of fungal burden in the kidneys of infected animals during infection onset. The results obtained propose tempol as a promising new antifungal compound and open new opportunities for the future development of novel therapies.

Cryptococcosis, caused by the basidiomycete Cryptococcus neoformans, is a life-threatening disease affecting approximately one million people per year worldwide. Infection can occur when C. neoformans cells are inhaled by immunocompromised people. In order to establish infection, the yeast must bypass recognition and clearance by immune cells guarding the tissue. Using in vitro infections, we characterized the role of mast cells (MCs) in cryptococcosis. We found that MCs recognize C. neoformans and release inflammatory mediators such as tryptase and cytokines. From the latter group MCs released mainly CCL-2/MCP-1, a strong chemoattractant for monocytic cells. We demonstrated that supernatants of infected MCs recruit monocytes but not neutrophils. During infection with C. neoformans, MCs have a limited ability to kill the yeast depending on the serotype. C. neoformans, in turn, modulates the lifespan of MCs both, by presence of its polysaccharide capsule and by secreting soluble modulators. Taken together, MCs might have important contributions to fungal clearance during early stages of cryptocococis where these cells regulate recruitment of monocytes to mucosal tissues.

Terpenes are naturally occurring compounds produced by plants that are of great commercial interest in the food, agricultural, cosmetic, and pharmaceutical industries due to their broad spectra of antibacterial, antifungal, anthelmintic, membrane permeation enhancement, and antioxidant biological activities. Applications of terpenes are often limited by their volatility and the need for surfactants or alcohols to produce stable, soluble (non-precipitated) products. Yeast particles (YPs) are hollow, porous microspheres that have been used for the encapsulation of terpenes (YP terpenes) by passive diffusion of terpenes through the porous YP cell walls. We here report the development of a second generation YP encapsulated terpene technology that incorporates the stimuli-responsive control of terpene release using biodegradable pro-terpene compounds (YP pro-terpenes). YP terpenes and YP pro-terpenes were both produced, in which high levels of carvacrol, eugenol, thymol and geraniol were encapsulated. The YP pro-terpenes show higher encapsulation stability than YP terpenes due to pro-terpenes being non-volatile solids at room temperature and stable in suspensions at neutral pH. YP pro-terpenes and YP terpenes were evaluated for biological activity in antibacterial, antifungal and anthelmintic assays. The YP pro-terpenes retained the full biological activity of the parent terpene compound.

The screening of candidate compounds and natural products for anthelmintic activity is important for discovering new drugs against human and animal parasites. We previously validated in Caenorhabditis elegans a microfluidic device (‘chip’) that records non-invasively the tiny electrophysiological signals generated by rhythmic contraction (pumping) of the worm's pharynx. These electropharyngeograms (EPGs) are recorded simultaneously from multiple worms per chip, providing a medium-throughput readout of muscular and neural activity that is especially useful for compounds targeting neurotransmitter receptors and ion channels. Microfluidic technologies have transformed C. elegans research and the goal of the current study was to validate hookworm and Ascaris suum host-stage larvae in the microfluidic EPG platform. Ancylostoma ceylanicum and A. caninum infective L3s (iL3s) that had been activated in vitro generally produced erratic EPG activity under the conditions tested. In contrast, A. ceylanicum L4s recovered from hamsters exhibited robust, sustained EPG activity, consisting of three waveforms: (1) conventional pumps as seen in other nematodes; (2) rapid voltage deflections, associated with irregular contractions of the esophagus and openings of the esophogeal-intestinal valve (termed a ‘flutter’); and (3) hybrid waveforms, which we classified as pumps. For data analysis, pumps and flutters were combined and termed EPG ‘events.’ EPG waveform identification and analysis were performed semi-automatically using custom-designed software. The neuromodulator serotonin (5-hydroxytryptamine; 5HT) increased EPG event frequency in A. ceylanicum L4s at an optimal concentration of 0.5 mM. The anthelmintic drug ivermectin (IVM) inhibited EPG activity in a concentration-dependent manner. EPGs from A. suum L3s recovered from pig lungs exhibited robust pharyngeal pumping in 1 mM 5HT, which was inhibited by IVM. These experiments validate the use of A. ceylanicum L4s and A. suum L3s with the microfluidic EPG platform, providing a new tool for screening anthelmintic candidates or investigating parasitic nematode feeding behavior.

ABSTRACT Microbial colonizers of humans have evolved to adapt to environmental cues and to sense nutrient availability. Oxygen is a constantly changing environmental parameter in different host tissues and in different types of infection. We describe how Candida albicans, an opportunistic fungal pathogen, can modulate the host response under hypoxia and anoxia. We found that high infiltration of polymorphonuclear leukocytes (PMNs) to the site of infection contributes to a low oxygen milieu in a murine subdermal abscess. A persistent hypoxic environment did not affect viability or metabolism of PMNs. Under oxygen deprivation, however, infection with C. albicans disturbed specific PMN responses. PMNs were not able to efficiently phagocytose, produce ROS, or release extracellular DNA traps. Failure to launch an adequate response was caused by C. albicans cell wall masking of β-glucan upon exposure to low oxygen levels which hindered PAMP sensing by Dectin-1 on the surfaces of PMNs. This in turn contributed to immune evasion and enhanced fungal survival. The cell wall masking effect is prolonged by the accumulation of lactate produced by PMNs under low oxygen conditions. Finally, adaptation to oxygen deprivation increased virulence of C. albicans which we demonstrated using a Caenorhabditis elegans infection model. IMPORTANCE Successful human colonizers have evolved mechanisms to bypass immune surveillance. Infiltration of PMNs to the site of infection led to the generation of a low oxygen niche. Exposure to low oxygen levels induced fungal cell wall masking, which in turn hindered pathogen sensing and antifungal responses by PMNs. The cell wall masking effect was prolonged by increasing lactate amounts produced by neutrophil metabolism under oxygen deprivation. In an invertebrate infection model, C. albicans was able to kill infected C. elegans nematodes within 2 days under low oxygen conditions, whereas the majority of uninfected controls and infected worms under normoxic conditions survived. These results suggest that C. albicans benefited from low oxygen niches to increase virulence. The interplay of C. albicans with innate immune cells under these conditions contributed to the overall outcome of infection. Adaption to low oxygen levels was in addition beneficial for C. albicans by reducing susceptibility to selected antifungal drugs. Hence, immunomodulation of host cells under low oxygen conditions could provide a valuable approach to improve current antifungal therapies.

Neutrophils are crucial for the human innate immunity and constitute the majority of leukocytes in circulation. Thus, blood neutrophil counts serve as a measure for the immune system's functionality. Hematological patients often have low neutrophil counts due to disease or chemotherapy. To increase neutrophil counts and thereby preventing infections in high-risk patients, recombinant G-CSF is widely used as adjunct therapy to stimulate the maturation of neutrophils. In addition, G-CSF is utilized to recruit stem cells (SCs) into the peripheral blood of SC donors. Still, the actual functionality of neutrophils resulting from G-CSF treatment remains insufficiently understood. We tested the ex vivo functionality of neutrophils isolated from blood of G-CSF-treated healthy SC donors. We quantified chemotaxis, oxidative burst, and phagocytosis before and after treatment and detected significantly reduced chemotactic activity upon G-CSF treatment. Similarly, in vitro treatment of previously untreated neutrophils with G-CSF led to reduced chemotactic activity. In addition, we revealed that this effect persists in the allogeneic SC recipients up to 4 weeks after neutrophil engraftment. Our data indicates that neutrophil quantity, as a sole measure of immunocompetence in high-risk patients should be considered cautiously as neutrophil functionality might be affected by the primary treatment.

Nutritional immunity describes the host-driven manipulation of essential micronutrients, including iron, zinc and manganese. To withstand nutritional immunity and proliferate within their hosts, pathogenic microbes must express efficient micronutrient uptake and homeostatic systems. Here we have elucidated the pathway of cellular zinc assimilation in the major human fungal pathogen Candida albicans. Bioinformatics analysis identified nine putative zinc transporters: four cytoplasmic-import Zip proteins (Zrt1, Zrt2, Zrt3 and orf19.5428) and five cytoplasmic-export ZnT proteins (orf19.1536/Zrc1, orf19.3874, orf19.3769, orf19.3132 and orf19.52). Only Zrt1 and Zrt2 are predicted to localise to the plasma membrane and here we demonstrate that Zrt2 is essential for C. albicans zinc uptake and growth at acidic pH. In contrast, ZRT1 expression was found to be highly pH-dependent and could support growth of the ZRT2-null strain at pH 7 and above. This regulatory paradigm is analogous to the distantly related pathogenic mould, Aspergillus fumigatus, suggesting that pH-adaptation of zinc transport may be conserved in fungi and we propose that environmental pH has shaped the evolution of zinc import systems in fungi. Deletion of C. albicans ZRT2 reduced kidney fungal burden in wild type, but not in mice lacking the zinc-chelating antimicrobial protein calprotectin. Inhibition of zrt2Δ growth by neutrophil extracellular traps was calprotectin-dependent. This suggests that, within the kidney, C. albicans growth is determined by pathogen-Zrt2 and host-calprotectin. As well as serving as an essential micronutrient, zinc can also be highly toxic and we show that C. albicans deals with this potential threat by rapidly compartmentalising zinc within vesicular stores called zincosomes. In order to understand mechanistically how this process occurs, we created deletion mutants of all five ZnT-type transporters in C. albicans. Here we show that, unlike in Saccharomyces cerevisiae, C. albicans Zrc1 mediates zinc tolerance via zincosomal zinc compartmentalisation. This novel transporter was also essential for virulence and liver colonisation in vivo. In summary, we show that zinc homeostasis in a major human fungal pathogen is a multi-stage process initiated by Zrt1/Zrt2-cellular import, followed by Zrc1-dependent intracellular compartmentalisation.

Neutrophils have the ability to capture and kill microbes extracellularly through the formation of neutrophil extracellular traps (NETs). These are DNA and protein structures that neutrophils release extracellularly and are believed to function as a defense mechanism against microbes. The classic NET formation process, triggered by, e.g., bacteria, fungi, or by direct stimulation of protein kinase C through phorbol myristate acetate, is an active process that takes several hours and relies on the production of reactive oxygen species (ROS) that are further modified by myeloperoxidase (MPO). We show here that NET-like structures can also be formed by neutrophils after interaction with phenol-soluble modulin α (PSMα) that are cytotoxic membrane-disturbing peptides, secreted from community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA). The PSMα-induced NETs contained the typical protein markers and were able to capture microbes. The PSMα-induced NET structures were disintegrated upon prolonged exposure to DNase-positive S. aureus but not on exposure to DNase-negative Candida albicans. Opposed to classic NETosis, PSMα-triggered NET formation occurred very rapidly, independently of ROS or MPO, and was also manifest at 4°C. These data indicate that rapid NETs release may result from cytotoxic membrane disturbance by PSMα peptides, a process that may be of importance for CA-MRSA virulence.

By computational high-throughput screening, the spontaneous magnetization M s , uniaxial magnetocrystalline anisotropy constant K 1 , anisotropy field H a , and maximum energy product ( B H ) max are estimated for ferromagnetic intermetallic phases with a tetragonal 1-13-X structure related to the LaCo 9 Si 4 structure type. For SmFe 13 N, a ( B H ) max as high as that of Nd 2 Fe 14 B and a comparable K 1 are predicted. Further promising candidates of composition SmFe 12 AN with A = Co, Ni, Cu, Zn, Ga, Ti, V, Al, Si, or P are identified which potentially reach (BH) max values higher than 400 kJ/m 3 combined with significant K 1 values, while containing almost 50% less rare-earth atoms than Nd 2 Fe 14 B.

In the originally published version of this Article, the affiliation details for Dorina Avram incorrectly included “Department of Infectious Diseases and Immunology, College of Veterinary Medicine, University of Florida, 2015 SW 16th Ave, Gainesville, FL, 32608, USA”, instead of “UF Health Cancer Center, University of Florida, Gainesville, FL 32610, USA”. This has now been corrected in both the PDF and HTML versions of the Article.