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Purpose: This case report seeks to highlight impressive photographs of amyloid staining with trypan blue that persisted after DMEK surgery with 3 years follow-up. This has only been reported in the literature twice previously, and may have contributed to visual symptoms in the early post-operative period of this patient. Observations: We report a case of an 82-year-old patient with concurrent Fuchs' corneal endothelial dystrophy and lattice corneal dystrophy who suffered permanent trypan blue staining of the amyloid deposits after descemet's membrane endothelial keratoplasty (DMEK) surgery. Conclusions and importance: Trypan blue staining of amyloid deposits in the corneal stroma is permanent, with research suggesting potential stimulation of progression or recurrence of amlyoid deposition. This is relevant for all anterior segment surgeons using trypan blue in the setting of primary (eg. lattice corneal dystrophy) or secondary amyloid deposition (eg. polymorphous amyloid degeneration, chronic inflammation, systemic disease). Trypan blue staining should be limited or alternative surgical options such as descemet's stripping automated endothelial keratoplasty (DSAEK) considered.

Toxoplasma gondii is a pervasive apicomplexan parasite that can cause severe disease and death in immunocompromised individuals and the developing foetus. The treatment of toxoplasmosis often leads to serious side effects and novel drugs and drug targets are therefore actively sought. In 2014, Mageed and colleagues suggested that the T. gondii pantothenate synthetase, the enzyme responsible for the synthesis of the vitamin B5 (pantothenate), the precursor of the important cofactor, coenzyme A, is a good drug target. Their conclusion was based on the ability of potent inhibitors of the M. tuberculosis pantothenate synthetase to inhibit the proliferation of T. gondii tachyzoites. They also reported that the inhibitory effect of the compounds could be antagonised by supplementing the medium with pantothenate, supporting their conclusion that the compounds were acting on the intended target. Contrary to these observations, we find that compound SW314, one of the compounds used in the Mageed et al. study and previously shown to be active against M. tuberculosis pantothenate synthetase in vitro, is inactive against the T. gondii pantothenate synthetase and does not inhibit tachyzoite proliferation, despite gaining access into the parasite in situ. Furthermore, we validate the recent observation that the pantothenate synthetase gene in T. gondii can be disrupted without detrimental effect to the survival of the tachyzoite-stage parasite in the presence or absence of extracellular pantothenate. We conclude that the T. gondii pantothenate synthetase is not essential during the tachyzoite stage of the parasite and it is therefore not a target for drug discovery against T. gondii tachyzoites.

Summary: Gastrointestinal immune cells, particularly muscularis macrophages (MM) interact with the enteric nervous system and influence gastrointestinal motility. Here we determine the human gastric muscle immunome and its changes in patients with idiopathic gastroparesis (IG). Single cell sequencing was performed on 26,000 CD45+ cells obtained from the gastric tissue of 20 subjects. We demonstrate 11 immune cell clusters with T cells being most abundant followed by myeloid cells. The proportions of cells belonging to the 11 clusters were similar between IG and controls. However, 9/11 clusters showed 578-11,429 differentially expressed genes. In IG, MM had decreased expression of tissue-protective and microglial genes and increased the expression of monocyte trafficking and stromal activating genes. Furthermore, in IG, IL12 mediated JAK-STAT signaling involved in the activation of tissue-resident macrophages and Eph-ephrin signaling involved in monocyte chemotaxis were upregulated. Patients with IG had a greater abundance of monocyte-like cells. These data further link immune dysregulation to the pathophysiology of gastroparesis.

Summary: Branched-chain amino acid (BCAA) metabolism is linked to glucose homeostasis, but the underlying signaling mechanisms are unclear. We find that gluconeogenesis is reduced in mice deficient of Ppm1k, a positive regulator of BCAA catabolism, which protects against obesity-induced glucose intolerance. Accumulation of branched-chain keto acids (BCKAs) inhibits glucose production in hepatocytes. BCKAs suppress liver mitochondrial pyruvate carrier (MPC) activity and pyruvate-supported respiration. Pyruvate-supported gluconeogenesis is selectively suppressed in Ppm1k-deficient mice and can be restored with pharmacological activation of BCKA catabolism by BT2. Finally, hepatocytes lack branched-chain aminotransferase that alleviates BCKA accumulation via reversible conversion between BCAAs and BCKAs. This renders liver MPC most susceptible to circulating BCKA levels hence a sensor of BCAA catabolism.

The L-arginine biosynthesis pathway consists of eight enzymes that catalyse the conversion of L-glutamate to L-arginine. Arginine auxotrophs (argB/argF deletion mutants) of Mycobacterium tuberculosis are rapidly sterilised in mice, while inhibition of ArgJ with Pranlukast was found to clear chronic M. tuberculosis infection in a mouse model. Enzymes in the arginine biosynthetic pathway have therefore emerged as promising targets for anti-tuberculosis drug discovery. In this work, the ligandability of four enzymes of the pathway ArgB, ArgC, ArgD and ArgF is assessed using a fragment-based approach. We identify several hits against these enzymes validated with biochemical and biophysical assays, as well as X-ray crystallographic data, which in the case of ArgB were further confirmed to have on-target activity against M. tuberculosis. These results demonstrate the potential for more enzymes in this pathway to be targeted with dedicated drug discovery programmes.

Summary: Heart disease is the leading cause of death with no method to repair damaged myocardium due to the limited proliferative capacity of adult cardiomyocytes. Curiously, mouse neonates and zebrafish can regenerate their hearts via cardiomyocyte de-differentiation and proliferation. However, a molecular mechanism of why these cardiomyocytes can re-enter cell cycle is poorly understood. Here, we identify a unique metabolic state that primes adult zebrafish and neonatal mouse ventricular cardiomyocytes to proliferate. Zebrafish and neonatal mouse hearts display elevated glutamine levels, predisposing them to amino-acid-driven activation of TOR, and that TOR activation is required for zebrafish cardiomyocyte regeneration in vivo. Through a multi-omics approach with cellular validation we identify metabolic and mitochondrial changes during the first week of regeneration. These data suggest that regeneration of zebrafish myocardium is driven by metabolic remodeling and reveals a unique metabolic regulator, TOR-primed state, in which zebrafish and mammalian cardiomyocytes are regeneration competent.

ShakerMaker is an open-source python framework which simplifies the generation of synthetic broad-band seismograms, produced by finite-fault kinematic representations of earthquake ruptures, using a 1-D layered model of the crust and the frequency–wavenumber (f–k) method. It is designed to bring closer the engineering seismology and earthquake engineering communities, by catering to the earthquake simulation needs of both disciplines. One particular goal of this framework is to provide a simple way to produce high-fidelity earthquake motions for use with the domain-reduction method, simplifying the setup of physically accurate finite-element simulations of multi-scale seismological and earthquake engineering problems through the use of a new specialized file format.ShakerMaker’s core is composed of a high-performance Fortran implementation of the f–k method, that is exposed to the user as a python framework. Its software architecture emphasizes simplicity, extensibility, and performance, allowing users to specify complex simulation scenarios with short scripts. The message passing interface is used to achieve scalability from simple single-processor machines to HPC clusters.

Objectives: Adverse childhood experience is thought to be associated with risk of coronary heart disease, but it is not clear which experiences are cardiotoxic, and whether risk increases with the accumulation of adverse childhood experiences. Methods: Participants were 5149 adults (72.6% men) in the Whitehall II cohort study. Parental death was recorded at phase 1 (median age in years 44.3), and 13 other adverse childhood experiences at phase 5 (55.3). We applied Cox proportional hazards regression with person-time from phase 5 to examine associations of adverse childhood experiences with incident coronary heart disease. We predicted hazard ratios according to count of the experiences, and examined dose-response effect. We finally estimated reduction of coronary heart disease in a hypothetical scenario, the absence of adverse childhood experiences. Results: Among study participants, 62.9% had at least one adversity, with “financial problems” having the highest prevalence (26.1%). There were 509 first episodes of coronary heart disease during an average 12.9 years follow-up. Among 14 adverse childhood experiences in a multiply adjusted model, “parental unemployment” showed the highest hazard of coronary heart disease incidence (hazard ratio; 95% confidence interval: 1.53; 1.16 to 2.02). No dose-response effect was observed (constant for proportionality in hazard ratio: 1.05, 0.99 to 1.11). Based on the estimates of final model, in the absence of childhood adversities, we estimated a 6.0% reduction in coronary heart disease (0.94; 0.87 to 1.01), but the confidence interval includes one. Conclusion: Although individual adverse childhood experiences show some association with coronary heart disease, there is no clear relationship with the number of adverse experiences. Further research is required to quantify effects of multiple and combinations of adverse childhood experiences considering timing, duration, and severity.

The first epithelial-to-mesenchymal transition (EMT) occurs in trophoblast stem (TS) cells during implantation. Inactivation of the serine/threonine kinase MAP3K4 in TS cells (TSKI4 cells) induces an intermediate state of EMT, where cells retain stemness, lose epithelial markers, and gain mesenchymal characteristics. Investigation of relationships among MAP3K4 activity, stemness, and EMT in TS cells may reveal key regulators of EMT. Here, we show that MAP3K4 activity controls EMT through the ubiquitination and degradation of HDAC6. Loss of MAP3K4 activity in TSKI4 cells results in elevated HDAC6 expression and the deacetylation of cytoplasmic and nuclear targets. In the nucleus, HDAC6 deacetylates the promoters of tight junction genes, promoting the dissolution of tight junctions. Importantly, HDAC6 knockdown in TSKI4 cells restores epithelial features, including cell-cell adhesion and barrier formation. These data define a role for HDAC6 in regulating gene expression during transitions between epithelial and mesenchymal phenotypes.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are an established cause of inherited Parkinson's disease (PD). LRRK2 is expressed in both neurons and glia in the central nervous system, but its physiological function(s) in each of these cell types is uncertain. Through sequential screens, we report a functional interaction between LRRK2 and Clathrin adaptor protein complex 2 (AP2). Analysis of LRRK2 KO tissue revealed a significant dysregulation of AP2 complex components, suggesting LRRK2 may act upstream of AP2. In line with this hypothesis, expression of LRRK2 was found to modify recruitment and phosphorylation of AP2. Furthermore, expression of LRRK2 containing the R1441C pathogenic mutation resulted in impaired clathrin-mediated endocytosis (CME). A decrease in activity-dependent synaptic vesicle endocytosis was also observed in neurons harboring an endogenous R1441C LRRK2 mutation. Alongside LRRK2, several PD-associated genes intersect with membrane-trafficking pathways. To investigate the genetic association between Clathrin-trafficking and PD, we used polygenetic risk profiling from IPDGC genome wide association studies (GWAS) datasets. Clathrin-dependent endocytosis genes were found to be associated with PD across multiple cohorts, suggesting common variants at these loci represent a cumulative risk factor for disease. Taken together, these findings suggest CME is a LRRK2-mediated, PD relevant pathway.

Summary: RNAP II switching from the paused to the productive transcription elongation state is a pivotal regulatory step that requires specific phosphorylations catalyzed by the P-TEFb kinase. Nucleosolic P-TEFb activity is inhibited by its interaction with the ribonuclear protein complex built around the 7SK small nuclear RNA (7SK snRNP). MePCE is the RNA methyltransferase that methylates and stabilizes 7SK in the nucleosol. Here, we report that MePCE also binds chromatin through the histone H4 tail to serve as a P-TEFb activator at specific genes important for cellular identity. Notably, this histone binding abolishes MePCE’s RNA methyltransferase activity toward 7SK, which explains why MePCE-bound P-TEFb on chromatin may not be associated with the full 7SK snRNP and is competent for RNAP II activation. Overall, our results suggest that crosstalk between the histone-binding and RNA methylation activities of MePCE regulates P-TEFb activation on chromatin in a 7SK- and Brd4-independent manner.

The data reported here support the manuscript Nuske et al. (2017) [1]. Searches were made for quantitative data on the occurrence of fungi within dietary studies of Australian mammal species. The original location reported in each study was used as the lowest grouping variable within the dataset. To standardise the data and compare dispersal events from populations of different mammal species that might overlap, data from locations were further pooled and averaged across sites if they occurred within 100 km of a random central point. Three locations in Australia contained data on several (>7) mycophagous mammals, all other locations had data on 1–3 mammal species. Within these three locations, the identity of the fungi species was compared between mammal species’ diets. A list of all fungi species found in Australian mammalian diets is also provide along with the original reference and fungal synonym names.

Carotid endarterectomy is a commonly performed vascular surgical procedure with well-known complications, such as stroke and nerve injury. Neovascular glaucoma (NVG) is an exceedingly rare complication after carotid endarterectomy that can result in loss of vision. All previous reports of NVG after carotid endarterectomy have occurred on the same side as the carotid surgery; in this report, we present a case of left-sided NVG after right carotid endarterectomy for contralateral ocular ischemic syndrome. We aim to emphasize the importance of early recognition and treatment of this serious complication as rapid intervention has the potential to save sight.

Summary: O-GalNAc glycosylation is initiated in the Golgi by glycosyltransferases called GALNTs. Proteomic screens identified >600 O-GalNAc-modified proteins, but the biological relevance of these modifications has been difficult to determine. We have discovered a conserved function for GALNT3 in trophoblast stem (TS) cells, blastocyst trophectoderm, and human mammary epithelial cells (HMECs). The loss of GALNT3 expression in these systems reduces O-GalNAc glycosylation and induces epithelial-mesenchymal transition. Furthermore, Galnt3 expression is reduced in aggressive, mesenchymal claudin-low breast cancer cells. We show that GALNT3 expression controls the O-GalNAc glycosylation of multiple proteins, including E-cadherin in both TS cells and HMECs. The loss of GALNT3 results in the intracellular retention of E-cadherin in the Golgi. Significantly, re-expression of GALNT3 in TS cells increases O-GalNAc glycosylation and restores the epithelial state. Together, these data demonstrate the critical biological role of GALNT3 O-GalNAc glycosylation to promote the epithelial phenotype in TS cells, blastocyst trophectoderm, and HMECs. : Raghu et al. demonstrate that O-GalNAc glycosylation is critical for epithelial state maintenance in trophoblast stem cells and HMECs. MAP3K4 promotes GALNT3 O-GalNAc modification of E-cadherin. Loss of GALNT3 results in the retention of E-cadherin in the Golgi. GALNT3 re-expression restores cell surface localization of E-cadherin, protecting the epithelial state. Keywords: epithelial-to-mesenchymal transition, EMT, MAP3K4, trophoblast stem cells, blastocyst, GALNT3, O-GalNAc glycosylation, E-cadherin, histone deacetylase, HDAC6

Summary: Background: Health inequalities persist into old age. We aimed to investigate risk factors for socioeconomic differences in frailty that could potentially be modified through policy measures. Methods: In this multi-wave longitudinal cohort study (Whitehall II study), we assessed participants' socioeconomic status, behavioural and biomedical risk factors, and disease status at age 45–55 years, and frailty (defined according to the Fried phenotype) at baseline and at one or more of three clinic visits about 18 years later (mean age 69 years [SD 5·9]). We used logistic mixed models to examine the associations between socioeconomic status and risk factors at age 50 years and subsequent prevalence of frailty (adjusted for sex, ethnic origin, and age), with sensitivity analyses and multiple imputation for missing data. Findings: Between Sept 9, 2007, and Dec 8, 2016, 6233 middle-aged adults were measured for frailty. Frailty was present in 562 (3%) of 16 164 person-observations, and varied by socioeconomic status: 145 (2%) person-observations had high socioeconomic status, 241 (4%) had intermediate status, and 176 (7%) had low socioeconomic status, adjusting for sex and age. Risk factors for frailty included cardiovascular disease, depression, smoking, high or abstinent alcohol consumption, low fruit and vegetable consumption, physical inactivity, poor lung function, hypertension, and overweight or obesity. Cardiometabolic markers for future frailty were high ratio of total to high-density lipoprotein cholesterol, and raised interleukin-6 and C-reactive protein concentrations. The five most important factors contributing to the frailty gradient, assessed by percent attenuation of the association between socioeconomic status and frailty, were physical activity (13%), interleukin-6 (13%), body-mass index category (11%), C-reactive protein (11%), and poor lung function (10%). Overall, socioeconomic differences in frailty were reduced by 40% in the maximally-adjusted model compared with the minimally-adjusted model. Interpretation: Behavioural and cardiometabolic risk factors in midlife account for more than a third of socioeconomic differences in frailty. Our findings suggest that interventions targeting physical activity, obesity, smoking, and low-grade inflammation in middle age might reduce socioeconomic differences in later-life frailty. Funding: British Heart Foundation and British Medical Research Council.

Feet and legs issues are some of the main causes for sow removal in the US swine industry. More timely lameness detection among breeding herd females will allow better treatment decisions and outcomes. Producers will be able to treat lame females before the problem becomes too severe and cull females while they still have salvage value. The objective of this study was to compare the predictive abilities and accuracies of weight distribution and gait measures relative to each other and to a visual lameness detection method when detecting induced lameness among multiparous sows. Developing an objective lameness diagnosis algorithm will benefit animals, producers and scientists in timely and effective identification of lame individuals as well as aid producers in their efforts to decrease herd lameness by selecting animals that are less prone to become lame. In the early stages of lameness, weight distribution and gait are impacted. Lameness was chemically induced for a short time period in 24 multiparous sows and their weight distribution and walking gait were measured in the days following lameness induction. A linear mixed model was used to determine differences between measurements collected from day to day. Using a classification tree analysis, it was determined that the mean weight being placed on each leg was the most predictive measurement when determining whether the leg was sound or lame. The classification tree’s predictive ability decreased as the number of days post-lameness induction increased. The weight distribution measurements had a greater predictive ability compared with the gait measurements. The error rates associated with the weight distribution trees were 29.2% and 31.3% at 6 days post-lameness induction for front and rear injected feet, respectively. For the gait classification trees, the error rates were 60.9% and 29.8% at 6 days post-lameness induction for front and rear injected feet, respectively. More timely lameness detection can improve sow lifetime productivity as well as animal welfare.

Rat adipose tissue homogenate contains a peptidase or group of peptidases which abolish the adipokinetic activity of ACTH, α-MSH, Β-MSH, vasopressin, and pituitary fraction H. The peptidase system is attached to an insoluble component in the homogenate. Homogenized rabbit adipose tissue inactivates only α-MSH, and homogenized guinea pig adipose tissue does not reduce the adipokinetic activity of any of the above hypophyseal peptides. Operation of the peptidase system in surviving slices of rat adipose tissue is suggested by these characteristics of this tissue: (a) rapid disappearance of adipokinetic activity from peptide-containing incubation medium; (b) absence of response to Β-MSH, vasopressin, or fraction H; (c) cessation of response to ACTH within 1 hr after exposure to the peptide is terminated. In contrast, slices of adipose tissue from the rabbit or guinea pig do not cause the disappearance of a detectable amount of activity from the medium; these slices are responsive to Β-MSH, vasopressin, and fraction H; their response to ACTH continues for at least 2 hr after exposure to the hormone is terminated. Differences between species in responsiveness of their adipose tissue to hypophyseal peptides appear to be related to differences in peptidase content of the fat cell.

In rats fed a stock diet, the oral administration of 17α-methyltestosterone (0.05% of the diet) produced a 46% average reduction of serum total cholesterol concentration. The ratio between the cholesterol content of the α- and β-lipoprotein fractions remained unchanged. In rats made mildly hypercholesterolemic by the addition of 1% cholesterol to the stock diet, the oral administration of 0.1% methyltestosterone prevented the rise in serum cholesterol concentration. This effect resulted primarily from a reduction of the cholesterol content of the α-lipoprotein fraction. The production of a more intense hypercholesterolemia by the administration of 1% cholesterol plus 0.5% cholic acid abolished the hypocholesterolemic action of 0.1% methyltestosterone. Analyses of the cholesterol content of the liver and intestinal wall indicated, in animals on both low and high cholesterol diets, that methyltestosterone did not exert its effect by producing a redistribution of the cholesterol between the serum and the tissues examined.

1 7α-Methyltestosterone-4-14C was fed to two dogs in an experiment to determine tissue localization and metabolic disposition of this hypocholesterolemic steroid. No accumulation of the drug was found in any tissue, although a small amount of radioactivity was detected in the liver and the ileal mucosa of one animal. Most of the administered radioactivity was excreted in urine and feces.The urinary metabolites consisted largely of highly polar compounds which appeared resistant to glucuronidase treatment or solvolysis procedures. Analysis of the fecal metabolites showed the presence of unchanged methyltestosterone, of four isomeric methylandrostanediols, and of labeled unidentified polar compounds. Of the four identified methylandrostanediols, the predominating fecal diols were 17α-methyl-5α-androstane-3β,17β-diol (45-62%) and 17α-methyl-5β-androstane-3α,l7β-diol (12-28%); 17α-methyl-5α-androstane-3α,-17β-diol and the 5β:3β isomer were found in very small amounts only.

The hypocholesterolemic activities of 17amethyltestosterone and its major identified fecal metabolite, 17α-methyl-5α-androstane-3β,17β-diol, were compared in dogs. The dose-response curves indicated that the two compounds had similar effects, although at doses below 1 mg/kg per day the diol appeared to be more active than the parent compound.