The hypoxia response pathway in the Antarctic fish Notothenia coriiceps is functional despite a poly Q/E insertion mutation in HIF-1α.

Antarctic notothenioid fishes, inhabiting the oxygen-rich Southern Ocean, possess a polyglutamine and glutamic acid (poly Q/E) insertion mutation in the master transcriptional regulator of oxygen homeostasis, hypoxia- inducible factor-1α (HIF-1α). To determine if this mutation impairs the ability of HIF-1 to regulate gene expression in response to hypoxia, we exposed Notothenia coriiceps, with a poly Q/E insertion mutation in HIF-1α that is 9 amino acids long, to hypoxia (2.3 mg L-1 O2) or normoxia (10 mg L -1 O2) for 12 h. Heart ventricles, brain, liver, and gill tissue were harvested and changes in gene expression quantified using RNA sequencing. Levels of glycogen and lactate were also quantified to determine if anaerobic metabolism increases in response to hypoxia. Exposure to hypoxia resulted in 818 unique differentially expressed genes (DEGs) in liver tissue of N. coriiceps. Many hypoxic genes were induced, including ones involved in the MAP kinase and FoxO pathways, glycolytic metabolism, and vascular remodeling. In contrast, there were fewer than 104 unique DEGs in each of the other tissues sampled. Lactate levels significantly increased in liver in response to hypoxia, indicating that anaerobic metabolism increases in response to hypoxia in this tissue. Overall, our results indicate that the hypoxia response pathway is functional in N. coriiceps despite a poly Q/E mutation in HIF-1α, and confirm that Antarctic fishes are capable of altering gene expression in response to hypoxia.

Why taxi tracking trumps tracking passengers with apps in planning for the electrification of Africa's paratransit.

Decarbonisation of Africa's informal paratransit through electrification requires adequate data captured correctly. Field workers getting on-board as passengers with tracked phones are extensively used to measure flow rates and volumes of passengers and vehicles on sections of roads in transport planning applications. Although this method is acceptable for transport planning, it is inadequate for planning for electrification. Combustion engine vehicles have long ranges and refill fast. Drivers and fuel outlets have existed in a symbiotic relationship without the bondage of needing detailed mobility information and planning. With electrification, battery-powered vehicles have become inextricably coupled to roadside infrastructure through their mobility patterns. We compare the current state of public transport data with vehicle tracking data for forecasting the electrification of Africa's paratransit. Discrepancies between them highlight the problem with using incomplete and/or unreliable data to estimate a city's peak load, pointing to a need for vehicle-based data acquisition.

Characterization of the hypoxia-inducible factor-1 pathway in hearts of Antarctic notothenioid fishes.

The ability of Antarctic notothenioid fishes to mount a robust molecular response to hypoxia is largely unknown. The transcription factor, hypoxia-inducible factor-1 (HIF-1), a heterodimer of HIF-1α and HIF-1ß subunits, is the master regulator of oxygen homeostasis in most metazoans. We sought to determine if, in the hearts of Antarctic notothenioids, HIF-1 is activated and functional in response to either an acute heat stress or hypoxia. The red-blooded Notothenia coriiceps and the hemoglobinless icefish, Chaenocephalus aceratus, were exposed to their critical thermal maximum (CTMAX) or hypoxia (5.0 ± 0.3 mg of O2 L-1) for 2 h. Additionally, N. coriiceps was exposed to 2.3 ± 0.3 mg of O2 L-1 for 12 h, and red-blooded Gobionotothen gibberifrons was exposed to both levels of hypoxia. Levels of HIF-1α were quantified in nuclei isolated from heart ventricles using western blotting. Transcript levels of genes involved in anaerobic metabolism, and known to be regulated by HIF-1, were quantified by real-time PCR, and lactate levels were measured in heart ventricles. Protein levels of HIF-1α increase in nuclei of hearts of N. coriiceps and C. aceratus in response to exposure to CTMAX and in hearts of N. coriiceps exposed to severe hypoxia, yet mRNA levels of anaerobic metabolic genes do not increase in any species, nor do lactate levels increase, suggesting that HIF-1 does not stimulate metabolic remodeling in hearts of notothenioids under these conditions. Together, these data suggest that Antarctic notothenioids may be vulnerable to hypoxic events, which are likely to increase with climate warming.

Dual CTLA-4 and PD-L1 Blockade Inhibits Tumor Growth and Liver Metastasis in a Highly Aggressive Orthotopic Mouse Model of Colon Cancer.

Immune checkpoint inhibitors have shown clinical benefit in several cancer entities including metastatic microsatellite instable colorectal carcinomas. However, for the majority of metastatic colorectal carcinomas the potential and limitations of immune checkpoint inhibition is not fully understood. In this study, the effects of sole and dual CTLA-4 and PD-L1 blockade were investigated in a microsatellite stable highly aggressive orthotopic mouse model of colon cancer. Dual CTLA-4 and PD-L1 inhibition resulted in tumor growth stagnation and completely blocked liver metastasis. Sole CTLA-4 and PD-L1 inhibition only moderately reduced metastatic spread of the colon cancer cells, though CTLA-4 blockade being superior to PD-L1 inhibition. Dual immune checkpoint blockade and sole CTLA-4 inhibition significantly increased intratumoral CD8+ and CD4+ T cells and reduced FOXP3+/CD4+ Treg cells. This was associated with increased expression levels of the pro-inflammatory Th1/M1-related cytokines IFN-γ, IL-1α, IL-2, and IL-12. Moreover, tumors treated with combined immune checkpoint blockade showed the strongest increase in intratumoral iNOS+ macrophages, reduction of PD-L1+ and Tie2+ macrophages and the lowest expression of M2/Th2-related IL-4, TARC and COX-2. The assessment of further microenvironmental changes by DCE-MRI and immunohistology revealed no alterations in functional tumor vascularization upon combined immune checkpoint blockade, but a significant increase in intratumoral fibroblasts and collagen I deposition. Thus, the synergistic inhibitory effects of dual immune checkpoint inhibition can be explained by anti-tumorigenic T cell responses mediated by CTLA-4 inhibition and M1 macrophage polarization predominantly induced by PD-L1 blockade. This was accompanied by pronounced fibroblast activation highlighting the interconnection between immunogenicity and desmoplasia.

Hypoxia-Inducible Factor-1α in Antarctic notothenioids contains a polyglutamine and glutamic acid insert that varies in length with phylogeny.

The long evolution of the Antarctic perciform suborder of Notothenioidei in the icy, oxygen-rich waters of the Southern Ocean may have reduced selective pressure to maintain a hypoxic response. To test this hypothesis, cDNA of the key transcriptional regulator of hypoxic genes, hypoxia-inducible factor-1α (HIF-1α), was sequenced in heart ventricles of the red-blooded notothenioid, Notothenia coriiceps, and the hemoglobinless icefish, Chaenocephalus aceratus. HIF-1α cDNA is 4500 base pairs (bp) long and encodes 755 amino acids in N. coriiceps, and in C. aceratus, HIF-1α is 3576 bp long and encodes 779 amino acids. All functional domains of HIF-1α are highly conserved compared to other teleosts, but HIF-1α contains a polyglutamine/glutamic acid (polyQ/E) insert 9 amino acids long in N. coriiceps and 34 amino acids long in C. aceratus. Sequencing of this region in four additional species, representing three families of notothenioids, revealed that the length of the polyQ/E insert varies with phylogeny. Icefishes, the crown family of notothenioids, contain the longest polyQ/E inserts, ranging between16 and 34 amino acids long, whereas the basal, cold-temperate notothenioid, Eleginops maclovinus, contains a polyQ/E insert only 4 amino acids long. PolyQ/E inserts may affect dimerization of HIF-1α and HIF-1ß, HIF-1 translocation into the nucleus and/or DNA binding.

Recommendation for severity assessment following liver resection and liver transplantation in rats: Part I.

Score sheets were first introduced 30 years ago to assess pain, distress and suffering in animals. To date, however, there is still no general agreement on their use in research practice, and only a few publications can be found on this topic. In the present work, we demonstrate the use of a special score sheet for severity assessment in the first three postoperative days in two showcased studies performed on Wistar and Lewis rats undergoing liver resection or orthotopic liver transplantation, respectively. Scoring of different criteria and the total score were evaluated within each intervention. Additionally, both procedures were compared regarding their degree of severity. Suitability of these score sheets was evaluated for assessing severity of the procedures and these showed a minor severity within each investigated study. A comparison of both studies showed slightly higher scores involving liver transplantation. In contradiction to the common classification of these procedures as a moderate severity grade the score sheets applied here indicates a minor severity grade within each investigated study. Also, limitations and possible improvements in the design of our score sheets for defined interventions are reconsidered.

[Molecular ultrasound imaging: Clinical applications]. / Molekulare Ultraschallbildgebung: Chancen für die Klinik.

BACKGROUND: Contrast-enhanced ultrasound imaging is increasingly being used in clinical applications, particularly for cardiovascular and liver diagnostics. In this context the availability of new molecular contrast agents and the initiation of clinical translation promises new options for pathomechanistic diagnostics. MATERIAL AND METHODS: Analysis of the current literature on the development of molecular ultrasound contrast agents, the detection methods as well as the applications in preclinical and clinical studies. RESULTS: Molecular contrast agents have become established in preclinical research for the detection of inflammation and angiogenesis and have been continuously refined over recent years. They consist of gas filled microbubbles with a diameter of 1-5 µm and the gas core is stabilized by a shell made of lipids, proteins or polymers to which biomolecules are conjugated that determine the target specificity. The agent BR55 is the first clinically evaluated molecular ultrasound contrast agent. It binds to the angiogenesis marker vascular endothelial growth factor receptor 2 (VEGFR2) and has been studied in several preclinical and clinical phase I and II studies on tumor diagnostics and characterization. CONCLUSION: Molecular ultrasound imaging is rapidly evolving in preclinical research for a broad field of applications. Translation to clinical practice is conceivable for many indications and is already ongoing for BR55.

The nucleolar targeting signal (NTS) of parathyroid hormone related protein mediates endocytosis and nucleolar translocation.

Previous work has identified the parathyroid hormone-related protein (PTHrP) nucleolar targeting signal (NTS) as both necessary and sufficient for localization of PTHrP to the nucleus and nucleolus of a variety of cells where it is believed to participate in the regulation of cell proliferation, differentiation, and apoptotic cell death. The mechanism whereby a secreted peptide, such as PTHrP, gains access to the nuclear compartment remains a question of debate. The current work examines the possibility that exogenous PTHrP is internalized and transported to the nuclear compartment by a mechanism that is dependent on preservation of the PTHrP NTS. Transiently expressed, PTHrP(1-141) was detected at the cell surface as well as in the cytoplasmic and nuclear compartments of COS-1 cells. Deletion of the NTS, or mutation of the conserved GxKKxxK motif within the NTS, effectively prevented both cell-surface binding and nuclear/nucleolar accumulation of PTHrP(1-141). A biotinylated peptide corresponding to the PTHrP NTS (PTHrP-NTS-biotin) was internalized and translocated to the nucleus and nucleolus in a time-, temperature-, and concentration-dependent manner, whereas a peptide representing a similar bipartite NTS from Nucleolin was not. Internalization and nucleolar targeting of PTHrP-NTS-biotin were indistinguishable in CFK2 cells, which express the common PTH/PTHrP receptor, and in 27m21 cells, which do not. In addition, pretreatment with a saturating dose of synthetic PTHrP(74-113) was capable of abrogating nucleolar accumulation of the PTHrP-NTS peptide, whereas pretreatment with PTHrP(1-34) or PTHrP(67-86) was not. These observations demonstrate that binding of exogenous, full-length PTHrP to the cell surface is mediated through a conserved motif embedded in the NTS and suggest that internalization and nucleolar targeting of an NTS peptide are mediated through binding to a cell surface protein distinct from the PTH/PTHrP receptor. In total, the data support the hypothesis that secreted PTHrP(1-141) can be endocytosed and targeted to the nucleolus through a mechanism that is dependent on preservation of a core motif within the PTHrP NTS.

Referral for 'prostatism': developing a 'performance indicator' for the threshold between primary and secondary care?

OBJECTIVE: We aimed to define a performance indicator at the gateway between primary and secondary care. METHOD: We carried out an analysis of referral letters sent to an urological department within the catchment area of a teaching hospital in Cardiff, Wales. The subjects were 221 sequential referral letters from 221 GPs. The main outcome measures were the information content of referral letters analysed. Letters were stratified into referral threshold groups by the presence of history, examination, routine investigations and specialized investigations. RESULTS: Three distinct categories of referral practice were identified: referrals which contained history alone; those providing history examination and a selection of routine investigations; and those providing history, examination data and the results of routine and specialized investigations. The study demonstrated that more than a third of GPs do not report the results of digital rectal examination in their referrals and only 4% record urinary flow rates and post-micturition residual urine volume. CONCLUSIONS: The majority (60%) of generalist referrals to an urology department for prostatism provide enough information for specialists to be able to prioritize appointments, but more than a third (36%) of the referrals contain inadequate information. The method has the potential of being developed into a gateway performance indicator in clinical practice.

Pit and fissure sealant application using a drying agent.

This clinical investigation assessed the retention of pit and fissure sealants with and without the use of a post-etching drying agent in pediatric dental patients. Twenty eight healthy patients with four fully erupted, non-carious first permanent molars participated. Sixteen males and 12 females, 5-12 years of age, each received four sealants, two with and two without a drying agent. Sealants were placed by three dental operators. Assignment for sealant application (with and without drying agent) was based on a computer-generated randomization table. Sealant application involved cleansing of the teeth with a non-fluoridated prophylaxis paste followed by rinsing 60 seconds with air/water spray, enamel conditioning with 40% phosphoric acid for 60 seconds, rinsing with air/water spray for 60 seconds, application of drying agent for 5 seconds, application of sealant material, and light curing for 60 seconds. Modified USPHS (Ryge) criteria were used to evaluate interfacial staining, secondary caries, marginal integrity, and surface texture. Sealant retention was evaluated as fully retained or lost (failed). All 28 participants returned for the 12-month recall visit. Four evaluators reported a 91% overall retention rate. Of the 56 teeth sealed without a drying agent, there was a 13% (n = 7) failure rate compared to 5% (n = 3) failure rate for the 56 teeth sealed with a drying agent. These results were not statistically significant (P > 0.05) based on the log-rank test.

Evaluation of an experimental anticholinergic drug, elantrine, in treating the tremor of parkinsonism.

An experimental anticholinergic drug, elantrine, had shown significant improvement in tremor of parkinsonism in 89 patients not taking L-dopa. A double-blind study of 22 parkinsonian patients stabilized on L-dopa showed marked improvement in tremor and moderate improvement in rigidity and bradykinesia when elantrine was added to their treatment program. Nine of 15 patients taking L-dopa (or Sinemet) and elantrine had cessation of all tremor and have continued free of tremor to date, over two years.