Modeling post-implantation stages of human development into early organogenesis with stem-cell-derived peri-gastruloids.

In vitro stem cell models that replicate human gastrulation have been generated, but they lack the essential extraembryonic cells needed for embryonic development, morphogenesis, and patterning. Here, we describe a robust and efficient method that prompts human extended pluripotent stem cells to self-organize into embryo-like structures, termed peri-gastruloids, which encompass both embryonic (epiblast) and extraembryonic (hypoblast) tissues. Although peri-gastruloids are not viable due to the exclusion of trophoblasts, they recapitulate critical stages of human peri-gastrulation development, such as forming amniotic and yolk sac cavities, developing bilaminar and trilaminar embryonic discs, specifying primordial germ cells, initiating gastrulation, and undergoing early neurulation and organogenesis. Single-cell RNA-sequencing unveiled transcriptomic similarities between advanced human peri-gastruloids and primary peri-gastrulation cell types found in humans and non-human primates. This peri-gastruloid platform allows for further exploration beyond gastrulation and may potentially aid in the development of human fetal tissues for use in regenerative medicine.

Mitochondrial uncouplers induce proton leak by activating AAC and UCP1.

Mitochondria generate heat due to H+ leak (IH) across their inner membrane1. IH results from the action of long-chain fatty acids on uncoupling protein 1 (UCP1) in brown fat2-6 and ADP/ATP carrier (AAC) in other tissues1,7-9, but the underlying mechanism is poorly understood. As evidence of pharmacological activators of IH through UCP1 and AAC is lacking, IH is induced by protonophores such as 2,4-dinitrophenol (DNP) and cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP)10,11. Although protonophores show potential in combating obesity, diabetes and fatty liver in animal models12-14, their clinical potential for treating human disease is limited due to indiscriminately increasing H+ conductance across all biological membranes10,11 and adverse side effects15. Here we report the direct measurement of IH induced by DNP, FCCP and other common protonophores and find that it is dependent on AAC and UCP1. Using molecular structures of AAC, we perform a computational analysis to determine the binding sites for protonophores and long-chain fatty acids, and find that they overlap with the putative ADP/ATP-binding site. We also develop a mathematical model that proposes a mechanism of uncoupler-dependent IH through AAC. Thus, common protonophoric uncouplers are synthetic activators of IH through AAC and UCP1, paving the way for the development of new and more specific activators of these two central mediators of mitochondrial bioenergetics.

Hardwiring tissue-specific AAV transduction in mice through engineered receptor expression.

The development of transgenic mouse models that express genes of interest in specific cell types has transformed our understanding of basic biology and disease. However, generating these models is time- and resource-intensive. Here we describe a model system, SELective Expression and Controlled Transduction In Vivo (SELECTIV), that enables efficient and specific expression of transgenes by coupling adeno-associated virus (AAV) vectors with Cre-inducible overexpression of the multi-serotype AAV receptor, AAVR. We demonstrate that transgenic AAVR overexpression greatly increases the efficiency of transduction of many diverse cell types, including muscle stem cells, which are normally refractory to AAV transduction. Superior specificity is achieved by combining Cre-mediated AAVR overexpression with whole-body knockout of endogenous Aavr, which is demonstrated in heart cardiomyocytes, liver hepatocytes and cholinergic neurons. The enhanced efficacy and exquisite specificity of SELECTIV has broad utility in development of new mouse model systems and expands the use of AAV for gene delivery in vivo.

Fazirsiran for Liver Disease Associated with Alpha1-Antitrypsin Deficiency.

BACKGROUND: Alpha1-antitrypsin (AAT) deficiency results from carriage of a homozygous SERPINA1 "Z" mutation (proteinase inhibitor [PI] ZZ). The Z allele produces a mutant AAT protein called Z-AAT, which accumulates in hepatocytes and can lead to progressive liver disease and fibrosis. This open-label, phase 2 trial investigated the safety and efficacy of fazirsiran, an RNA interference therapeutic, in patients with liver disease associated with AAT deficiency. METHODS: We assigned adults with the PI ZZ genotype and liver fibrosis to receive fazirsiran at a dose of 200 mg (cohorts 1 [4 patients] and 2 [8 patients]) or 100 mg (cohort 1b [4 patients]) subcutaneously on day 1 and week 4 and then every 12 weeks. The primary end point was the change from baseline to week 24 (cohorts 1 and 1b) or week 48 (cohort 2) in liver Z-AAT concentrations, which were measured by means of liquid chromatography-mass spectrometry. RESULTS: All the patients had reduced accumulation of Z-AAT in the liver (median reduction, 83% at week 24 or 48). The nadir in serum was a reduction of approximately 90%, and treatment was also associated with a reduction in histologic globule burden (from a mean score of 7.4 [scores range from 0 to 9, with higher scores indicating a greater globule burden] at baseline to 2.3 at week 24 or 48). All cohorts had reductions in liver enzyme concentrations. Fibrosis regression was observed in 7 of 15 patients and fibrosis progression in 2 of 15 patients after 24 or 48 weeks. There were no adverse events leading to trial or drug discontinuation. Four serious adverse events (viral myocarditis, diverticulitis, dyspnea, and vestibular neuronitis) resolved. CONCLUSIONS: In this small trial, fazirsiran was associated with a strong reduction of Z-AAT concentrations in the serum and liver and concurrent improvements in liver enzyme concentrations. (Funded by Arrowhead Pharmaceuticals; AROAAT-2002 ClinicalTrials.gov number, NCT03946449.).

Fazirsiran for Adults With Alpha-1 Antitrypsin Deficiency Liver Disease: A Phase 2 Placebo Controlled Trial (SEQUOIA).

BACKGROUND & AIMS: Homozygous ZZ alpha-1 antitrypsin (AAT) deficiency produces mutant AAT (Z-AAT) proteins in hepatocytes, leading to progressive liver fibrosis. We evaluated the safety and efficacy of an investigational RNA interference therapeutic, fazirsiran, that degrades Z-AAT messenger RNA, reducing deleterious protein synthesis. METHODS: This ongoing, phase 2 study randomized 40 patients to subcutaneous placebo or fazirsiran 25, 100, or 200 mg. The primary endpoint was percent change in serum Z-AAT concentration from baseline to week 16. Patients with fibrosis on baseline liver biopsy received treatment on day 1, at week 4, and then every 12 weeks and had a second liver biopsy at or after weeks 48, 72, or 96. Patients without fibrosis received 2 doses on day 1 and at week 4. RESULTS: At week 16, least-squares mean percent declines in serum Z-AAT concentration were -61%, -83%, and -94% with fazirsiran 25, 100, and 200 mg, respectively, vs placebo (all P < .0001). Efficacy was sustained through week 52. At postdose liver biopsy, fazirsiran reduced median liver Z-AAT concentration by 93% compared with an increase of 26% with placebo. All fazirsiran-treated patients had histologic reduction from baseline in hepatic globule burden. Portal inflammation improved in 5 of 12 and 0 of 8 patients with a baseline score of >0 in the fazirsiran and placebo groups, respectively. Histologic meta-analysis of histologic data in viral hepatitis score improved by >1 point in 7 of 14 and 3 of 8 patients with fibrosis of >F0 at baseline in the fazirsiran and placebo groups, respectively. No adverse events led to discontinuation, and pulmonary function tests remained stable. CONCLUSIONS: Fazirsiran reduced serum and liver concentrations of Z-AAT in a dose-dependent manner and reduced hepatic globule burden. (ClinicalTrials.gov, Number NCT03945292).

Long-term hepatitis B surface antigen response after finite treatment of ARC-520 or JNJ-3989.

BACKGROUND AND AIMS: RNA interference has been extensively explored in patients with chronic hepatitis B (CHB) infection. We aimed to characterise the long-term efficacy of small interfering RNA (siRNA) on hepatitis B surface antigen (HBsAg) suppression. METHODS: We prospectively followed up participants with CHB who received siRNA, either ARC-520 or JNJ-73763989 (JNJ-3989), in combination with nucleoside analogue (NUC) in our centre. Participants enrolled included 15 receiving 4 monthly injections of ARC-520, 38 receiving 3 injections of JNJ-3989 at 1, 2 or 4 weekly intervals and 5 receiving placebo in previous clinical trials. Serial blood sampling was performed according to the original protocols and on completion every 24 weeks until last follow-up (LFU) with mean duration of 52.5 months. RESULTS: Among the 53 NUC+siRNA-treated participants (mean age 46.8, baseline HBsAg 3.08 log, 83% previously on NUC, 34% hepatitis B e antigen+), the proportion of patients achieving HBsAg seroclearance or <100 IU/mL at LFU was 1.9% and 32.1%, respectively, compared with 0% and 0% for placebo. Among siRNA-recipients, 48.5% and 5.0% of those with HBsAg <100 IU/mL and >100 IU/mL at nadir or ≤24 weeks from last dose could maintain or achieve HBsAg <100 IU/mL at LFU, respectively. Compared with placebo recipients, siRNA-recipients demonstrated faster overall annual decline of HBsAg (0.08 vs 0.21 log IU/mL/year) contributed predominantly by changes in the first year. Age was negatively correlated with HBsAg reduction at LFU (r=-0.427, p=0.001). CONCLUSION: Short-duration siRNA treatment suppressed HBsAg expression with a prolonged effect for up to 6 years in some participants.

A phase I/II study of ARO-HSD, an RNA interference therapeutic, for the treatment of non-alcoholic steatohepatitis.

BACKGROUND & AIMS: Loss-of-function HSD17ß13 mutations protect against the development of chronic liver disease. HSD17ß13 inhibition represents a potential approach to treat liver diseases, such as non-alcoholic steatohepatitis (NASH). ARO-HSD is an RNA interference (RNAi) therapeutic designed to selectively reduce expression of HSD17ß13 mRNA in hepatocytes. In this study, we evaluated the effects of ARO-HSD in normal healthy volunteers (NHVs) and patients with confirmed or clinically suspected NASH. METHODS: The safety, tolerability, and pharmacodynamics of ARO-HSD were evaluated in 32 NHVs and 18 patients with confirmed/clinically suspected NASH. Double-blind NHV cohorts received single escalating doses of ARO-HSD (25, 50, 100, or 200 mg) or placebo subcutaneously on Day 1. Open-label patient cohorts received ARO-HSD (25, 100, or 200 mg) subcutaneously on Days 1 and 29. Liver biopsy was performed pre-dose and on Day 71 to evaluate expression levels of HSD17ß13 mRNA and protein. RESULTS: ARO-HSD treatment was well tolerated with no treatment-related serious adverse events or drug discontinuations. The most frequently reported treatment-emergent adverse events were mild injection site reactions, which were short in duration. Mean changes in hepatic HSD17ß13 mRNA from baseline to Day 71 were: -56.9% (25 mg), -85.5% (100 mg), and -93.4% (200 mg). The mean HSD17ß13 mRNA reduction was 78.6% (p <0.0001) across pooled cohorts. Hepatic HSD17ß13 protein levels were similarly reduced across doses. In patients, mean changes in alanine aminotransferase from baseline to Day 71 were -7.7% (25 mg), -39.3% (100 mg), and -42.3% (200 mg) (p <0.001 for pooled cohorts). CONCLUSIONS: ARO-HSD was well tolerated at doses ≤200 mg. This proof-of-concept study demonstrated that short-term treatment with ARO-HSD reduces hepatic HSD17ß13 mRNA and protein expression, which is accompanied by reductions in alanine aminotransferase. GOV NUMBER: NCT04202354. IMPACTS AND IMPLICATIONS: There is an unmet medical need for new therapies to treat alcohol-related and non-alcoholic liver disease. ARO-HSD is a small-interfering RNA designed to silence HSD17ß13 expression and hence to phenocopy the protective effect seen in individuals with HSD17ß13 loss-of-function. The reductions in HSD17ß13 expression and in transaminases seen with ARO-HSD administration represent an initial step towards clinical validation of HSD17ß13, a drug target with substantial genetic validation, as an important modulator of human liver disease.

Odour of domestic dogs infected with Leishmania infantum is attractive to female but not male sand flies: Evidence for parasite manipulation.

Globally visceral leishmaniasis (VL) causes thousands of human deaths every year. In South America, the etiologic agent, Leishmania infantum, is transmitted from an infected canine reservoir to human hosts by the bite of the sand fly vector; predominantly Lutzomyia longipalpis. Previous evidence from model rodent systems have suggested that the odour of infected hosts is altered by the parasite making them more attractive to the vector leading to an increased biting rate and improved transmission prospects for the pathogen. However, there has been no assessment of the effect of Le infantum infection on the attractiveness of dogs, which are the natural reservoirs for human infection. Hair collected from infected and uninfected dogs residing in a VL endemic city in Brazil was entrained to collect the volatile chemical odours present in the headspace. Female and male Lu. longipalpis sand flies were offered a choice of odour entrained from infected and uninfected dogs in a series of behavioural experiments. Odour of uninfected dogs was equally attractive to male or female Lu. longipalpis when compared to a solvent control. Female Lu. longipalpis were significantly more attracted to infected dog odour than uninfected dog odour in all 15 experimental replicates (average 45.7±0.87 females attracted to infected odour; 23.9±0.82 to uninfected odour; paired T-test, P = 0.000). Male Lu. longipalpis did not significantly prefer either infected or uninfected odour (average 36.1±0.4 males to infected odour; 35.7±0.6 to uninfected odour; paired T-test, P = 0.722). A significantly greater proportion of females chose the infected dog odour compared to the males (paired T-test, P = 0.000). The results showed that the odour of dogs infected with Le. infantum was significantly more attractive to blood-seeking female sand flies than it was to male sand flies. This is strong evidence for parasite manipulation of the host odour in a natural transmission system and indicates that infected dogs may have a disproportionate significance in maintaining infection in the canine and human population.

Hepatic Steatosis in the Mouse Model of Wilson Disease Coincides with a Muted Inflammatory Response.

Wilson disease (WND) is caused by inactivation of the copper transporter ATP7B and copper accumulation in tissues. WND presentations vary from liver steatosis to inflammation, fibrosis, and liver failure. Diets influence the liver phenotype in WND, but findings are inconsistent. To better understand the impact of excess calories on liver phenotype in WND, the study compared C57BL/6J Atp7b-/- and C57BL/6J mice fed for 12 weeks with Western diet or normal chow. Serum and liver metabolites, body fat content, liver histology, hepatic proteome, and copper content were analyzed. Wild-type and Atp7b-/- livers showed striking similarities in their responses to Western diet, most notably down-regulation of cholesterol biosynthesis, altered nuclear receptor signaling, and changes in cytoskeleton. Western diet increased body fat content and induced liver steatosis in males and females regardless of genotype; however, the effects were less pronounced in Atp7b-/- mice compared with those in the wild type mice. Although hepatic copper remained elevated in Atp7b-/- mice, liver inflammation was reduced. The diet diminished signaling by Rho GTPases, integrin, IL8, and reversed changes in cell cycle machinery and cytoskeleton. Overall, high calories decreased inflammatory response in favor of steatosis without improving markers of cell viability. Similar changes of cellular pathways during steatosis development in wild-type and Atp7b-/- mice explain histologic overlap between WND and non-alcoholic fatty liver disease despite opposite copper changes in these disorders.

Critical interactions between race and the highly granular area deprivation index in liver transplant evaluation.

Neighborhood socioeconomic deprivation may have important implications on disparities in liver transplant (LT) evaluation. In this retrospective cohort study, we constructed a novel dataset by linking individual patient-level data with the highly granular Area Deprivation Index (ADI), which is advantageous over other neighborhood measures due to: specificity of Census Block-Group (versus Census Tract, Zip code), scoring, and robust variables. Our cohort included 1377 adults referred to our center for LT evaluation 8/1/2016-12/31/2019. Using modified Poisson regression, we tested for effect measure modification of the association between neighborhood socioeconomic status (nSES) and LT evaluation outcomes (listing, initiating evaluation, and death) by race and ethnicity. Compared to patients with high nSES, those with low nSES were at higher risk of not being listed (aRR = 1.14; 95%CI 1.05-1.22; p < .001), of not initiating evaluation post-referral (aRR = 1.20; 95%CI 1.01-1.42; p = .03) and of dying without initiating evaluation (aRR = 1.55; 95%CI 1.09-2.2; p = .01). While White patients with low nSES had similar rates of listing compared to White patients with high nSES (aRR = 1.06; 95%CI .96-1.17; p = .25), Underrepresented patients from neighborhoods with low nSES incurred 31% higher risk of not being listed compared to Underrepresented patients from neighborhoods with high nSES (aRR = 1.31; 95%CI 1.12-1.5; p < .001). Interventions addressing neighborhood deprivation may not only benefit patients with low nSES but may address racial and ethnic inequities.

Ultrasound Elastography Detects Age-Related Changes in Adult False Vocal Folds.

PURPOSE: To investigate the feasibility of ultrasound elastography for assessing the symmetry in stiffness values and movements of both false vocal folds (FVFs). METHODS: After Institutional Review Board's approval and written informed consent obtained, we measured ultrasound strain and shear wave velocity (SWV) of the bilateral FVF in vocal fold abduction and adduction in 30 participants using a linear array transducer (4-10 MHz). Twenty-eight participants met inclusion criteria as healthy subjects for analysis. Mean strain of FVF produced by FVF movement from abduction to adduction was analyzed using 2D speckle-tracking software offline. A SWV ratio ([SWVadduction - SWabduction ]/SWabduction ) was developed to test the contractibility of FVF. Statistical analysis included one-way ANOVA to test the difference in mean strain, SWV (adduction and abduction), and SWV ratio among the three age groups; linear regression to analyze correlations of stiffness and movement between the right and left FVF; and intraclass correlation coefficient (ICC) to examine intra- and interobserver reliability in performing shear wave elastography (SWE) of FVF. RESULTS: The 28 healthy participants were divided into 3 age groups (10 of young 20-44 years; 9 of mid-age 45-64 years; and 9 of senior ≥65 years). The SWV in FVF abduction was higher and the SWV ratio was lower in seniors compared to young participants (P < .05). Good to excellent correlation of mean strain and SWV between both FVFs (R2 > 0.89). The reliability of performing SWE of FVF was moderate to excellent. CONCLUSION: Ultrasound elastography is feasible to assess the stiffness, dynamic movement, and symmetry of adult FVF, and healthy seniors may exhibit increased FVF stiffness.

Locking mixed-lineage kinase domain-like protein in its auto-inhibited state prevents necroptosis.

As an alternative pathway of controlled cell death, necroptosis can be triggered by tumor necrosis factor via the kinases RIPK1/RIPK3 and the effector protein mixed-lineage kinase domain-like protein (MLKL). Upon activation, MLKL oligomerizes and integrates into the plasma membrane via its executioner domain. Here, we present the X-ray and NMR costructures of the human MLKL executioner domain covalently bound via Cys86 to a xanthine class inhibitor. The structures reveal that the compound stabilizes the interaction between the auto-inhibitory brace helix α6 and the four-helix bundle by stacking to Phe148. An NMR-based functional assay observing the conformation of this helix showed that the F148A mutant is unresponsive to the compound, providing further evidence for the importance of this interaction. Real-time and diffusion NMR studies demonstrate that xanthine derivatives inhibit MLKL oligomerization. Finally, we show that the other well-known MLKL inhibitor Necrosulfonamide, which also covalently modifies Cys86, must employ a different mode of action.

Observed transmissions and ocean-ice-acoustic coupled modelling in the Beaufort Sea.

An ocean-ice-acoustic coupled model is configured for the Beaufort Sea. The model uses outputs from a data assimilating global scale ice-ocean-atmosphere forecast to drive a bimodal roughness algorithm for generating a realistic ice canopy. The resulting range-dependent ice cover obeys observed roughness, keel number density, depth, and slope, and floe size statistics. The ice is inserted into a parabolic equation acoustic propagation model as a near-zero impedance fluid layer along with a model defined range-dependent sound speed profile. Year-long observations of transmissions at 35 Hz from the Coordinated Arctic Acoustic Thermometry Experiment and 925 Hz from the Arctic Mobile Observing System source were recorded over the winter of 2019-2020 on a free-drifting, eight-element vertical line array designed to vertically span the Beaufort duct. The ocean-ice-acoustic coupled model predicts receive levels that reasonably agree with the measurements over propagation ranges of 30-800 km. At 925 Hz, seasonal and sub-seasonal ocean and ice driven variations of propagation loss are captured in the data and reproduced in the model.

Global burden of disease: acute-on-chronic liver failure, a systematic review and meta-analysis.

BACKGROUND AND AIMS: Acute-on-chronic liver failure (ACLF) is characterised by acute decompensation of cirrhosis associated with organ failures. We systematically evaluated the geographical variations of ACLF across the world in terms of prevalence, mortality, aetiology of chronic liver disease (CLD), triggers and organ failures. METHODS: We searched EMBASE and PubMed from 3/1/2013 to 7/3/2020 using the ACLF-EASL-CLIF (European Association for the Study of the Liver-Chronic Liver Failure) criteria. Two investigators independently conducted the abstract selection/abstraction of the aetiology of CLD, triggers, organ failures and prevalence/mortality by presence/grade of ACLF. We grouped countries into Europe, East/South Asia and North/South America. We calculated the pooled proportions, evaluated the methodological quality using the Newcastle-Ottawa Scale and statistical heterogeneity, and performed sensitivity analyses. RESULTS: We identified 2369 studies; 30 cohort studies met our inclusion criteria (43 206 patients with ACLF and 140 835 without ACLF). The global prevalence of ACLF among patients admitted with decompensated cirrhosis was 35% (95% CI 33% to 38%), highest in South Asia at 65%. The global 90-day mortality was 58% (95% CI 51% to 64%), highest in South America at 73%. Alcohol was the most frequently reported aetiology of underlying CLD (45%, 95% CI 41 to 50). Infection was the most frequent trigger (35%) and kidney dysfunction the most common organ failure (49%). Sensitivity analyses showed regional estimates grossly unchanged for high-quality studies. Type of design, country health index, underlying CLD and triggers explained the variation in estimates. CONCLUSIONS: The global prevalence and mortality of ACLF are high. Region-specific variations could be explained by the type of triggers/aetiology of CLD or grade. Health systems will need to tailor early recognition and treatment of ACLF based on region-specific data.

Long-term serological, virological and histological responses to RNA inhibition by ARC-520 in Chinese chronic hepatitis B patients on entecavir treatment.

OBJECTIVE: We examined the serological, virological (in serum and liver) and histological profiles in chronic hepatitis B virus (HBV) patients during and after completion of multiple dose (MD) ARC-520. DESIGN: The present phase 1b study was a multidose, open-label extension cohort of patients that had received single dose ARC-520 in our previous study. Eight patients received 4-9 4 weekly doses of MD ARC-520 and entecavir. Liver biopsies were performed in six patients. Intrahepatic and serum HBV DNA, HBV RNA and viral antigens were measured. RESULTS: All patients had 28.9-30.4 months of follow-up after the last MD. All three hepatitis B e antigen (HBeAg)-positive patients had profound reductions in hepatitis B surface antigen (HBsAg), HBeAg, hepatitis B core-related antigen and HBV RNA with two undergoing HBeAg seroconversion. One further achieved HBsAg seroconversion (anti-HBs level of 25.1 IU/L) and the remaining two had HBsAg reductions of -1.7 and -3.5 log IU/mL >30 months after MD. Among the five HBeAg-negative patients, four had modest HBsAg reduction >29 months after completion of MD and one achieved HBsAg seroconversion (anti-HBs level of 152.5 IU/L) and was negative for liver HBsAg staining. Entecavir was successfully stopped in this patient 12 months after HBsAg seroconversion. Temporally related alanine aminotransferase elevations preceded by HBsAg reductions were observed in three patients suggesting immune activation. HBcAg staining was negative in all six biopsied patients. Two patients with <10% HBsAg positive staining of hepatocytes had correspondingly low serum HBsAg levels of 1.5 and 11.5 IU/mL. CONCLUSIONS: MD ARC-520 therapy achieved sustained and profound reductions of viral antigens and HBV RNA. HBsAg seroclearance was achievable. TRIAL REGISTRATION NUMBER: NCT02065336.

The effect of mitochondrial calcium uniporter and cyclophilin D knockout on resistance of brain mitochondria to Ca2+-induced damage.

The mitochondrial calcium uniporter (MCU) and cyclophilin D (CyD) are key players in induction of the permeability transition pore (PTP), which leads to mitochondrial depolarization and swelling, the major signs of Ca2+-induced mitochondrial damage. Mitochondrial depolarization inhibits ATP production, whereas swelling results in the release of mitochondrial pro-apoptotic proteins. The extent to which simultaneous deletion of MCU and CyD inhibits PTP induction and prevents damage of brain mitochondria is not clear. Here, we investigated the effects of MCU and CyD deletion on the propensity for PTP induction using mitochondria isolated from the brains of MCU-KO, CyD-KO, and newly created MCU/CyD-double knockout (DKO) mice. Neither deletion of MCU nor of CyD affected respiration or membrane potential in mitochondria isolated from the brains of these mice. Mitochondria from MCU-KO and MCU/CyD-DKO mice displayed reduced Ca2+ uptake and diminished extent of PTP induction. The Ca2+ uptake by mitochondria from CyD-KO mice was increased compared with mitochondria from WT mice. Deletion of CyD prevented mitochondrial swelling and resulted in transient depolarization in response to Ca2+, but it did not prevent Ca2+-induced delayed mitochondrial depolarization. Mitochondria from MCU/CyD-DKO mice did not swell in response to Ca2+, but they did exhibit mild sustained depolarization. Dibucaine, an inhibitor of the Ca2+-activated mitochondrial phospholipase A2, attenuated and bovine serum albumin completely eliminated the sustained depolarization. This suggests the involvement of phospholipase A2 and free fatty acids. Thus, in addition to induction of the classical PTP, alternative deleterious mechanisms may contribute to mitochondrial damage following exposure to elevated Ca2+.

Mutant huntingtin does not cross the mitochondrial outer membrane.

Mutant huntingtin (mHTT) is associated with mitochondria, but the exact mitochondrial location of mHTT has not been definitively established. Recently, it was reported that mHTT is present in the intermembrane space and inhibits mitochondrial protein import by interacting with TIM23, a major component of mitochondrial protein import machinery, but evidence for functional ramifications were not provided. We assessed mHTT location using synaptic and nonsynaptic mitochondria isolated from brains of YAC128 mice and subjected to alkali treatment or limited trypsin digestion. Mitochondria were purified either with discontinuous Percoll gradient or with anti-TOM22-conjugated iron microbeads. We also used mitochondria isolated from postmortem brain tissues of unaffected individuals and HD patients. Our results demonstrate that mHTT is located on the cytosolic side of the mitochondrial outer membrane (MOM) but does not cross it. This refutes the hypothesis that mHTT may interact with TIM23 and inhibit mitochondrial protein import. The levels of expression of nuclear-encoded, TIM23-transported mitochondrial proteins ACO2, TUFM, IDH3A, CLPP and mitochondrially encoded and synthesized protein mtCO1 were similar in mitochondria from YAC128 mice and their wild-type littermates as well as in mitochondria from postmortem brain tissues of unaffected individuals and HD patients, supporting the lack of deficit in mitochondrial protein import. Regardless of purification technique, mitochondria from YAC128 and WT mice had similar respiratory activities and mitochondrial membrane potentials. Thus, our data argue against mHTT crossing the MOM and entering into the mitochondrial intermembrane space, making it highly unlikely that mHTT interacts with TIM23 and inhibits protein import in intact mitochondria.

Urine DNA biomarkers for hepatocellular carcinoma screening.

BACKGROUND: Hepatocellular carcinoma (HCC) occurs in a well-defined high-risk patient population, but better screening tests are needed to improve sensitivity and efficacy. Therefore, we investigated the use of urine circulating tumour DNA (ctDNA) as a screening test. METHODS: Candidate markers in urine were selected from HCC and controls. We then enrolled 609 patients from five medical centres to test the selected urine panel. A two-stage model was developed to combine AFP and urine panel as a screening test. RESULTS: Mutated TP53, and methylated RASSF1a, and GSTP1 were selected as the urine panel markers. Serum AFP outperformed the urine panel among all cases of HCC, but the urine panel identified 49% of HCC cases with low AFP < 20 ng/ml. Using the two-stage model, the combined AFP and urine panel identified 148 of the 186 HCC cases (79.6% sensitivity at 90% specificity), which was 30% more than the cases detected with serum AFP alone. It also increased early-stage HCC detection from 62% to 92% (BCLC stage 0), and 40% to 77% (BCLC stage A). CONCLUSION: Urine ctDNA has promising diagnostic utility in patients in HCC, especially in those with low AFP and can be used as a potential non-invasive HCC screening test.

HOPE in action: A prospective multicenter pilot study of liver transplantation from donors with HIV to recipients with HIV.

Liver transplantation (LT) from donors-with-HIV to recipients-with-HIV (HIV D+/R+) is permitted under the HOPE Act. There are only three international single-case reports of HIV D+/R+ LT, each with limited follow-up. We performed a prospective multicenter pilot study comparing HIV D+/R+ to donors-without-HIV to recipients-with-HIV (HIV D-/R+) LT. We quantified patient survival, graft survival, rejection, serious adverse events (SAEs), human immunodeficiency virus (HIV) breakthrough, infections, and malignancies, using Cox and negative binomial regression with inverse probability of treatment weighting. Between March 2016-July 2019, there were 45 LTs (8 simultaneous liver-kidney) at 9 centers: 24 HIV D+/R+, 21 HIV D-/R+ (10 D- were false-positive). The median follow-up time was 23 months. Median recipient CD4 was 287 cells/µL with 100% on antiretroviral therapy; 56% were hepatitis C virus (HCV)-seropositive, 13% HCV-viremic. Weighted 1-year survival was 83.3% versus 100.0% in D+ versus D- groups (p = .04). There were no differences in one-year graft survival (96.0% vs. 100.0%), rejection (10.8% vs. 18.2%), HIV breakthrough (8% vs. 10%), or SAEs (all p > .05). HIV D+/R+ had more opportunistic infections, infectious hospitalizations, and cancer. In this multicenter pilot study of HIV D+/R+ LT, patient and graft survival were better than historical cohorts, however, a potential increase in infections and cancer merits further investigation.

Combination treatments including the small-interfering RNA JNJ-3989 induce rapid and sometimes prolonged viral responses in patients with CHB.

BACKGROUND & AIMS: RNA interference therapy has been shown to reduce hepatitis B surface antigen (HBsAg) levels in preclinical models, which could confer functional cure in patients with chronic hepatitis B. This phase IIa trial (ClinicalTrials.gov Identifier: NCT03365947) assessed the safety and efficacy of the small-interfering RNA JNJ-73763989 (JNJ-3989) plus a nucleos(t)ide analogue (NA), with/without the capsid assembly modulator JNJ-56136379 (JNJ-6379) in patients with chronic hepatitis B. METHODS: Treatment-naïve and NA-suppressed patients received 3 subcutaneous JNJ-3989 doses every week (QW; 100, 200, or 300 mg), 2 weeks (Q2W; 100 mg) or 4 weeks (Q4W; 25, 50, 100, 200, 300, or 400 mg), or JNJ-3989 Q4W (200 mg) plus oral JNJ-6379 250 mg daily for 12 weeks. Patients received NAs throughout. RESULTS: Eighty-four patients were recruited. All treatments were well tolerated, with all 5 serious adverse events considered unrelated to study drugs. JNJ-3989 100 to 400 mg Q4W resulted in HBsAg reductions ≥1 log10 IU/ml from baseline in 39/40 (97.5%) patients at the nadir. All patients receiving the triple combination (n = 12) had HBsAg reductions ≥1 log10 IU/ml from baseline at the nadir. HBsAg reductions were similar for HBeAg-positive (n = 21) and HBeAg-negative (n = 47) patients in all JNJ-3989 Q4W treatment arms, including the triple combination (n = 68). Smaller HBsAg reductions were seen with 25 mg (n = 8) and 50 mg (n = 8) than with 100 to 400 mg (n = 40). Shorter dosing intervals (QW [n = 12] and Q2W [n = 4]) did not improve response vs. Q4W dosing. HBsAg reductions ≥1 log10 IU/ml from baseline persisted in 38% of patients 336 days after the last JNJ-3989 dose. CONCLUSIONS: JNJ-3989 plus an NA, with/without JNJ-6379, was well tolerated and resulted in HBsAg reductions up to 336 days after the last JNJ-3989 Q4W dose. CLINICAL TRIAL NUMBER: NCT03365947. LAY SUMMARY: Hepatitis B virus affects people's livers and produces particles called hepatitis B surface antigen (HBsAg) that damage a person's liver and can help the virus infect a person for a long time, known as chronic hepatitis B (CHB). In this study, a new treatment called JNJ-3989 was assessed (in combination with normal treatment known as nucleos(t)ide analogues), for its safety and effectiveness in reducing the number of HBsAg particles in people with CHB. The results of this study showed that treatment with JNJ-3989 could be safe for people with CHB, lowered their HBsAg levels, and kept HBsAg levels lowered for 336 days in 38% of patients after receiving their last dose of JNJ-3989.