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Cellular senescence is linked to chronic age-related diseases including atherosclerosis, diabetes, and neurodegeneration. Compared to proliferating cells, senescent cells express distinct subsets of proteins. In this study, we used cultured human diploid fibroblasts rendered senescent through replicative exhaustion or ionizing radiation to identify proteins differentially expressed during senescence. We identified acid ceramidase (ASAH1), a lysosomal enzyme that cleaves ceramide into sphingosine and fatty acid, as being highly elevated in senescent cells. This increase in ASAH1 levels in senescent cells was associated with a rise in the levels of ASAH1 mRNA and a robust increase in ASAH1 protein stability. Furthermore, silencing ASAH1 in pre-senescent fibroblasts decreased the levels of senescence proteins p16, p21, and p53, and reduced the activity of the senescence-associated β-galactosidase. Interestingly, depletion of ASAH1 in pre-senescent cells sensitized these cells to the senolytics Dasatinib and Quercetin (D+Q). Together, our study indicates that ASAH1 promotes senescence, protects senescent cells, and confers resistance against senolytic drugs. Given that inhibiting ASAH1 sensitizes cells towards senolysis, this enzyme represents an attractive therapeutic target in interventions aimed at eliminating senescent cells.

The role of brain cholesterol metabolism in Alzheimer’s disease (AD) remains unclear. Peripheral and brain cholesterol levels are largely independent due to the impermeability of the blood brain barrier (BBB), highlighting the importance of studying the role of brain cholesterol homeostasis in AD. We first tested whether metabolite markers of brain cholesterol biosynthesis and catabolism were altered in AD and associated with AD pathology using linear mixed-effects models in two brain autopsy samples from the Baltimore Longitudinal Study of Aging (BLSA) and the Religious Orders Study (ROS). We next tested whether genetic regulators of brain cholesterol biosynthesis and catabolism were altered in AD using the ANOVA test in publicly available brain tissue transcriptomic datasets. Finally, using regional brain transcriptomic data, we performed genome-scale metabolic network modeling to assess alterations in cholesterol biosynthesis and catabolism reactions in AD. We show that AD is associated with pervasive abnormalities in cholesterol biosynthesis and catabolism. Using transcriptomic data from Parkinson’s disease (PD) brain tissue samples, we found that gene expression alterations identified in AD were not observed in PD, suggesting that these changes may be specific to AD. Our results suggest that reduced de novo cholesterol biosynthesis may occur in response to impaired enzymatic cholesterol catabolism and efflux to maintain brain cholesterol levels in AD. This is accompanied by the accumulation of nonenzymatically generated cytotoxic oxysterols. Our results set the stage for experimental studies to address whether abnormalities in cholesterol metabolism are plausible therapeutic targets in AD.

Aptamer-based proteomics revealed differentially abundant proteins in Alzheimer’s disease (AD) brains in the Baltimore Longitudinal Study of Aging and Religious Orders Study (mean age, 89 ± 9 years...

Description, Brain proteins altered in young APOE ε4 carriers are found decades later in Alzheimer’s disease and present novel drug targets., Aptamer-based proteomics revealed differentially abundant proteins in Alzheimer’s disease (AD) brains in the Baltimore Longitudinal Study of Aging and Religious Orders Study (mean age, 89 ± 9 years). A subset of these proteins was also differentially abundant in the brains of young APOE ε4 carriers relative to noncarriers (mean age, 39 ± 6 years). Several of these proteins represent targets of approved and experimental drugs for other indications and were validated using orthogonal methods in independent human brain tissue samples as well as in transgenic AD models. Using cell culture–based phenotypic assays, we showed that drugs targeting the cytokine transducer STAT3 and the Src family tyrosine kinases, YES1 and FYN, rescued molecular phenotypes relevant to AD pathogenesis. Our findings may accelerate the development of effective interventions targeting the earliest molecular triggers of AD.

Black-knobbed map turtles (Graptemys nigrinoda Cagle) and Alabama map turtles (Graptemys pulchra Baur) were infected with several blood flukes in Alabama (southeastern North America). Spirorchis paraminutus Roberts & Bullard n. sp. differs from its congeners by having a body that is 12-24× longer than wide, a testicular column of 10 testes that is 1/5-1/4 of the body length and located far posterior to the caecal bifurcation (the anterior-most testis is located in the posterior body half), and a common genital pore that is ventral to the ovary and 1/4-1/3 of the body length from the posterior extremity. These turtles and an Escambia map turtle (Graptemys ernsti Lovich & McCoy) were infected with Spirorchis elegans Stunkard, 1923, Spirorchis scripta Stunkard, 1923 and two innominate species of Spirorchis MacCallum, 1918. Phylogenetic analyses of the internal transcribed spacer region (ITS2) and large subunit rDNA (28S) recovered a monophyletic Spirorchis and the new species sister to Spirorchis collinsi Roberts & Bullard, 2016.

Background While Alzheimer disease (AD) and vascular dementia (VaD) may be accelerated by hypercholesterolemia, the mechanisms underlying this association are unclear. We tested whether dysregulation of cholesterol catabolism, through its conversion to primary bile acids (BAs), was associated with dementia pathogenesis. Methods and findings We used a 3-step study design to examine the role of the primary BAs, cholic acid (CA), and chenodeoxycholic acid (CDCA) as well as their principal biosynthetic precursor, 7α-hydroxycholesterol (7α-OHC), in dementia. In Step 1, we tested whether serum markers of cholesterol catabolism were associated with brain amyloid accumulation, white matter lesions (WMLs), and brain atrophy. In Step 2, we tested whether exposure to bile acid sequestrants (BAS) was associated with risk of dementia. In Step 3, we examined plausible mechanisms underlying these findings by testing whether brain levels of primary BAs and gene expression of their principal receptors are altered in AD. Step 1: We assayed serum concentrations CA, CDCA, and 7α-OHC and used linear regression and mixed effects models to test their associations with brain amyloid accumulation (N = 141), WMLs, and brain atrophy (N = 134) in the Baltimore Longitudinal Study of Aging (BLSA). The BLSA is an ongoing, community-based cohort study that began in 1958. Participants in the BLSA neuroimaging sample were approximately 46% male with a mean age of 76 years; longitudinal analyses included an average of 2.5 follow-up magnetic resonance imaging (MRI) visits. We used the Alzheimer’s Disease Neuroimaging Initiative (ADNI) (N = 1,666) to validate longitudinal neuroimaging results in BLSA. ADNI is an ongoing, community-based cohort study that began in 2003. Participants were approximately 55% male with a mean age of 74 years; longitudinal analyses included an average of 5.2 follow-up MRI visits. Lower serum concentrations of 7α-OHC, CA, and CDCA were associated with higher brain amyloid deposition (p = 0.041), faster WML accumulation (p = 0.050), and faster brain atrophy mainly (false discovery rate [FDR] p = 4 million registrants from general practice clinics in the United Kingdom, we tested whether patients using BAS (BAS users; 3,208 with ≥2 prescriptions), which reduce circulating BAs and increase cholesterol catabolism, had altered dementia risk compared to those on non-statin lipid-modifying therapies (LMT users; 23,483 with ≥2 prescriptions). Patients in the study (BAS/LMT) were approximately 34%/38% male and with a mean age of 65/68 years; follow-up time was 4.7/5.7 years. We found that BAS use was not significantly associated with risk of all-cause dementia (hazard ratio (HR) = 1.03, 95% confidence interval (CI) = 0.72–1.46, p = 0.88) or its subtypes. We found a significant difference between the risk of VaD in males compared to females (p = 0.040) and a significant dose–response relationship between BAS use and risk of VaD (p-trend = 0.045) in males. Step 3: We assayed brain tissue concentrations of CA and CDCA comparing AD and control (CON) samples in the BLSA autopsy cohort (N = 29). Participants in the BLSA autopsy cohort (AD/CON) were approximately 50%/77% male with a mean age of 87/82 years. We analyzed single-cell RNA sequencing (scRNA-Seq) data to compare brain BA receptor gene expression between AD and CON samples from the Religious Orders Study and Memory and Aging Project (ROSMAP) cohort (N = 46). ROSMAP is an ongoing, community-based cohort study that began in 1994. Participants (AD/CON) were approximately 56%/36% male with a mean age of 85/85 years. In BLSA, we found that CA and CDCA were detectable in postmortem brain tissue samples and were marginally higher in AD samples compared to CON. In ROSMAP, we found sex-specific differences in altered neuronal gene expression of BA receptors in AD. Study limitations include the small sample sizes in the BLSA cohort and likely inaccuracies in the clinical diagnosis of dementia subtypes in primary care settings. Conclusions We combined targeted metabolomics in serum and amyloid positron emission tomography (PET) and MRI of the brain with pharmacoepidemiologic analysis to implicate dysregulation of cholesterol catabolism in dementia pathogenesis. We observed that lower serum BA concentration mainly in males is associated with neuroimaging markers of dementia, and pharmacological lowering of BA levels may be associated with higher risk of VaD in males. We hypothesize that dysregulation of BA signaling pathways in the brain may represent a plausible biologic mechanism underlying these results. Together, our observations suggest a novel mechanism relating abnormalities in cholesterol catabolism to risk of dementia., Vijay Varma and co-workers study cholesterol and bile acid metabolism in dementia., Author summary Why was this study done? Hypercholesterolemia is associated with increased risk of Alzheimer disease (AD) and vascular dementia (VaD). However, cholesterol is impermeable to the blood–brain barrier (BBB), and it is unclear how peripheral cholesterol mediates risk of dementia. While prior research has examined the relationship between de novo cholesterol biosynthesis and dementia, few studies have assessed the role of cholesterol catabolism and its principal breakdown products, oxysterols, and primary bile acids (BAs) in dementia. What did the researchers do and find? We examined the role of cholesterol catabolism in dementia pathogenesis by first testing the association between serum oxysterols and BAs and neuroimaging markers of dementia. We also tested whether exposure to bile acid sequestrants (BAS) was associated with risk of dementia in a large, real-world clinical dataset. Finally, we tested plausible mechanisms underlying these associations by examining whether primary BAs and mRNA of their receptors were altered in the brain in dementia. We found that lower serum levels of 7α-hydroxycholesterol (7α-OHC) and primary BAs were associated with higher brain amyloid deposition, faster WML accumulation, and faster brain atrophy mainly in males. Consistent with this finding, we observed a sex difference in the association between use of BAS and risk of VaD. We found that primary BAs were detectable in the brain, and levels of gene expression of BA receptors were altered in AD mainly in males. What do these findings mean? Our findings suggest that cholesterol catabolism and BA synthesis may impact dementia progression through sex-specific effects on signaling pathways in the brain. These results set the stage for experimental studies to test whether BA signaling in the brain may be a novel therapeutic target in dementia.

Rapid lifestyle and dietary changes have contributed to a rise in the global prevalence of metabolic syndrome (MetS), which presents a potential healthcare crisis, owing to its association with an increased burden of multiple cardiovascular and neurological diseases. Prior work has identified the role that genetic, lifestyle, and environmental factors can play in the prevalence of MetS. Metabolomics is an important tool to study alterations in biochemical pathways intrinsic to the pathophysiology of MetS. We undertook a metabolomic study of MetS in serum samples from two ethnically distinct, well-characterized cohorts&mdash, the Baltimore Longitudinal Study of Aging (BLSA) from the U.S. and the Tsuruoka Metabolomics Cohort Study (TMCS) from Japan. We used multivariate logistic regression to identify metabolites that were associated with MetS in both cohorts. Among the top 25 most significant (lowest p-value) metabolite associations with MetS in each cohort, we identified 18 metabolites that were shared between TMCS and BLSA, the majority of which were classified as amino acids. These associations implicate multiple biochemical pathways in MetS, including branched-chain amino acid metabolism, glutathione production, aromatic amino acid metabolism, gluconeogenesis, and the tricarboxylic acid cycle. Our results suggest that fundamental alterations in amino acid metabolism may be central features of MetS.

While the relationship between obesity and reproductive dysfunction is well known, the physiological mechanism behind obesity-related infertility remains unclear. Previous work suggests that follicle development prior to ovulation is disrupted in obese individuals. Follicle-stimulating hormone (FSH) and anti-Mullerian hormone (AMH) are two key regulators of follicle development, and the poorest reproductive outcomes have been recorded when these hormones are imbalanced. In order to understand how obesity impacts the reproductive axis, the present study induces reproductive dysfunction in female rats using a high-fat, high-sugar diet (HFHS).

River cooters (Pseudemys concinna [Le Conte, 1830]) from the Pascagoula River (30°37′07.67″N; 88°36′44.53″W; Mississippi, U.S.A.) were infected by Spirorchis scripta Stunkard, 1923, and Spirorchis testiplexus n. sp. The new species resembles Spirorchis artericola (Ward, 1921) Stunkard, 1921, and Spirorchis innominatus Ward, 1921 (type species), by having a median esophageal diverticulum, ceca that extend slightly posterior to the genitalia, 10 testes, and a testicular column that extends anteriad to near the cecal bifurcation. Based on a comparative morphological study of existing type materials, vouchers, and published descriptions of similar congeners, S. testiplexus is unique by the combination of having a proportionally wider body, laterally directed cecal diverticula, deeply lobed testes, a testicular column that fills the intercecal space, and an external seminal vesicle that occupies the space between the posterior-most testis and the ovary and that passes dorsal or dorsolateral to the ovary. The large-subunit rDNA (28S) Bayesian inference phylogenetic analysis (including 16 turtle blood fluke species of 7 genera) showed a strongly supported monophyletic Spirorchis MacCallum, 1918, grouping sister to Spirhapalum siamensis Tkach, Snyder, and Vaughn, 2009.

Diet-induced obesity has been associated with various metabolic and reproductive disorders, including polycystic ovary syndrome. However, the mechanisms by which obesity influences the reproductive system are still not fully known. Studies have suggested that impairments in hormone signaling are associated with the development of symptoms such as acyclicity and ovarian cysts. However, these studies have often failed to address how these hormonal changes arise and how they might contribute to the progression of reproductive diseases. In the present study, we used a high-fat, high-sugar (HFHS) diet to induce obesity in a female rodent model to determine the changes in critical reproductive hormones that might contribute to the development of irregular estrous cycling and reproductive cycle termination. The HFHS animals exhibited impaired estradiol, progesterone (P4), and luteinizing hormone (LH) surges before ovulation. The HFHS diet also resulted in altered basal levels of testosterone (T) and LH. Furthermore, alterations in the basal P4/T ratio correlated strongly with ovarian cyst formation in HFHS rats. Thus, this model provides a method to assess the underlying etiology of obesity-related reproductive dysfunction and to examine an acyclic reproductive phenotype as it develops., A high-fat, high-sugar diet led to disrupted preovulatory and basal hormone levels and induced cystic ovaries in a rodent model of diet-induced obesity.

Acipensericola glacialis n. sp. infects the heart of lake sturgeon, Acipenser fulvescens (Rafinesque), in the Lake Winnebago System and differs from its only congener, Acipensericola petersoni Bullard, Snyder, Jensen & Overstreet, 2008, by having a dendritic intestine, deeply-lobed testes, a post-ovarian ootype, and a common genital pore that is medial to the dextral caecum. Acipensericola petersoni has a non-dendritic intestine, testes that are not deeply lobed, an ootype that is at level of the ovary (ventral to the ovary), and a common genital pore that is dorsal to the dextral caecum. Comparison of the large (28S) and small (18S) sub-unit ribosomal DNA and internal transcribed spacer 2 (ITS2) regions between specimens of A. glacialis n. sp. and A. petersoni revealed 13 (of 1,621 nt; 99.2% similarity in the 28S), 8 (of 1,841 nt; 99.9% similarity in the 18S), and 11 (of 442 nt; 97.5% similarity in the ITS2) nucleotide differences. Collectively, these results comprise an unexpectedly high degree of morphological and molecular similarity given the geographical (Mississippi River Basin vs Great Lakes Basin) and phylogenetic (Polyodontidae vs Acipenseridae) separation of these hosts but seemingly did not reject a previous hypothesis concerning lake sturgeon dispersal from the Mississippi Refugium following the Wisconsin glaciation ~18,000 years ago. The new species is the first nominal blood fluke described from a sturgeon.

Aims Products of lipid oxidation, such as 4-hydroxynonenal (4-HNE), are key activators of hepatic stellate cells (HSC) to a pro-fibrogenic phenotype. Isolevuglandins (IsoLG) are a family of acyclic γ-ketoaldehydes formed through oxidation of arachidonic acid or as by-products of the cyclooxygenase pathway. IsoLGs are highly reactive aldehydes which are efficient at forming protein adducts and cross-links at concentrations 100-fold lower than 4-hydroxynonenal. Since the contribution of IsoLGs to liver injury has not been studied, we synthesized 15-E 2 -IsoLG and used it to investigate whether IsoLG could induce activation of HSC. Results Primary human HSC were exposed to 15-E 2 -IsoLG for up to 48 h. Exposure to 5 μM 15-E 2 -IsoLG in HSCs promoted cytotoxicity and apoptosis. At non-cytotoxic doses (50 pM-500 nM) 15-E 2 -IsoLG promoted HSC activation, indicated by increased expression of α-SMA, sustained activation of ERK and JNK signaling pathways, and increased mRNA and/or protein expression of cytokines and chemokines, which was blocked by inhibitors of JNK and NF-kB. In addition, IsoLG promoted formation of reactive oxygen species, and induced an early activation of ER stress, followed by autophagy. Inhibition of autophagy partially reduced the pro-inflammatory effects of IsoLG, suggesting that it might serve as a cytoprotective response. Innovation This study is the first to describe the biological effects of IsoLG in primary HSC, the main drivers of hepatic fibrosis. Conclusions IsoLGs represent a newly identified class of activators of HSC in vitro , which are biologically active at concentrations as low as 500 pM, and are particularly effective at promoting a pro-inflammatory response and autophagy.

Two new genera and species of freshwater turtle blood flukes (TBFs) are described herein based on specimens infecting the nephritic and mesenteric blood vessels of "matamatas" (a side-necked turtle

High-fat, high-sugar (HFHS) diet induces a number of metabolic dysfunctions. Furthermore, there is evidence that diet-induced obesity correlates with diminished reproductive outcomes, though the mechanism remains unclear. We utilized a female, HFHS rat model to determine the effects of adiposity on ovarian morphology and hormonal balance. Because animals were on an ad libitum diet, we observed a gradient of obesity and adiposity, as well as ovarian dysfunction. We explored the distribution of these variables in order to establish a potential mechanism for reproductive dysfunction in our HFHS model. To determine the effect of a HFHS diet on ovarian structures, adiposity, and follicle-relevant hormones, female Sprague-Dawley rats were separated into two diet groups. The control group (n=14) received standard chow and water ad libitum while the HFHS group (n=16) received 60% of calories from fat, 30% sucrose solution, and water ad libitum. Weights were recorded every other day, and estrous cycles were monitored through daily vaginal cytology. During the course of the study, a fraction of animals ceased cycling. Terminal blood samples were taken on diestrus-2. AMH and FSH levels in the plasma were measured using ELISA. Ovaries and abdominal fat pads were collected and sectioned for histological analysis. At the conclusion of the study, HFHS rats gained significantly more weight than controls and displayed larger abdominal and gonadal fat pads. Rats were divided into three groups based on estrous cycle status: non-cycling (n=7), HFHS cycling (n=8), and control cycling (n=7). Non-cycling and HFHS rats displayed different distributions in adipocyte size relative to controls, indicating a shift towards larger cell size. On average, non-cyclers and HFHS had more cysts then controls, and non-cyclers had fewer corpora lutea compared to HFHS and controls. While AMH and FSH levels did not differ between groups, the ratio of FSH to AMH significantly correlated with follicular structures. The FSH/AMH ratio displayed strong negative correlations with total follicle number (r=-0.72, p

Parasite prevalence is thought to be positively related to host population density owing to enhanced contagion. However, the relationship between prevalence and local abundance of multiple host species is underexplored. We surveyed birds and their haemosporidian parasites (genera Plasmodium and Haemoproteus) at multiple sites across eastern North America to test whether the prevalence of these parasites in a host species at a particular site is related to that host's local abundance. Prevalence was positively related to host abundance within most sites, although the effect was stronger and more consistent for Plasmodium than for Haemoproteus. In contrast, prevalence was not related to variation in the abundance of most individual host species among sites across the region. These results suggest that parasite prevalence partly reflects the relative abundances of host species in local assemblages. However, three nonnative host species had low prevalence despite being relatively abundant at one site, as predicted by the enemy release hypothesis.

Baracktrema obamai n. gen., n. sp. infects the lung of geoemydid turtles (black marsh turtle, Siebenrockiella crassicollis [type host] and southeast Asian box turtle, Cuora amboinensis ) in the Malaysian states of Perak, Perlis, and Selangor. Baracktrema and Unicaecum Stunkard, 1925 are the only accepted turtle blood fluke genera having the combination of a single cecum, single testis, oviducal seminal receptacle, and uterine pouch. Baracktrema differs from Unicaecum by having a thread-like body approximately 30-50× longer than wide and post-cecal terminal genitalia. Unicaecum has a body approximately 8-12× longer than wide and terminal genitalia that are anterior to the distal end of the cecum. The new genus further differs from all other accepted turtle blood fluke genera by having a cecum that is highly convoluted for its entire length, a spindle-shaped ovary between the cirrus sac and testis, a uterine pouch that loops around the primary vitelline collecting duct, a Laurer's canal, and a dorsal common genital pore. Phylogenetic analysis of the D1-D3 domains of the nuclear large subunit ribosomal DNA (28S) revealed, with high nodal support and as predicted by morphology, that Baracktrema and Unicaecum share a recent common ancestor and form a clade sister to the freshwater turtle blood flukes of Spirorchis, paraphyletic Spirhapalum, and Vasotrema and that, collectively, these flukes were sister to all other tetrapod blood flukes (Hapalorhynchus + Griphobilharzia plus the marine turtle blood flukes and schistosomes). Pending a forthcoming emended morphological diagnosis of the family, the clade including Spirorchis spp., paraphyletic Spirhapalum, Vasotrema, Baracktrema, and Unicaecum is a likely placeholder for "Spirorchiidae Stunkard, 1921 " (type genus Spirorchis MacCallum, 1918 ; type species Spirorchis innominatus Ward, 1921 ). The present study comprises the 17th blood fluke known to infect geoemydid turtles and the first proposal of a new genus of turtle blood fluke in 21 yr.

Haemosporidian parasites (genera Plasmodium and Haemoproteus) are common blood parasites of birds transmitted by dipteran insect vectors. We analyzed blood samples from 329 individuals of 43 bird species in eastern Tennessee to better understand the relationship between the local community of birds and their blood parasites, including the distribution of parasites across hosts and the underlying ecological factors and life -history traits that influence parasite prevalence across host species. Using molecular methods, we found 144 individuals of 25 species to be infected with haemosporidian parasites (overall prevalence of 44 %). We distinguished 22 genetic lineages, including 11 in the genus Haemoproteus and 11 in Plasmodium. Fourteen percent of infected individuals harbored more than one parasite lineage. Across species, total prevalence increased with local abundance and decreased with incubation period, but did not vary with nesting or foraging height, average annual survival of host species, migratory or flocking behavior, sexual dimorphism, average species mass, or among sites. The prevalence of Haemoproteus was higher in species that nest 1–5 m above ground than in species that nest below 1 m or above 5 m, and the prevalence of Plasmodium was marginally higher in species with open-cup nests. Infection status did not vary with age, sex, or body condition. Our research reveals substantial variation in prevalence and richness of haemosporidian parasites, some of which is related to specific avian life history traits.

The drivers of regional parasite distributions are poorly understood, especially in comparison with those of free-living species. For vector-transmitted parasites, in particular, distributions might be influenced by host-switching and by parasite dispersal with primary hosts and vectors. We surveyed haemosporidian blood parasites (Plasmodium and Haemoproteus) of small land birds in eastern North America to characterize a regional parasite community. Distributions of parasite populations generally reflected distributions of their hosts across the region. However, when the interdependence between hosts and parasites was controlled statistically, local host assemblages were related to regional climatic gradients, but parasite assemblages were not. Moreover, because parasite assemblage similarity does not decrease with distance when controlling for host assemblages and climate, parasites evidently disperse readily within the distributions of their hosts. The degree of specialization on hosts varied in some parasite lineages over short periods and small geographic distances independently of the diversity of available hosts and potentially competing parasite lineages. Nonrandom spatial turnover was apparent in parasite lineages infecting one host species that was well-sampled within a single year across its range, plausibly reflecting localized adaptations of hosts and parasites. Overall, populations of avian hosts generally determine the geographic distributions of haemosporidian parasites. However, parasites are not dispersal-limited within their host distributions, and they may switch hosts readily.

We used microscopy and molecular biology to provide the first documentation of infections of Myxobolus cerebralis (Myxozoa: Myxobolidae), the etiological agent of whirling disease, in trout (Salmonidae) from North Carolina (USA) river basins. A total of 1085 rainbow trout Oncorhynchus mykiss, 696 brown trout Salmo trutta, and 319 brook trout Salvelinus fontinalis from 43 localities across 9 river basins were screened. Myxospores were observed microscopically in pepsin-trypsin digested heads of rainbow and brown trout from the Watauga River Basin. Those infections were confirmed using the prescribed nested polymerase chain reaction (PCR; 18S rDNA), which also detected infections in rainbow, brown, and brook trout from the French Broad River Basin and the Yadkin Pee-Dee River Basin. Myxospores were 9.0-10.0 µm (mean ± SD = 9.6 ± 0.4; N = 119) long, 8.0-10.0 µm (8.8 ± 0.6; 104) wide, and 6.0-7.5 µm (6.9 ± 0.5; 15) thick and had polar capsules 4.0-6.0 µm (5.0 ± 0.5; 104) long, 2.5-3.5 µm (3.1 ± 0.3; 104) wide, and with 5 or 6 polar filament coils. Myxospores from these hosts and rivers were morphologically indistinguishable and molecularly identical, indicating conspecificity, and the resulting 18S rDNA and ITS-1 sequences derived from these myxospores were 99.5-100% and 99.3-99.8% similar, respectively, to published GenBank sequences ascribed to M. cerebralis. This report comprises the first taxonomic circumscription and molecular confirmation of M. cerebralis in the southeastern USA south of Virginia.

Gulf Coast spiny softshell turtles, Apalone spinifera aspera (Agassiz, 1857) (Testudines: Trionychidae) from Canoe Lake (33°47′56.16″N, 86°29′25.02″W; Springville, Alabama) and Round Lake (32°41′50.91″N, 87°14′30.39″W; Perry Lakes State Park, Marion, Alabama), were infected by V. robustum Stunkard, 1928, Vasotrema longitestis Byrd, 1939, and Vasotrema rileyae n. sp. The new species differs from its congeners by having papillate suckers, a short testis, an ovary dextral to the oviduct, and a pre-ovarian genital pore that is lateral to the ventral sucker. We studied the newly collected specimens and museum specimens of all congeners to revise the diagnosis of Vasotrema Stunkard, 1926 and redescribe and provide an updated dichotomous key to all species of the genus.

Hapalorhynchus Stunkard, 1922 is emended based on morphological study of existing museum specimens (type and voucher specimens) and newly-collected specimens infecting musk turtles (Testudines: Kinosternidae: Sternotherus spp.) from rivers in Alabama and Florida (USA). Hapalorhynchus conecuhensis n. sp. is described from an innominate musk turtle, Sternotherus cf. minor, (type host) from Blue Spring (31°5'27.64″N, 86°30'53.21″W; Pensacola Bay Basin, Alabama) and the loggerhead musk turtle, Sternotherus minor (Agassiz, 1857) from the Wacissa River (30°20'24.73″N, 83°59'27.56″W; Apalachee Bay Basin, Florida). It differs from congeners by lacking a body constriction at level of the ventral sucker, paired anterior caeca, and a transverse ovary as well as by having a small ventral sucker, proportionally short posterior caeca, nearly equally-sized anterior and posterior testes, a small cirrus sac, and a uterus extending dorsal to the ovary and the anterior testis. Specimens of Hapalorhynchus reelfooti Byrd, 1939 infected loggerhead musk turtles, stripe-necked musk turtles (Sternotherus peltifer Smith and Glass, 1947), Eastern musk turtles (Sternotherus odoratus [Latreille in Sonnini and Latreille, 1801]), and S. cf. minor. Those of Hapalorhynchus cf. stunkardi infected S. minor and S. odoratus. Sternothorus minor, S. peltifer, and S. cf. minor plus S. minor and S. odoratus are new host records for H. reelfooti and H. cf. stunkardi, respectively. This is the first report of an infected musk turtle from the Coosa and Tallapoosa Rivers (Mobile-Tensaw River Basin), Pensacola Bay Basin, or Apalachee Bay Basin. Sequence analysis of the large subunit rDNA (28S) showed a strongly-supported clade for Hapalorhynchus.

Salmincola californiensis infected 25 of 31 (prevalence 0.8; intensity 2-35 [mean 6.6 ± standard deviation 7.7; n = 25]) rainbow trout, Oncorhynchus mykiss, from a private trout farm connected to the Watauga River, North Carolina. Salmincola edwardsii infected all of 9 (1.0; 2-43 [9.3 ± 13.0; 9]) brook trout, Salvelinus fontinalis, from Big Norton Prong, a tributary of the Little Tennessee River, North Carolina. Both lernaeopodids are well-known salmonid pathogens, but neither is native to, nor has been previously taxonomically confirmed from, the southeastern United States. Herein, we (1) use light and scanning electron microscopy to identify and provide supplemental morphological observations of these lernaeopodids, (2) furnish complementary molecular sequence data from the 28S rDNA (28S), and (3) document the pathological effects of gill infections. We identified and differentiated these lernaeopodids by the second antenna (exopod tip with large [S. californiensis] vs. slender [S. edwardsii] spines; endopod terminal segment with subequal ventral processes shorter than [S. californiensis] vs. longer than or equal to [S. edwardsii] dorsal hook), maxilliped palp (length typically ≤1/3 [S. californiensis] vs. 1/3-1/2 [S. edwardsii] subchela length exclusive of claw), and bulla (sub-circular and concave on manubrium's side [S. californiensis] vs. non-stellate [S. edwardsii]). Analysis of the 28S rDNA sequences confirmed our taxonomic assignments as demonstrated by 100% sequence similarity among the sympatric, morphologically-conspecific isolates. Histopathology revealed focal gill epithelial hyperplasia, obstruction of interlamellar water channels, lamellar fusion, and crypting of gill filaments. High intensity infections by either lernaeopodid are surveillance-worthy because they are potentially pathogenic to trout in the southeastern United States.

Chicken turtles, Deirochelys reticularia (Latreille in Sonnini et Latreille) (Testudines: Emydidae) from Alabama, USA were infected by Spirorchis collinsi Roberts et Bullard sp. n. and Spirorchis cf. scripta. The new species is most easily differentiated from its congeners by the combination of having caeca that extend far beyond the genitalia, intercaecal genitalia positioned in the middle portion of the body, a testicular column that nearly abuts the caecal bifurcation, a cirrus sac positioned between the testes and ovary, a massive Mehlis' gland, an elongate, longitudinal metraterm that extends anteriad beyond the level of the ovary, a pre-ovarian genital pore, and a prominent, intercaecal Manter's organ. The specimens of S. cf. scripta differed from the holotype and published descriptions of Spirorchis scripta Stunkard, 1923 by several subtle morphological features, perhaps comprising intraspecific variation, but collectively warranted a detailed description herein. Based on examinations of the aforementioned specimens plus the holotype, paratypes and vouchers of morphologically-similar congeners, Spirorchis MacCallum, 1918 is emended to include the presence of oral sucker spines, a pharynx, lateral oesophageal diverticula ('plicate organ') and a median oesophageal diverticulum ('oeseophageal pouch'). Phylogenetic analysis of the nuclear large subunit rDNA (28S) recovered S. collinsi sister to Spirorchis picta Stunkard, 1923, > 99% similarity between S. cf. scripta and S. scripta, and a monophyletic Spirorchis MacCallum, 1918. No blood fluke infection has been reported previously from these drainages, Alabama, or this turtle species. This is the first new species of Spirorchis to be described from North America in 26 years.

Two new genera and species of freshwater turtle blood flukes (TBFs) are described herein based on specimens infecting the nephritic and mesenteric blood vessels of “matamatas” (a side-necked turtle, Chelus fimbriata [Schneider, 1783] [Pleurodira: Chelidae]) from the Amazon River Basin, Peru. These taxa comprise the first-named species and the first-proposed genera of freshwater TBFs from the continent of South America. A new comparison of all TBF genera produced 6 morphologically diagnosed groups that are discussed in light of previous TBF classification schemes and a novel phylogenetic hypothesis based on the nuclear large subunit ribosomal DNA (28S). Considering external and internal anatomical features, species of the new genera (Atamatam Bullard and Roberts n. gen., Paratamatam Bullard and Roberts n. gen.) are most similar to each other and are together most similar to those of several marine TBF genera. The 28S phylogenetic analysis supported the monophyly of all 6 morphologically diagnosed groups of genera. Most notably, the freshwater TBFs of South America comprise a derived group nested within the clade that includes the paraphyletic marine TBFs. Not surprisingly in light of morphology, another marine TBF lineage (Neospirorchis Price, 1934) clustered with the freshwater TBFs of Baracktrema Roberts, Platt, and Bullard, 2016 and Unicaecum Stunkard, 1925. Our results, including an ancestral state reconstruction, indicated that (1) freshwater TBFs have colonized marine turtles twice independently and that (2) the South American freshwater TBFs comprise a marine-derived lineage. This is the first evidence that TBFs have twice independently transitioned from a marine to freshwater definitive host. Marine incursion is considered as a possible mechanism affecting the natural history of marine-derived freshwater TBFs in South America. A dichotomous key to accepted TBF genera is provided.

Coeuritrema Mehra, 1933, previously regarded as a junior subjective synonym of Hapalorhynchus Stunkard, 1922, herein is revised to include Coeuritrema lyssimus Mehra, 1933 (type species), Coeuritrema rugatus (Brooks et Sullivan, 1981) comb. n., and Coeuritrema platti Roberts et Bullard sp. n. These genera are morphologically similar by having a ventral sucker, non-fused caeca, two testes, a pre-testicular cirrus sac, an intertesticular ovary, and a common genital pore that opens dorsally and in the sinistral half of the body. Phylogenetic analysis of the D1-D3 domains of the nuclear large subunit ribosomal DNA (28S) suggested that Coeuritrema and Hapalorhynchus share a recent common ancestor. Coeuritrema is morphologically most easily differentiated from Hapalorhynchus by having ventrolateral tegumental papillae and a definitive metraterm that is approximately 3-7× longer than the uterus. Coeuritrema comprises species that reportedly infect Asiatic softshell turtles (Testudines: Trionychidae) only, whereas Hapalorhynchus (as currently defined) comprises blood flukes that reportedly infect those hosts plus North American musk turtles (Sternotherus Bell in Gray) and mud turtles (Kinosternon Spix), both Kinosternidae, North American snapping turtles (Chelydridae), Asiatic hard-shelled turtles (Geoemydidae) and African pleurodirans (Pelomedusidae). Coeuritrema platti sp. n. infects the blood of Chinese softshell turtles, Pelodiscus sinensis (Wiegmann), cultured in the Da Rang River Basin (Phu Yen Province, Vietnam). It differs from C. lyssimus by having a narrow hindbody (< 1.6× forebody width), ventrolateral tegumental papillae restricted to the hindbody, a short cirrus sac (< 10% of corresponding body length), a transverse ovary buttressing the caeca, a short, wholly pre-ovarian metraterm (~ 10% of corresponding body length), and a submarginal genital pore. It differs from C. rugatus by having small ventrolateral tegumental papillae, testes without deep lobes, and a Laurer's canal pore that opens posterior to the vitelline reservoir and dorsal to the oviducal seminal receptacle. The new species is only the second turtle blood fluke reported from Vietnam.

Adaptive immunity and especially T lymphocytes play a crucial role in the development of hypertension. Proteins that are oxidatively modified by highly reactive isolevuglandins (isoLGs) accumulate in dendritic cells & lead to subsequent activation of T lymphocytes. This process can be prevented by compounds known to scavenge isoLGs, such as 2-hydroxybenzylamine (2-HOBA). The overall goal of current study is to develop novel antihypertensive drugs based on this isoLG scavenging strategy, that will not only prevent but will also reverse hypertension and its associated inflammatory end-organ damage. Initially, the preventative effects of 10 putative isoLG scavengers were tested. C57Bl/6 mice received angiotensin (Ang) II (490 ng/kg/min) infusion for 14 days. Each compound was administered in the drinking water (1mg/ml) at the onset of Ang II infusion for 7 days to a half of the mice & for entire 14 day period to another half of the animals. Blood pressure was measured by tail cuff method. Among 10 compounds, we found that 2-HOBA, methyl 2HOBA (Me2HOBA) & 3-methoxy2-HOBA (3-Mo2HOBA) were most effective in lowering blood pressure. Interestingly, the pyridoxamine analogs were not effectively lowered blood pressure. We further examined the efficacy of our 3 most effective compounds in reversing established hypertension. Mice received Ang II infusion for 6 weeks and received each study drug (2 mg/ml) during the last 4 weeks. We employed radiotelemetry to monitor blood pressure. Compared with untreated mice, Me2-HOBA, 3-Mo2-HOBA & 2-HOBA were equally effective in lowering blood pressure by 20 mmHg (all p < 0.05 vs no drug). Six weeks of Ang II infusion caused 2- to 4-fold increases in renal T cell (CD3 + ) & monocyte/macrophage (F4/80 + ) infiltration as measured by immunohistochemistry & all three compounds markedly reduced these by 50%. These treatments also reduced aortic fibrosis as measured by Masson’s Trichrome blue staining. We conclude that these isoLG scavengers can be potentially used as new effective therapy for lowering blood pressure & attenuating hypertensive renal & vascular damage. Variability of in vivo effectiveness for the different scavengers likely reflects differences in bioavailability due to structure & lypophilicity.

Background Asthma is associated with oxidant stress and diminished antioxidant defenses. Yet, the mechanistic role of oxidant stress and antioxidant supplementation in human asthmatics remains uncertain. We determined the effect of high doses of the antioxidant natural-source d-α-tocopheryl acetate for 16 weeks on allergen-induced airway oxidant stress, inflammation, and bronchial responsiveness to methacholine and allergen in atopic asthmatics in vivo. Methods Thirty-three mild atopic asthmatics underwent bronchoscopy with baseline bronchoalveolar lavage and segmental allergen challenge. The allergen-challenged airway was lavaged 24 h later. At least 3 weeks later, patients underwent inhaled challenges with methacholine and specific allergen. Volunteers took 1500 IU of natural-source d-α-tocopheryl acetate daily for at least 16 weeks. At the end of the treatment, the two bronchoscopies and inhaled methacholine and allergen challenges were repeated. F2-isoprostanes, specific markers of oxidant stress, and selected Th1 and Th2 cytokines were analyzed in the lavage fluid. Results Following supplementation of natural-source d-α-tocopheryl acetate, plasma concentrations of α-tocopherol increased and γ-tocopherol decreased. Both baseline and allergen-induced F2-isoprostanes significantly decreased, providing biochemical evidence for an antioxidant effect. Natural-source d-α-tocopheryl acetate reduced allergen-provoked concentrations of interleukin 3 and interleukin 4 and augmented levels of interleukin 12 in bronchoalveolar lavage fluid. Natural-source d-α-tocopheryl acetate improved airway responsiveness to methacholine but did not alter airway reactivity to specific allergen. Conclusions Inhibition of oxidant stress by natural-source d-α-tocopheryl acetate modulates allergic inflammation and airway hyperresponsiveness in human atopic asthmatics in vivo. These results need to be confirmed by a randomized placebo-controlled trial.

Superoxide and inflammation contribute to the genesis of hypertension but the mechanisms involved are not fully understood. We examined the hypothesis that oxidative stress in dendritic cells (DCs) alters endogenous proteins via Isoketal-modification leading to formation of neo-antigens, T cell activation and blood pressure elevation. DCs isolated from mice with angiotensin II-induced hypertension had a significant increase in NADPH oxidase-dependent superoxide production when compared to sham-treated mice (334.0±49.7 versus 65.8±4.5 pmol/mg protein). This was associated with an exuberant DC accumulation of protein-isoketal adducts and activation of IL-6, IL-1β and IL-23 production. DCs from hypertensive mice but not sham mice promoted survival and proliferation of CD8 + T cells in culture. Scavenging of isoketals not only prevented activation and immunogenicity of DCs, but also markedly attenuated angiotensin II-induced hypertension (142.59 ± 8.98 mmHg versus 175.53 ± 5.19 mmHg in controls). Moreover, adaptive transfer of DCs from hypertensive mice primed development of hypertension in mice given a sub-pressor dose of angiotensin II (157.45 ± 33.86 mmHg versus 119.90 ± 17.33 mmHg in controls). These studies show that angiotensin II-induced hypertension activates DCs, in large part by causing superoxide production and formation of isoketals. We propose that Isoketal-modified proteins can be presented as neo-antigens by DCs, which in turn trigger T cell activation leading to hypertension.

The adaptive immune system contributes to development of hypertension but the mechanisms or antigens involved are not known. Isoketals (IsoKs) are products of arachidonic acid oxidation that can cross-link lysine residues on proteins which in turn can be immunogenic. We sought to determine if IsoK-modified proteins serve as neoantigens presented by dendritic cells (DCs) to activate T cells in hypertension. Superoxide production by DCs was increased 5-fold by angiotensin II infusion compared to sham treated mice (334.0±49.7 versus 65.8±4.5 pmol/mg protein). This was NADPH-dependent as it did not occur in the gp91 phox-/- mice. This increase in superoxide was associated with accumulation of IsoKs in DCs and activation of DC IL-6 production (1411.0±1330.2 versus 576.2±512.8 pg/ml). Treatment with a potent IsoK scavenger, 2-hydroxybenzylamine, markedly attenuated angiotensin II-induced hypertension (131.4 ± 9.4 mmHg versus 160.1 ± 5.1 mmHg in control mice). Finally we employed an immunization assay involving priming by adoptive transfer of DCs from either sham or angiotensin II-treated mice to naïve recipients. T cells from these recipients were then cultured for 10 days with DCs from either sham or angiotensin II treated mice. DCs from angiotensin II treated mice, but not sham mice, caused a striking activation of CD8 + cells in this assay, as reflected by proliferation and polarization to Tc1 and Tc17 phenotypes. These studies show that angiotensin II-induced hypertension activates DCs, in large part by causing superoxide production and formation of IsoKs. We propose that IsoK-modified proteins can be presented as neoantigens by DCs, which in turn trigger T cell activation leading to hypertension.

Mitochondria are the source of most oxygen-derived free radicals. Mutations in mitochondrial DNA can impair mitochondrial electron transport resulting in decreased ATP production and increased free radical-induced oxidant injury. The specific mitochondrial DNA mutation A8344G alters the TPsiC loop or the mitochondrial tRNA for lysine. We investigated a large five-generational family harboring this mutation to determine whether the degree of heteroplasmy (proportion of mutated mitochondrial genomes) for the mtA8344G mutation correlated with a marker of oxidant damage. We measured F2-isoprostanes because they are specific and reliable markers of oxidant injury formed when free radicals attack esterified arachidonate in cell membranes. Family members with high heteroplasmy (>40%) had significantly higher F2-isoprostane levels (62 +/- 39 pg/ml) than those with lower heteroplasmy (33 +/- 13 pg/ml, P < 0.001). The degree of heteroplasmy for the mtA8344G mutation in this family correlated positively with F2-isoprostane levels (P = 0.03). This study highlights the underappreciated role free radicals play in the complex pathophysiology of inherited mitochondrial DNA disorders. The most important novel finding from this family is that some currently asymptomatic individuals with moderate heteroplasmy have evidence of ongoing free-radical mediated oxidant injury.