Greater Protein Intake Emphasizing Lean Beef Does Not Affect Resistance Training-Induced Adaptations in Skeletal Muscle and Tendon of Older Women: A Randomized Controlled Feeding Trial.

BACKGROUND: Although experimental research supports that resistance training (RT), especially with greater dietary protein intake, improves muscle mass and strength in older adults, comparable research on tendons is needed. OBJECTIVES: We assessed the effects of a protein-rich diet emphasizing lean beef, compared with 2 control diets, on RT-induced changes in skeletal muscle and tendon size and strength in older women. METHODS: We randomly assigned women [age: 66 ± 1 y, body mass index (BMI): 28 ± 1] to groups that consumed 1) 0.8 g total protein/kg body weight/day from mixed food sources (normal protein control, n = 16); 2) 1.4 g/kg/d protein from mixed food sources (high protein control, n = 17); or 3) 1.4 g/kg/d protein emphasizing unprocessed lean beef (high protein experimental group, n = 16). Participants were provided with all foods and performed RT 3 times/wk, 70% of 1-repetition maximum for 12 wk. We measured quadriceps muscle volume via magnetic resonance imaging (MRI). We estimated patellar tendon biomechanical properties and cross-sectional area (CSA) using ultrasound and MRI. RESULTS: Dietary intake did not influence RT-induced increases in quadriceps strength (P < 0.0001) or muscle volume (P < 0.05). We noted a trend for an RT effect on mean tendon CSA (P = 0.07), with no differences among diets (P > 0.05). Proximal tendon CSA increased with RT (P < 0.05) with no difference between dietary groups (P > 0.05). Among all participants, midtendon CSA increased with RT (P ≤ 0.05). We found a decrease in distal CSA in the 0.8 g group (P < 0.05) but no change in the 1.4 g group (P > 0.05). Patellar tendon MRI signal or biomechanical properties were unchanged. CONCLUSIONS: Our findings indicated that greater daily protein intake, emphasizing beef, did not influence RT-induced changes in quadriceps muscle strength or muscle volume of older women. Although we noted trends in tendon CSA, we did not find a statistically significant impact of greater daily protein intake from beef on tendon outcomes. This trial was registered at clinicaltrials.gov as NCT04347447.

The Immune Response to Nematode Infection.

Nematode infection is a major threat to the health of humans, domestic animals and wildlife. Nematodes vary in their effect on the host and in the mechanisms underlying immunity but the general features are becoming clear. There is considerable variation among individuals in resistance to infection and much of this variation is due to genetic variation in the immune response. The major histocompatibility complex has a strong influence on resistance to infection but other genes are collectively more important. Resistant individuals produce more IgA, eosinophils, IgE and mast cells than susceptible individuals and this is a consequence of stronger type 2 (Th2) immune responses. A variety of factors promote Th2 responses including genetic background, diet, molecules produced by the parasite and the location of the infection. A variety of cells and molecules including proteins, glycolipids and RNA act in concert to promote responses and to regulate the response. Nematodes themselves also modulate the host response and over 20 parasite-derived immunomodulatory molecules have been identified. Different species of nematodes modulate the immune response in different ways and probably use multiple molecules. The reasons for this are unclear and the interactions among immunomodulators have still to be investigated.

The heritability of Nematodirus battus fecal egg counts.

Although Nematodirus battus is a serious threat to the health and survival of young lambs, there are few options to control this parasite. Bayesian Monte Carlo Markov Chain modelling with a zero-inflated Poisson distribution was used to estimate the heritability of egg counts in both June and July for each of five consecutive cohorts of 200 Scottish Blackface lambs. In one of the 10 analyses, the results failed the diagnostic tests. In seven of the analyses, there was no convincing evidence that the variation in egg counts was heritable. In the 2 years of high infection, the heritability was approximately 0.4 in June but the estimates lacked precision and the 95% highest posterior density credible intervals ranged from just above zero to 0.7. Selective breeding for resistance to N. battus will be difficult because genetically resistant or susceptible lambs cannot be consistently identified by phenotypic markers.

DRfit: a Java tool for the analysis of discrete data from multi-well plate assays.

BACKGROUND: Analyses of replicates in sets of discrete data, typically acquired in multi-well plate formats, is a recurring task in many contemporary areas in the Life Sciences. The availability of accessible cross-platform data analysis tools for such fundamental tasks in varied projects and environments is an important prerequisite to ensuring a reliable and timely turnaround as well as to provide practical analytical tools for student training. RESULTS: We have developed an easy-to-use, interactive software tool for the analysis of multiple data sets comprising replicates of discrete bivariate data points. For each dataset, the software identifies the replicate data points from a defined matrix layout and calculates their means and standard errors. The averaged values are then automatically fitted using either a linear or a logistic dose response function. CONCLUSIONS: DRfit is a practical and convenient tool for the analysis of one or multiple sets of discrete data points acquired as replicates from multi-well plate assays. The design of the graphical user interface and the built-in analysis features make it a flexible and useful tool for a wide range of different assays.

Identification of Fromiamycalin and Halaminol A from Australian Marine Sponge Extracts with Anthelmintic Activity against Haemonchus contortus.

There is an urgent need to discover and develop new anthelmintics for the treatment of parasitic nematodes of veterinary importance to circumvent challenges linked to drug resistant parasites. Being one of the most diverse natural ecosystems, the marine environment represents a rich resource of novel chemical entities. This study investigated 2000 extracts from marine invertebrates, collected from Australian waters, for anthelmintic activity. Using a well-established in vitro bioassay, these extracts were screened for nematocidal activity against Haemonchus contortus-a socioeconomically important parasitic nematode of livestock animals. Extracts (designated Mu-1, Ha-1 and Ha-2) from two marine sponges (Monanchora unguiculata and Haliclona sp.) each significantly affected larvae of H. contortus. Individual extracts displayed a dose-dependent inhibition of both the motility of exsheathed third-stage larvae (xL3s) and the development of xL3s to fourth-stage larvae (L4s). Active fractions in each of the three extracts were identified using bioassay-guided fractionation. From the active fractions from Monanchora unguiculata, a known pentacyclic guanidine alkaloid, fromiamycalin (1), was purified. This alkaloid was shown to be a moderately potent inhibitor of L4 development (half-maximum inhibitory concentration (IC50) = 26.6 ± 0.74 µM) and L4 motility (IC50 = 39.4 ± 4.83 µM), although it had a relatively low potency at inhibiting of xL3 motility (IC50 ≥ 100 µM). Investigation of the active fractions from the two Haliclona collections led to identification of a mixture of amino alcohol lipids, and, subsequently, a known natural product halaminol A (5). Anthelmintic profiling showed that 5 had limited potency at inhibiting larval development and motility. These data indicate that fromiamycalin, other related pentacyclic guanidine alkaloids and/or halaminols could have potential as anthelmintics following future medicinal chemistry efforts.

Deguelin exerts potent nematocidal activity via the mitochondrial respiratory chain.

As a result of limited classes of anthelmintics and an over-reliance on chemical control, there is a great need to discover new compounds to combat drug resistance in parasitic nematodes. Here, we show that deguelin, a plant-derived rotenoid, selectively and potently inhibits the motility and development of nematodes, which supports its potential as a lead candidate for drug development. Furthermore, we demonstrate that deguelin treatment significantly increases gene transcription that is associated with energy metabolism, particularly oxidative phosphorylation and mitoribosomal protein production before inhibiting motility. Mitochondrial tracking confirmed enhanced oxidative phosphorylation. In accordance, real-time measurements of oxidative phosphorylation in response to deguelin treatment demonstrated an immediate decrease in oxygen consumption in both parasitic (Haemonchus contortus) and free-living (Caenorhabditis elegans) nematodes. Consequently, we hypothesize that deguelin is exerting its toxic effect on nematodes as a modulator of oxidative phosphorylation. This study highlights the dynamic biologic response of multicellular organisms to deguelin perturbation.-Preston, S., Korhonen, P. K., Mouchiroud, L., Cornaglia, M., McGee, S. L., Young, N. D., Davis, R. A., Crawford, S., Nowell, C., Ansell, B. R. E., Fisher, G. M., Andrews, K. T., Chang, B. C. H., Gijs, M. A. M., Sternberg, P. W., Auwerx, J., Baell, J., Hofmann, A., Jabbar, A., Gasser, R. B. Deguelin exerts potent nematocidal activity via the mitochondrial respiratory chain.

Spatiotemporal Labeling of Melanocytes in Mice.

Melanocytes are pigment producing cells in the skin that give rise to cutaneous malignant melanoma, which is a highly aggressive and the deadliest form of skin cancer. Studying melanocytes in vivo is often difficult due to their small proportion in the skin and the lack of specific cell surface markers. Several genetically-engineered mouse models (GEMMs) have been created to specifically label the melanocyte compartment. These models give both spatial and temporal control over the expression of a cellular 'beacon' that has an added benefit of inducible expression that can be activated on demand. Two powerful models that are discussed in this review include the melanocyte-specific, tetracycline-inducible green fluorescent protein expression system (iDct-GFP), and the fluorescent ubiquitination-based cell cycle indicator (FUCCI) model that allows for the monitoring of the cell-cycle. These two systems are powerful tools in studying melanocyte and melanoma biology. We discuss their current uses and how they could be employed to help answer unresolved questions in the fields of melanocyte and melanoma biology.

Practical and low cost whole-organism motility assay: A step-by-step protocol.

Here, we provide a step-by-step protocol for a practical and low cost whole-organism assay for the screening of chemical compounds for activity against parasitic worms. This assay has considerable advantages over conventional methods, mainly in relation to ease of use, throughput, time and cost. It is readily suited to the screening of hundreds to thousands of compounds for subsequent hit-to-lead optimisation, and should be applicable to many different parasites and other organisms commensurate with the size of wells in the microtiter plates used for phenotypic screening.

Strongyle egg shedding and egg reappearance periods in horses with pituitary pars intermedia dysfunction.

Pituitary pars intermedia dysfunction (PPID) is the most common endocrine disorder of older horses. Immune dysfunction in horses with PPID could increase susceptibility to infectious diseases, including strongyle infections; however, few data are available. The aim of this study was to determine if horses with PPID had increased strongyle faecal egg counts (FEC) compared with control horses, over a fourteen-week period in Victoria, Australia. Clinical signs and plasma adrenocorticotropic hormone (ACTH) concentrations were used to categorise horses into PPID (n=14) or control (n=31) groups. Faecal samples were collected for FEC determination prior to anthelmintic treatment, and fortnightly post-treatment for each horse. Generalised linear mixed modelling, using a gamma distribution, was used to compare differences between groups in the repeated measures study. The confounding variable of age was controlled for as a fixed effect. Following anthelmintic treatment, mean FEC was greater for the PPID group compared to the control group on day 56 (405 ± 756 eggs per gram [EPG] vs 40 ± 85 EPG, p=0.05) and day 70 (753 ±1598 EPG vs 82 ±141 EPG, p=0.04). There were no differences in mean FEC between groups on days 84 and 98. Cumulative FEC (day 14 to day 98) was significantly greater for the PPID horses than control horses (2118 ± 4016 EPG vs 798 ± 768 EPG, p<0.0001). Group egg reappearance period was shorter for PPID horses (day 56 post-anthelmintic treatment) compared to control horses (day 70) and 30% of the PPID horses reached a FEC threshold of >200 EPG on day 42, compared to 0% of control horses (p=0.02). These results suggest that the rate of a re-established patent infection between groups could be different due to a comprised immune response in PPID horses or differences in the host-parasite relationship regarding encysted stage larvae. However, despite differences between groups, some horses with PPID consistently had no detectable or low FEC (<200 EPG) during the study period. These findings highlight the importance of individual FEC monitoring to determine if anthelmintic treatment is required, in line with sustainable parasite management practices.

Evaluation of the Role of Galectins in Parasite Immunity.

Galectin-11 (LGALS-11) and galectin-14 (LGALS-14) are ruminant specific galectins, first reported in sheep. Although their roles in parasite immunity are still being elucidated, it appears that they influence protection against parasites. In gastrointestinal infections with the nematode Haemonchus contortus, both galectin-11 and galectin-14 appear to be protective. However, in a chronic infection of liver fluke, Fasciola hepatica, these galectins may aid parasite survival. To unravel the structural, functional, and ligand profile of galectin-11 and galectin-14, recombinant production of these proteins is vital. Here we present the recombinant production of soluble galectin-11 and galectin-14 from domestic sheep for in vitro and structural biology studies. These methods include parasite cultivation and infection, galectin staining of host and parasite tissue, surface staining of parasites with recombinant galectins, pull-down assays to identify endogenous galectin binding proteins, and in vitro assays to monitor the effect of galectins on parasite development.

Quantifying the sources of variation in eosinophilia among Scottish blackface lambs with mixed, predominantly Teladorsagia circumcincta nematode infection.

Eosinophils play a key role in defence against gastrointestinal nematodes. There is considerable variation among animals in the intensity of eosinophilia following nematode infection. However, the statistical distribution of eosinophils among animals has still to be determined. A better description of the variation among animals could provide biological insight and determine the most appropriate way to analyse the effect of eosinophils. We estimated blood eosinophil numbers in a flock of Scottish Blackface sheep that were naturally exposed to mixed, predominantly Teladorsagia circumcincta infection. Three of the four eosinophil counts were better described by a gamma distribution than by a lognormal distribution. The scale and shape parameters of the gamma distribution varied over time. Eosinophil counts differed among animals kept on separate fields before weaning and between singletons and twins but were not significantly different between years and genders. Eosinophil counts also differed among offspring from different sires and dams. The parameters of the gamma distribution were used to enable a power analysis. Large numbers of animals were required to reliably detect even large differences between two groups. These results indicate that methods appropriate for gamma distributions, such as generalized linear mixed models, will provide more reliable inferences than traditional methods of analysis and experimental design.

Advances in the discovery and development of anthelmintics by harnessing natural product scaffolds.

Widespread resistance to currently-used anthelmintics represents a major obstacle to controlling parasitic nematodes of livestock animals. Given the reliance on anthelmintics in many control regimens, there is a need for the continued discovery and development of new nematocides. Enabling such a focus are: (i) the major chemical diversity of natural products; (ii) the availability of curated, drug-like extract-, fraction- and/or compound-libraries from natural sources; (iii) the utility and practicality of well-established whole-worm bioassays for Haemonchus contortus-an important parasitic nematodes of livestock-to screen natural product libraries; and (iv) the availability of advanced chromatographic (HPLC), spectroscopic (NMR) and spectrometric (MS) techniques for bioassay-guided fractionation and structural elucidation. This context provides a sound basis for the identification and characterisation of anthelmintic candidates from natural sources. This chapter provides a background on the importance and impact of helminth infections/diseases, parasite control and aspects of drug discovery, and reviews recent work focused on (i) screening well-defined compound libraries to establish the methods needed for large-scale screening of natural extract libraries; (ii) discovering plant and marine extracts with nematocidal or nematostatic activity, and purifying bioactive compounds and assessing their potential for further development; and (iii) synthesising analogues of selected purified natural compounds for the identification of possible 'lead' candidates. The chapter describes some lessons learned from this work and proposes future areas of focus for drug discovery. Collectively, the findings from this recent work show potential for selected natural product scaffolds as candidates for future development. Developing such candidates via future chemical optimisation, efficacy and safety evaluations, broad spectrum activity assessments, and target identification represents an exciting prospect and, if successful, could pave the way to subsequent pre-clinical and clinical evaluations.

1-Methyl-1H-pyrazole-5-carboxamide Derivatives Exhibit Unexpected Acute Mammalian Toxicity.

A series of 1-methyl-1H-pyrazole-5-carboxamides were synthesized as potent inhibitors of the parasitic nematode of sheep, Haemonchus contortus. These compounds did not show overt cytotoxicity to a range of mammalian cell lines under standard in vitro culture conditions, had high selectivity indices, and were progressed to an acute toxicity study in a rodent model. Strikingly, acute toxicity was observed in mice. Experiments measuring cellular respiration showed a dose-dependent inhibition of mitochondrial respiration. Under these conditions, potent cytotoxicity was observed for these compounds in rat hepatocytes suggesting that the potent acute mammalian toxicity of this chemotype is most likely associated with respiratory inhibition. In contrast, parasite toxicity was not correlated to acute toxicity or cytotoxicity in respiring cells. This paper highlights the importance of identifying an appropriate in vitro predictor of in vivo toxicity early on in the drug discovery pipeline, in particular assessment for in vitro mitochondrial toxicity.

Beta-glycoglycosphingolipid-induced alterations of the STAT signaling pathways are dependent on CD1d and the lipid raft protein flotillin-2.

Beta-glucosylceramide has been shown to affect natural killer T cell function in models of inflammation. We, therefore, investigated the effects of different beta-glycosphingolipids, including beta-glucosylceramide, on STAT (signal transducers and activators of transcription) signaling pathways and determined whether these effects were mediated by lipid raft microdomains and/or CD1d molecules. The effects of alpha- and beta-structured ligands on the lipid raft protein flotillin-2 were studied in both natural killer T hybridoma cells and leptin-deficient mice. To determine whether CD1d was involved in the effects of the beta-glycosphingolipids, an anti-CD1d blocking antibody was used in a cell proliferation assay system. The downstream effects on the protein phosphorylation levels of STAT1, STAT3, and STAT6 were examined in both immune-mediated hepatitis and hepatoma models. The effects of beta-glycosphingolipids on the STAT signaling pathways were found to be dependent on CD1d. Lipid rafts were affected by both the dose and ratio of the beta-glycosphingolipids and the acyl chain length, and these effects were followed by downstream effects on STAT proteins. Our results show that beta-glycosphingolipids have beneficial effects in natural killer T cell-dependent immune-mediated metabolic and malignant animal models in vivo.

A perspective on the discovery of selected compounds with anthelmintic activity against the barber's pole worm-Where to from here?

Parasitic roundworms (nematodes) cause substantial morbidity and mortality in animals worldwide. Anthelmintic treatment is central to controlling these worms, but widespread resistance to most of the commercially available anthelmintics for veterinary and agricultural use is compromising control, such that there is an urgency to discover new and effective drugs. The purpose of this article is to review information on parasitic nematodes, the treatment and control of parasitic nematode infections and aspects of discovering new anthelmintics in the context of anthelmintic resistance problems, and then to discuss some progress that our group has made in identifying selected compounds with activity against nematodes. The focus of our recent work has been on discovering new chemical entities and known drugs with anthelmintic activities against Haemonchus contortus as well as other socioeconomically important parasitic nematodes for subsequent development. Using whole worm-based phenotypic assays, we have been screening compound collections obtained via product-development-partnerships and/or collaborators, and active compounds have been assessed for their potential as anthelmintic candidates. Following the screening of 15,333 chemicals from five distinct compound collections against H. contortus, we have discovered one new chemical entity (designated SN00797439), two human kinase inhibitors (SNS-032 and AG-1295), 14 tetrahydroquinoxaline analogues, one insecticide (tolfenpyrad) and two tolfenpyrad (pyrazole-5-carboxamide) derivatives (a-15 and a-17) with anthelmintic activity in vitro. Some of these 20 'hit' compounds have selectivity against H. contortus in vitro when compared to particular human cell lines. In our opinion, some of these compounds could represent starting points for 'lead' development. Accordingly, the next research steps to be pursued include: (i) chemical optimisation of representative chemicals via structure-activity relationship (SAR) evaluations; (ii) assessment of the breadth of spectrum of anthelmintic activity on a range of other parasitic nematodes, such as strongyloids, ascaridoids, enoplids and filarioids; (iii) detailed investigations of the absorption, distribution, metabolism, excretion and toxicity (ADMET) of optimised chemicals with broad nematocidal or nematostatic activity; and (iv) establishment of the modes of action of lead candidates.

Synthesis and structure-activity relationship study of pyrrolidine-oxadiazoles as anthelmintics against Haemonchus contortus.

Parasitic roundworms (nematodes) are significant pathogens of humans and animals and cause substantive socioeconomic losses due to the diseases that they cause. The control of nematodes in livestock animals relies heavily on the use of anthelmintic drugs. However, their extensive use has led to a widespread problem of drug resistance in these worms. Thus, the discovery and development of novel chemical entities for the treatment of parasitic worms of humans and animals is needed. Herein, we describe our medicinal chemistry optimization efforts of a phenotypic hit against Haemonchus contortus based on a pyrrolidine-oxadiazole scaffold. This led to the identification of compounds with potent inhibitory activities (IC50 = 0.78-22.4 µM) on the motility and development of parasitic stages of H. contortus, and which were found to be highly selective in a mammalian cell counter-screen. These compounds could be used as suitable chemical tools for drug target identification or as lead compounds for further optimization.

The oligomeric assembly of galectin-11 is critical for anti-parasitic activity in sheep (Ovis aries).

Galectins are a family of glycan-binding molecules with a characteristic affinity for ß-D-glycosides that mediate a variety of important cellular functions, including immune and inflammatory responses. Galectin-11 (LGALS-11) has been recently identified as a mediator induced specifically in animals against gastrointestinal nematodes and can interfere with parasite growth and development. Here, we report that at least two natural genetic variants of LGALS-11 exist in sheep, and demonstrate fundamental differences in anti-parasitic activity, correlated with their ability to dimerise. This study improves our understanding of the role of galectins in the host immune and inflammatory responses against parasitic nematodes and provides a basis for genetic studies toward selective breeding of animals for resistance to parasites.

Modulation of intracellular machinery by beta-glycolipids is associated with alteration of NKT lipid rafts and amelioration of concanavalin-induced hepatitis.

UNLABELLED: The integrity of lipid rafts in cell membranes is important for signal transduction. AIM: To determine the distinct effects of beta-glycolipids on the composition of lipid rafts in natural killer T (NKT) cells and on the level of expression of flotillin-2, leukocyte-specific protein tyrosine kinase (Lck), and STAT1-associated pathways. METHODS: The effects of glycolipids were determined by composition analysis of the raft domains, FACS analysis of the distribution patterns for the raft ganglioside, GM1, and fluorescence microscopy of raft patching. To evaluate the effects of the immune environment on glycolipid-associated alteration of lipid rafts, hepatitis was induced by an intravenous injection of concanavalin A (ConA) in mice treated with various glycolipids. RESULTS: The administration of beta-glucosylceramide, beta-lactosylceramide, and a combination of both significantly altered GM1 content in lymphocyte membranes in an environment-dependent manner. These effects were associated with altered expression levels of flotillin-2, Lck, and STAT1, and with a significant decrease in intrahepatic CD8+ lymphocyte trapping and the alleviation of ConA-induced hepatitis. The administration of alpha-glycolipids failed to induce similar effects. CONCLUSIONS: The alteration in the expression levels of flotillin-2, Lck, and STAT1 that occurs concomitantly with changes in lipid raft composition and structure following the administration of beta-glycolipids in ConA-induced hepatitis is microenvironment-dependent and is associated with decreased intrahepatic CD8(+) T lymphocyte trapping and amelioration of immune-mediated hepatitis.

Macroenvironment-gene-microenvironment interactions in ultraviolet radiation-induced melanomagenesis.

Cutaneous malignant melanoma is one of the few major cancers that continue to exhibit a positive rate of increase in the developed world. A wealth of epidemiological data has undisputedly implicated ultraviolet radiation (UVR) from sunlight and artificial sources as the major risk factor for melanomagenesis. However, the molecular mechanisms of this cause-and-effect relationship remain murky and understudied. Recent efforts on multiple fronts have brought unprecedented expansion of our knowledge base on this subject and it is now clear that melanoma is caused by a complex interaction between genetic predisposition and environmental exposure, primarily to UVR. Here we provide an overview of the effects of the macroenvironment (UVR) on the skin microenvironment and melanocyte-specific intrinsic (mostly genetic) landscape, which conspire to produce one of the deadliest malignancies.

The current status of anthelmintic resistance in a temperate region of Australia; implications for small ruminant farm management.

Widespread anthelmintic resistance in small ruminants is a constraint on the profitability of the meat/wool industry. Limited published data is available on the prevalence and efficacy of anthelmintics, particularly in Australia where parasites affecting ruminant systems vary greatly between geographic regions. This paper reports on the anthelmintic resistance status in a temperate region of Victoria, Australia, a major sheep producing state largely affected by Trichostrongylus species and Teladorsagia circumcincta. The prevalence of anthelmintic resistance to any product was high (71%), with farms reporting varying levels of drug efficacies (21-100%). Resistance to older chemical groups (i.e. fenbendazole and levamisole) and single active macrocyclic lactone treatments was higher than newer chemical groups and combination treatments. This report provides clarity on anthelmintic resistance in the temperate region of Victoria and more importantly suggests that more comprehensive, regional specific anthelmintic resistance studies are required to understand the real level of chemical resistance threatening the effective control of worms.