FGF21 protects against ischaemia reperfusion injury in normal and fatty livers.

BACKGROUND: Liver ischaemia/reperfusion (I/R) injury, which is an inevitable clinical problem of liver resection, liver transplantation and haemorrhagic shock. Fibroblast growth factor 21 (FGF21) was intimately coupled with multiple metabolic processes and proved to protect against apoptosis and inflammatory response in hepatocytes during hepatic I/R injury. However, the regulatory mechanisms of FGF21 in hepatic I/R injury remains unknown. Therefore, we hypothesize that FGF21 protects hepatic tissues from I/R injury. METHODS: Blood samples were available from haemangiomas patients undergoing hepatectomy and murine liver I/R model and used to further evaluate the serum levels of FGF21 both in humans and mice. We further explored the regulatory mechanisms of FGF21 in murine liver I/R model by using FGF21-knockout mice (FGF21-KO mice) and FGF21-overexpression transgenic mice (FGF21-OE mice) fed a high-fat or ketogenic diet. RESULTS: Our results show that the circulating levels of FGF21 were robustly decreased after liver I/R in both humans and mice. Silencing FGF21 expression with FGF21-KO mice aggravates liver injury at 6 h after 75 min of partial liver ischaemia, while FGF21-OE mice display alleviated hepatic I/R injury and inflammatory response. Compared with chow diet mice, exogenous FGF21 decreases the levels of aminotransferase, histological changes, apoptosis and inflammatory response in hepatic I/R injury treatment mice with a high-fat diet. Meanwhile, ketogenic diet mice are not sensitive to hepatic I/R injury. CONCLUSIONS: The circulating contents of FGF21 are decreased during liver warm I/R injury and exogenous FGF21 exerts hepatoprotective effects on hepatic I/R injury. Thus, FGF21 regulates hepatic I/R injury and may be a key therapeutic target.

Workload Measurement in Subspecialty Placental Pathology in Canada.

BACKGROUND: Workload measurement is important to help determine optimal staffing and workload distribution for pathology laboratories. The Level 4 Equivalent (L4E) System is the most widely used Anatomical Pathology (AP) workload measurement tool in Canada. However, it was initially not developed with subspecialties in mind. METHODS: In 2016, a Pan-Canadian Pediatric-Perinatal Pathology Workload Committee (PCPPPWC) was organized to adapt the L4E System to assess Pediatric-Perinatal Pathology workload. Four working groups were formed. The Placental Pathology Working Group was tasked to develop a scheme for fair valuation of placental specimens signed out by subspecialists in the context of the L4E System. Previous experience, informal time and motion studies, a survey of Canadian Pediatric-Perinatal Pathologists, and interviews of Pathologists' Assistants (PA) informed the development of such scheme. RESULTS: A workload measurement scheme with average L4E workload values for examination and reporting of singleton and multiple gestation placentas was proposed. The proposal was approved by the Canadian Association of Pathologist - Association canadienne des pathologistes Workload and Human Resources Committee for adoption into the L4E System. CONCLUSION: The development of a workload measurement model for placental specimens provides an average and fair valuation of these specimen types, enabling its use for resource planning and workload distribution.

Adulteration of proprietary Chinese medicines and health products with undeclared drugs: experience of a tertiary toxicology laboratory in Hong Kong.

AIMS: Proprietary Chinese medicines (pCMs) and health products, generally believed to be natural and safe, are gaining popularity worldwide. However, the safety of pCMs and health products has been severely compromised by the practice of adulteration. The current study aimed to examine the problem of adulteration of pCMs and health products in Hong Kong. METHODS: The present study was conducted in a tertiary referral clinical toxicology laboratory in Hong Kong. All cases involving the use of pCMs or health products, which were subsequently confirmed to contain undeclared adulterants, from 2005 to 2015 were reviewed retrospectively. RESULTS: A total of 404 cases involving the use of 487 adulterated pCMs or health products with a total of 1234 adulterants were identified. The adulterants consisted of approved drugs, banned drugs, drug analogues and animal thyroid tissue. The six most common categories of adulterants detected were nonsteroidal anti-inflammatory drugs (17.7%), anorectics (15.3%), corticosteroids (13.8%), diuretics and laxatives (11.4%), oral antidiabetic agents (10.0%) and erectile dysfunction drugs (6.0%). Sibutramine was the most common adulterant (n = 155). The reported sources of these illicit products included over-the-counter drug stores, the internet and Chinese medicine practitioners. A significant proportion of patients (65.1%) had adverse effects attributable to these illicit products, including 14 severe and two fatal cases. Psychosis, iatrogenic Cushing syndrome and hypoglycaemia were the three most frequently encountered adverse effects. CONCLUSIONS: Adulteration of pCMs and health products with undeclared drugs poses severe health hazards. Public education and effective regulatory measures are essential to address the problem.

Aortico-left ventricular tunnel: the elusive diagnosis.

We present a case of an infant prenatally diagnosed with bilateral outflow-tract obstruction and severe aortic regurgitation who underwent cardiac transplantation at 45 days of life. Aortico-left ventricular tunnel was subsequently diagnosed on pathologic examination of the explant heart. Aortico-left ventricular tunnel is a rare congenital cardiac malformation and can remain undiagnosed if the clinician has a low level of suspicion. Aortico-left ventricular tunnel should be considered in any fetus or newborn with aortic regurgitation.

Epidermal growth factor receptor and Ink4a/Arf: convergent mechanisms governing terminal differentiation and transformation along the neural stem cell to astrocyte axis.

Ink4a/Arf inactivation and epidermal growth factor receptor (EGFR) activation are signature lesions in high-grade gliomas. How these mutations mediate the biological features of these tumors is poorly understood. Here, we demonstrate that combined loss of p16(INK4a) and p19(ARF), but not of p53, p16(INK4a), or p19(ARF), enables astrocyte dedifferentiation in response to EGFR activation. Moreover, transduction of Ink4a/Arf(-/-) neural stem cells (NSCs) or astrocytes with constitutively active EGFR induces a common high-grade glioma phenotype. These findings identify NSCs and astrocytes as equally permissive compartments for gliomagenesis and provide evidence that p16(INK4a) and p19(ARF) synergize to maintain terminal astrocyte differentiation. These data support the view that dysregulation of specific genetic pathways, rather than cell-of-origin, dictates the emergence and phenotype of high-grade gliomas.

Dysfunctional telomeres activate an ATM-ATR-dependent DNA damage response to suppress tumorigenesis.

The POT1 (protection of telomeres) protein binds the single-stranded G-rich overhang and is essential for both telomere end protection and telomere length regulation. Telomeric binding of POT1 is enhanced by its interaction with TPP1. In this study, we demonstrate that mouse Tpp1 confers telomere end protection by recruiting Pot1a and Pot1b to telomeres. Knockdown of Tpp1 elicits a p53-dependent growth arrest and an ATM-dependent DNA damage response at telomeres. In contrast to depletion of Trf2, which activates ATM, removal of Pot1a and Pot1b from telomeres initiates an ATR-dependent DNA damage response (DDR). Finally, we show that telomere dysfunction as a result of Tpp1 depletion promotes chromosomal instability and tumorigenesis in the absence of an ATM-dependent DDR. Our results uncover a novel ATR-dependent DDR at telomeres that is normally shielded by POT1 binding to the single-stranded G-overhang. In addition, our results suggest that loss of ATM can cooperate with dysfunctional telomeres to promote cellular transformation and tumor formation in vivo.

Idiopathic anaphylaxis: What you do not know may hurt you.

Idiopathic anaphylaxis (IA), like immunologic and nonimmunologic anaphylaxis, is a life-threatening, sometimes fatal allergic disease. Although the priority is immediate recognition and initiation of treatment, long-term care planning is important to help reduce anxiety and promote healthy growth and development. Learning to recognize, manage, and stabilize the child is an essential part of improving the family dynamics. Despite advancements in the management of anaphylaxis, research has shown a need for continued patient education and training to improve timely recognition and treatment. This article focuses on elucidating the clinical presentation, theories of pathogenesis, and diagnosis, treatment, and management of IA.

Screening for submicroscopic chromosomal rearrangements in Wilms tumor using whole-genome microarrays.

Wilms tumor is the fourth most common malignancy of childhood; its pathogenesis, however, remains largely unknown. With advancements in cytogenetic techniques, such as array comparative genomic hybridization (aCGH), there is new hope for uncovering small chromosomal microdeletions or microduplications that may contribute to our understanding of Wilms tumor. We performed aCGH on 10 samples of Wilms tumor with normal conventional cytogenetic and chromosomal CGH findings. Array CGH revealed abnormalities in 3 of the 10 samples, including microdeletions (2q37.1, 7q31 approximately q32, and 11q22.3), microduplication (18q21.1), and gains and losses of larger chromosomal areas (1q and 7q gain and loss of 7p, 11q, 14q, and 16q). Fluorescence in situ hybridization (FISH) analysis confirmed the abnormalities and revealed the majority of them existed only in a proportion cells (> or =30% of cells). We also performed aCGH on three samples of Wilms tumor with previously identified translocations between chromosomes 1 and 16, to determine the breakpoints. The breakpoints were seen in the pericentromeric regions of both chromosomes. Array CGH is useful for identifying submicroscopic changes in Wilms tumor and is more sensitive for detecting clonal abnormalities than conventional methods.

Glucose phosphorylation is required for insulin-dependent mTOR signalling in the heart.

OBJECTIVE: Insulin regulates both glucose uptake and postnatal cardiac growth. The anabolic effects of insulin are mediated by the mammalian target of rapamycin (mTOR), an evolutionarily conserved kinase which is also a convergence point between nutrient sensing and cell growth. We postulated that mTOR signalling in the heart requires the metabolism of glucose. METHODS: We interrogated the insulin-mediated mTOR signalling pathway in response to different metabolic interventions regulating substrate metabolism in the isolated working rat heart and in isolated cardiomyocytes. RESULTS: Although insulin enhanced Akt activity, phosphorylation of mTOR and its downstream targets (p70S6K and 4EBP1) required the addition of glucose. Glucose-dependent p70S6K phosphorylation was independent of the hexosamine biosynthetic pathway, the AMP kinase pathway, and the pentose phosphate pathway. However, inhibition of glycolysis downstream of hexokinase markedly enhanced p70S6K phosphorylation. Furthermore, 2-deoxyglucose activated p70S6K suggesting that phosphorylation of glucose is required for carbohydrate-mediated mTOR signalling in the heart. Lastly, we also found enhanced p70S6K phosphorylation in the hearts of diabetic rats. CONCLUSION: Phosphorylation of glucose is necessary for insulin-dependent mTOR activity in the heart, suggesting a link between intermediary metabolism and cardiac growth.

Acute hepatitis E superinfection leading to chronic hepatitis B reactivation.

Reactivation of chronic hepatitis B (CHB) can be associated with significant morbidity and mortality. There are many different causes of hepatitis B reactivation. This case describes an Asian woman with stable CHB presenting with significant hepatitis flare with markedly elevated serum aminotransferases and hepatitis B virus DNA level. The clinical symptoms were subtle with fatigue and vague right upper quadrant tenderness. We ruled out drug-associated hepatotoxicity and screened for common causes of acute hepatitis. Interestingly, she was noted to have reactive anti-hepatitis E virus (HEV) IgM at initial presentation followed by anti-HEV IgG positivity a month later. The serological pattern confirmed the diagnosis of acute hepatitis E. The combination of antiviral therapy for hepatitis B and resolution of acute hepatitis E resulted in normalisation of serum aminotransferases. This case illustrates the importance of taking a careful history and having a high index of suspicion for various aetiologies when evaluating patients with reactivation of CHB.

Evaluation of three rapid oral fluid test devices on the screening of multiple drugs of abuse including ketamine.

Rapid oral fluid testing (ROFT) devices have been extensively evaluated for their ability to detect common drugs of abuse; however, the performance of such devices on simultaneous screening for ketamine has been scarcely investigated. The present study evaluated three ROFT devices (DrugWipe® 6S, Ora-Check® and SalivaScreen®) on the detection of ketamine, opiates, methamphetamine, cannabis, cocaine and MDMA. A liquid chromatography tandem mass spectrometry (LCMS) assay was firstly established and validated for confirmation analysis of the six types of drugs and/or their metabolites. In the field test, the three ROFT devices were tested on subjects recruited from substance abuse clinics/rehabilitation centre. Oral fluid was also collected using Quantisal® for confirmation analysis. A total of 549 samples were collected in the study. LCMS analysis on 491 samples revealed the following drugs: codeine (55%), morphine (49%), heroin (40%), methamphetamine (35%), THC (8%), ketamine (4%) and cocaine (2%). No MDMA-positive cases were observed. Results showed that the overall specificity and accuracy were satisfactory and met the DRUID standard of >80% for all 3 devices. Ora-Check® had poor sensitivities (ketamine 36%, methamphetamine 63%, opiates 53%, cocaine 60%, THC 0%). DrugWipe® 6S showed good sensitivities in the methamphetamine (83%) and opiates (93%) tests but performed relatively poorly for ketamine (41%), cocaine (43%) and THC (22%). SalivaScreen® also demonstrated good sensitivities in the methamphetamine (83%) and opiates (100%) tests, and had the highest sensitivity for ketamine (76%) and cocaine (71%); however, it failed to detect any of the 28 THC-positive cases. The test completion rate (proportion of tests completed with quality control passed) were: 52% (Ora-Check®), 78% (SalivaScreen®) and 99% (DrugWipe® 6S).

Deficiency of fibroblast growth factor 21 (FGF21) promotes hepatocellular carcinoma (HCC) in mice on a long term obesogenic diet.

OBJECTIVE: Non-alcoholic fatty liver (NAFL) associated with obesity is a major cause of liver diseases which can progress to non-alcoholic steatohepatitis, cirrhosis, and hepatocellular carcinoma (HCC). Fibroblast growth factor 21 (FGF21) plays an important role in liver metabolism and is also a potential marker for NAFL. Here we aimed to test the effect of FGF21 deficiency on liver pathology in mice consuming a conventional high fat, high sucrose (HFHS) obesogenic diet for up to 52 weeks. METHODS: C57BL6 WT and FGF21 KO mice were fed a conventional obesogenic diet and were evaluated at 16 and 52 weeks. Evaluation included metabolic assessment, liver pathology, and transcriptomic analysis. RESULTS: With consumption of HFHS diet, FGF21 deficient mice (FGF21 KO) develop excess fatty liver within 16 weeks. Hepatic pathology progresses and at 52 weeks FGF21 KO mice show significantly worse fibrosis and 78% of mice develop HCC; in contrast only 6% of WT mice develop HCC. Well differentiated hepatocellular carcinomas in FGF21 KO mice were characterized by expanded hepatic plates, loss of reticulin network, cytologic atypia, and positive immunostaining for glutamine synthetase. Microarray analysis reveals enrichment of several fibroblast growth factor signaling pathways in the tumors. CONCLUSIONS: In addition to attenuating inflammation and fibrosis in mice under a number of dietary challenges, we show here that FGF21 is required to limit the progression from NAFL to HCC in response to prolonged exposure to an obesogenic diet. The induction of hepatic FGF21 in response to the high fat, high sucrose obesogenic diet may play an important role in limiting progression of liver pathology from NAFL to HCC.

p16(INK4a) and p53 deficiency cooperate in tumorigenesis.

The combined impact of mutations in p16(INK4a) and p53 was examined in cellular growth,transformation, and tumor formation. In cultured cells, p16(INK4a) loss enhanced growth at high density and conferred susceptibility to oncogene-induced transformation. In vivo, mice doubly deficient for p16(INK4a) and p53 showed an increased rate of tumor formation with particular susceptibility to aggressive angiosarcomas. Furthermore, p16(INK4a) silencing by promoter methylation was detected in tumors derived from p16(INK4a+/-) and (+/+) mice, independent of p53 status. These data suggest at least one general feature of malignancy, resistance to density-mediated growth arrest depends on p16(INK4a) rather than p53. This cooperation between p16(INK4a) and p53 loss in tumorigenesis is consistent with the view that these genes function in distinct anticancer pathways.

Inhibitors of tripeptidyl peptidase II. 3. Derivation of butabindide by successive structure optimizations leading to a potential general approach to designing exopeptidase inhibitors.

The cholecystokinin-8 (CCK-8)-inactivating peptidase is a serine peptidase that has been shown to be a membrane-bound isoform of tripeptidyl peptidase II (EC 3.4.14.10). It cleaves the neurotransmitter CCK-8 sulfate at the Met-Gly bond to give Asp-Tyr(SO3H)-Met-OH + Gly-Trp-Met-Asp-Phe-NH2. Starting from Val-Pro-NHBu, a dipeptide of submicromolar affinity that had previously been generated to serve as a lead, successive optimization at P3, P1, and then P2 gave Abu-Pro-NHBu (18, Ki = 80 nM). Further transformation (by making a benzologue) gave the indoline analogue, butabindide (33) as a reversible inhibitor having nanomolar affinity (Ki = 7 nM). Retrospective analysis suggested the possibility of a general approach to designing exopeptidase inhibitors starting from the structure of the first hydrolysis product. Application of this approach to CCK-8 led to Abu-Phe-NHBu (37), but this only had Ki = 9.4 microM. Molecular modeling, to determine the minimum energy conformations and explain the 1000-fold better affinity of butabindide, indicated that 37 cannot access the likely active conformation of butabindide.

Heat shock and cold shock in Deinococcus radiodurans.

On the basis of acquired thermotolerance and cryotolerance, the optimal heat shock and cold shock temperatures have been determined for Deinococcus radiodurans. A heat shock at 42 degrees C maximized survival at the lethal temperature of 52 degrees C and a cold shock at 20 degrees C maximized survival after repeated freeze-thawing. Enhanced survival from heat shock was found to be strongly dependent on growth stage, with its greatest effect shortly after phase. Increased synthesis of a total of 67 proteins during heat shock and 42 proteins during cold shock were observed by two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and autoradiography. Eight of the most highly induced heat shock proteins shown by 2D PAGE were identified by MALDI-MS as Hsp20, GroEL, DnaK, SodA, Csp, Protease I, and two proteins of unknown function.

Impact of low-temperature plasmas on Deinococcus radiodurans and biomolecules.

The effects of cold plasma on Deinococcus radiodurans, plasmid DNA, and model proteins were assessed using microbiological, spectrometric, and biochemical techniques. In low power O(2) plasma (approximately 25 W, approximately 45 mTorr, 90 min), D. radiodurans, a radiation-resistant bacterium, showed a 99.999% reduction in bioburden. In higher power O(2) plasma (100 W and 500 mTorr), the reduction rate increased about 10-fold and observation by atomic force microscopy showed significant damage to the cell. Damage to cellular lipids, proteins, and chromosome was indicated by losses of infrared spectroscopic peaks at 2930, 1651, 1538, and 1245 cm(-1), respectively. In vitro experiments show that O(2) plasmas induce DNA strand scissions and cross-linking as well as reduction of enzyme activity. The observed degradation and removal of biomolecules was power-dependent. Exposures to 200 W at 500 mTorr removed biomolecules to below detection limits in 60 s. Emission spectroscopy indicated that D. radiodurans cells were volatilized into CO(2), CO, N(2), and H(2)O, confirming that these plasmas were removing complex biological matter from surfaces. A CO(2) plasma was not as effective as the O(2) plasma, indicating the importance of plasma composition and the dominant role of chemical degradation. Together, these findings have implications for NASA planetary protection schemes and for the contamination of Mars.

Near-infrared detection of potential evidence for microscopic organisms on Europa.

The possibility of an ocean within the icy shell of Jupiter's moon Europa has established that world as a primary candidate in the search for extraterrestrial life within our Solar System. This paper evaluates the potential to detect evidence for microbial life by comparing laboratory studies of terrestrial microorganisms with measurements from the Galileo Near Infrared Imaging Spectrometer (NIMS). If the interior of Europa at one time harbored life, some evidence may remain in the surface materials. Examination of laboratory spectra of terrestrial extremophiles measured at cryogenic temperatures reveals distorted, asymmetric nearinfrared absorption features due to water of hydration. The band centers, widths, and shapes of these features closely match those observed in the Europa spectra. These features are strongest in reddish-brown, disrupted terrains such as linea and chaos regions. Narrow spectral features due to amide bonds in the microbe proteins provide a means of constraining the abundances of such materials using the NIMS data. The NIMS data of disrupted terrains exhibit distorted, asymmetric near-infrared absorption features consistent with the presence of water ice, sulfuric acid octahydrate, hydrated salts, and possibly as much as 0.2 mg cm(-3) of carbonaceous material that could be of biological origin. However, inherent noise in the observations and limitations of spectral sampling must be taken into account when discussing these findings.

Defending the end zone: studying the players involved in protecting chromosome ends.

The linear nature of eukaryotic chromosomes leaves natural DNA ends susceptible to triggering DNA damage responses. Telomeres are specialized nucleoprotein structures that comprise the "end zone" of chromosomes. Besides having specialized sequences and structures, there are six resident proteins at telomeres that play prominent roles in protecting chromosome ends. In this review, we discuss this team of proteins, termed shelterin, and how it is involved in regulating DNA damage signaling, repair and replication at telomeres.

The rosettazyme: a synthetic cellulosome.

Cellulose is an attractive feedstock for biofuel production because of its abundance, but the cellulose polymer is extremely stable and its constituent sugars are difficult to access. In nature, extracellular multi-enzyme complexes known as cellulosomes are among the most effective ways to transform cellulose to useable sugars. Cellulosomes consist of a diversity of secreted cellulases and other plant cell-wall degrading enzymes bound to a protein scaffold. These scaffold proteins have cohesin modules that bind conserved dockerin modules on the enzymes. It is thought that the localization of these diverse enzymes on the scaffold allows them to function synergistically. In order to understand and harness this synergy smaller, simplified cellulosomes have been constructed, expressed, and reconstituted using truncated cohesin-containing scaffolds. Here we show that an 18-subunit protein complex called a rosettasome can be genetically engineered to bind dockerin-containing enzymes and function like a cellulosome. Rosettasomes are thermostable, group II chaperonins from the hyperthermo-acidophilic archaeon Sulfolobus shibatae, which in the presence of ATP/Mg(2+) assemble into 18-subunit, double-ring structures. We fused a cohesin module from Clostridium thermocellum to a circular permutant of a rosettasome subunit, and we demonstrate that the cohesin-rosettasomes: (1) bind dockerin-containing endo- and exo-gluconases, (2) the bound enzymes have increased cellulose-degrading activity compared to their activity free in solution, and (3) this increased activity depends on the number and ratio of the bound glucanases. We call these engineered multi-enzyme structures rosettazymes.