Ledipasvir/sofosbuvir and ribavirin for the treatment of ribavirin-refractory persistent hepatitis E virus infection.

Persistent Hepatitis E Virus infection (HEV) is a rare but increasingly recognised condition in immunocompromised individuals. Untreated, this infection can rapidly progress to cirrhosis. Ribavirin is recommended as the first line treatment and the majority achieve sustained viral clearance. However, treatment options are limited for those who fail ribavirin. We report a case of a patients with ribavirin-refractory persistent HEV who responded to ledipasvir/sofosbuvir and ribavirin treatment. This patients had failed 2 course of ribavirin and 1 course of PEG-Interferon and ribavirin and he was known to harbour ribavirin-associated mutations (G1634R, D1384G and K1383N) in the RNA dependent RNA polymerase. He was treated with ledipasvir/sofosbuvir (LDV/SOF; Harvoni 90/400 mg) and ribavirin (R) 400 mg twice daily for 32 weeks. At treatment initiation his HEV RNA was 1.1 × 106 IU/ML and reduced to 1.8 × 104 IU/ML and 43 IU/ML at one and four weeks of treatment, respectively, becoming not detected in blood and stool by week eight. His blood HEV RNA remained undetectable for seven months after treatment completion. Unfortunately, at eight months post-treatment, his blood HEV RNA became detectable at a low level (35 IU/ML). His stool HEV RNA was also detectable at 620 IU/ML consistent with a late relapse. He restarted LDV/SOF+R and by week four of treatment HEV RNA was not detected in blood and stool. He remains on treatment. In conclusion, this is the first report demonstrating the antiviral activity of LDV/SOF+R in the treatment of persistent HEV infection.

Improved outcomes following the implementation of a decompensated cirrhosis discharge bundle.

Introduction: Mortality from liver disease is increasing and management of decompensated cirrhosis (DC) is inconsistent across the UK. Patients with DC have complex medical needs when discharged from hospital and early readmissions are common. Our aims were: (1) to develop a Decompensated Cirrhosis Discharge Bundle (DCDB) to optimise ongoing care and (2) evaluate the impact of the DCDB. Methods: A baseline review of the management of patients with DC was conducted in Newcastle in 2017. The DCCB was developed and implemented in 2018. Impact of the DCDB was evaluated in two cycles, first a paper version (November 2018-October 2019) and then an electronic version (November 2020-March 2021). Key clinical data were collected from the time of discharge. Results: Overall, 192 patients (62% male; median age 55; median model for end-stage liver disease 17; 72% alcohol related) were reviewed in three cycles. At baseline, management was suboptimal, particularly ascites/diuretic management and provision of follow-up for alcohol misuse and 12% of patients had a potentially avoidable readmission within 30 days. After DCDB introduction, care improved across most domains, particularly electrolyte monitoring (p=0.012) and provision of community alcohol follow-up (p=0.026). Potentially preventable readmissions fell to 5% (p=0.055). Conclusions: Use of a care bundle for patients with DC can standardise care and improve patient management. If used more widely this could improve outcomes and reduce variability in care for patients with DC.

The effectiveness and acceptability of Mediterranean diet and calorie restriction in non-alcoholic fatty liver disease (NAFLD): A systematic review and meta-analysis.

BACKGROUND & AIMS: Lifestyle interventions that focus on reduced energy intake and improved dietary pattern are the mainstay of non-alcoholic fatty liver disease (NAFLD) management. However, it remains unclear which dietary approaches are most beneficial and promote greatest adherence. We aimed to synthesise data from randomised and clinical controlled trials, describing the effects of Mediterranean Diet and Calorie Restriction interventions on NAFLD surrogate markers, in adults. METHODS: We searched MEDLINE, Embase, Scopus, Web of Science, and Cochrane Central Register of Controlled Trials (October 2021). Study quality was assessed using the Cochrane Collaboration's tools: risk of bias for randomised controlled trials, and risk of bias in non-randomised studies of interventions. Meta-analyses were performed using a random effects model, and the I2 statistic was used to assess heterogeneity. RESULTS: Of 4041 records identified, 26 articles with 3037 participants met the inclusion criteria, including studies on calorie-restricted interventions (CRI) (n 9), Mediterranean diet (MD) interventions (n 13) and MD component interventions (n 4). Studies were heterogeneous regarding intervention components, assessment of liver status and diet outcomes. 3 studies reported zero attrition and mean attrition rate for the remaining 23 studies was 14%. Post-intervention meta-analyses revealed that dietary interventions reduced alanine aminotransferase (ALT) (P < 0.001), aspartate aminotransferase (AST) (P = 0.004), Fatty Liver Index (FLI) (P < 0.001), hepatic steatosis (HS) (P = 0.02), and liver stiffness (P = 0.01). CRI had favourable effects on ALT (P < 0.001), HS (P < 0.001) and liver stiffness (P = 0.009). MD reduced ALT (P = 0.02), FLI (P < 0.001) and liver stiffness (P = 0.05). There was a dose-response relationship between degree of calorie restriction and beneficial effects on liver function and weight loss, suggesting that this approach should remain the cornerstone of NAFLD management. In addition, diet composition changes have potential for improving NAFLD and the limited data suggest that MD may be an effective diet therapy. CONCLUSION: These results support the current guidelines in NAFLD. However, further studies, which robustly evaluate the effects of interventions on dietary intake, acceptability and sustainability of the interventions, and quality of life and other patient-related outcomes are needed to support effective care delivery.

Crystal structure of a ligand-free stable TSH receptor leucine-rich repeat domain.

The crystal structures of the thyroid-stimulating hormone receptor (TSHR) leucine-rich repeat domain (amino acids 22-260; TSHR260) in complex with a stimulating human monoclonal autoantibody (M22TM) and in complex with a blocking human autoantibody (K1-70™) have been solved. However, attempts to purify and crystallise free TSHR260, that is not bound to an autoantibody, have been unsuccessful due to the poor stability of free TSHR260. We now describe a TSHR260 mutant that has been stabilised by the introduction of six mutations (H63C, R112P, D143P, D151E, V169R and I253R) to form TSHR260-JMG55TM, which is approximately 900 times more thermostable than wild-type TSHR260. These six mutations did not affect the binding of human TSHR monoclonal autoantibodies or patient serum TSHR autoantibodies to the TSHR260. Furthermore, the response of full-length TSHR to stimulation by TSH or human TSHR monoclonal autoantibodies was not affected by the six mutations. Thermostable TSHR260-JMG55TM has been purified and crystallised without ligand and the structure solved at 2.83 Å resolution. This is the first reported structure of a glycoprotein hormone receptor crystallised without ligand. The unbound TSHR260-JMG55TM structure and the M22 and K1-70 bound TSHR260 structures are remarkably similar except for small changes in side chain conformations. This suggests that neither the mutations nor the binding of M22TM or K1-70TM change the rigid leucine-rich repeat domain structure of TSHR260. The solved TSHR260-JMG55TM structure provides a rationale as to why the six mutations have a thermostabilising effect and provides helpful guidelines for thermostabilisation strategies of other soluble protein domains.

A mutagenesis and screening strategy to generate optimally thermostabilized membrane proteins for structural studies.

The thermostability of an integral membrane protein (MP) in detergent solution is a key parameter that dictates the likelihood of obtaining well-diffracting crystals that are suitable for structure determination. However, many mammalian MPs are too unstable for crystallization. We developed a thermostabilization strategy based on systematic mutagenesis coupled to a radioligand-binding thermostability assay that can be applied to receptors, ion channels and transporters. It takes ∼6-12 months to thermostabilize a G-protein-coupled receptor (GPCR) containing 300 amino acid (aa) residues. The resulting thermostabilized MPs are more easily crystallized and result in high-quality structures. This methodology has facilitated structure-based drug design applied to GPCRs because it is possible to determine multiple structures of the thermostabilized receptors bound to low-affinity ligands. Protocols and advice are given on how to develop thermostability assays for MPs and how to combine mutations to make an optimally stable mutant suitable for structural studies. The steps in the procedure include the generation of ∼300 site-directed mutants by Ala/Leu scanning mutagenesis, the expression of each mutant in mammalian cells by transient transfection and the identification of thermostable mutants using a thermostability assay that is based on binding of an (125)I-labeled radioligand to the unpurified, detergent-solubilized MP. Individual thermostabilizing point mutations are then combined to make an optimally stable MP that is suitable for structural biology and other biophysical studies.

The 2.1 Å resolution structure of cyanopindolol-bound ß1-adrenoceptor identifies an intramembrane Na+ ion that stabilises the ligand-free receptor.

The ß1-adrenoceptor (ß1AR) is a G protein-coupled receptor (GPCR) that is activated by the endogenous agonists adrenaline and noradrenaline. We have determined the structure of an ultra-thermostable ß1AR mutant bound to the weak partial agonist cyanopindolol to 2.1 Å resolution. High-quality crystals (100 µm plates) were grown in lipidic cubic phase without the assistance of a T4 lysozyme or BRIL fusion in cytoplasmic loop 3, which is commonly employed for GPCR crystallisation. An intramembrane Na+ ion was identified co-ordinated to Asp872.50, Ser1283.39 and 3 water molecules, which is part of a more extensive network of water molecules in a cavity formed between transmembrane helices 1, 2, 3, 6 and 7. Remarkably, this water network and Na+ ion is highly conserved between ß1AR and the adenosine A2A receptor (rmsd of 0.3 Å), despite an overall rmsd of 2.4 Å for all Cα atoms and only 23% amino acid identity in the transmembrane regions. The affinity of agonist binding and nanobody Nb80 binding to ß1AR is unaffected by Na+ ions, but the stability of the receptor is decreased by 7.5°C in the absence of Na+. Mutation of amino acid side chains that are involved in the co-ordination of either Na+ or water molecules in the network decreases the stability of ß1AR by 5-10°C. The data suggest that the intramembrane Na+ and associated water network stabilise the ligand-free state of ß1AR, but still permits the receptor to form the activated state which involves the collapse of the Na+ binding pocket on agonist binding.