The Detection of Thoracolumbar Spine Injuries in Horses with Chronic Laminitis Using a Novel Clinical-Assessment Protocol and Ultrasonographic Examination.

Postural adaptation is a prominent feature in horses affected by laminitis. Laminitis induces intense pain, especially in the forelimbs, prompting affected horses to assume a caudally displaced trunk posture, resulting in the hyperflexion of the thoracolumbar spine. This study assessed the nature and prevalence of thoracolumbar injuries in horses with chronic laminitis compared to horses without it. Sixty horses were used (thirty laminitic and thirty non-laminitic) of different athletic purposes and ages (2-20 years). The experimental protocol entailed a single assessment of horses' thoracolumbar spines, utilizing physical examination by MACCTORE, a scoring system developed specifically for this study. Additional evaluations included the Grimace Equine Pain Scale (HGS) and ultrasound exams. Statistical tests were used to compare values (Mann-Whitney or t-test) and lesions prevalences (Fisher) between groups (p < 0.05). The results showed a higher pain manifestation (HGS and heart rate, p < 0.0001) and thoracolumbar-spine-injury levels in chronic laminitis horses, both in MACCTORE clinical examinations (11.7 ± 4.8 vs. 4.2 ± 3.3, p < 0.0001) and general ultrasonographic indices (39.6 ± 12.0 vs. 20.7 ± 7.1, p < 0.0001), including specific examination approaches for various spinal elements. Horses with laminitis presented with a 14-fold higher prevalence of ultrasound-relevant lesions in the thoracolumbar spine (CI: 4.4 to 50.6, p < 0.0001) compared to controls. These findings constitute new evidence of an association between chronic laminitis and the presence of thoracolumbar spine injuries in horses, which may be confirmed by more sophisticated study designs.

Amodiaquine Nonspecifically Binds Double Stranded and Three-Way Junction DNA Structures.

The 4-aminoquinoline class of compounds includes the important antimalarial compounds amodiaquine and chloroquine. Despite their medicinal importance, the mode of action of these compounds is poorly understood. In a previous study we observed these compounds, as well as quinine and mefloquine, tightly bind the DNA cocaine-binding aptamer. Here, we further explore the range of nucleic acid structures bound by these compounds. To gauge a wide range of binding affinities, we used isothermal titration calorimetry to explore high affinity binding (nM to tens of mM) and NMR spectroscopy to assay weak binding biding in the hundreds of micromolar range.   We find that amodiaquine tightly binds all double stranded DNA structures explored. Mefloquine binds double stranded DNA duplex molecules tightly and weakly associates with a three-way junction DNA construct. Quinine and chloroquine only weakly bind duplex DNA but do not tightly bind any of the DNA constructs explored. A simulation of the free energy of binding of these ligands to the Dickerson-Drew dodecamer resulted in an excellent agreement between the simulated and experimental free energy.These results provide new insight into the DNA binding of clinically important antimalarial compounds and may play a role in future development of new antimalarials.

Transient X-Ray Absorption Near Edge Structure Spectroscopy Using Broadband Free-Electron Laser Pulses.

A new method for time-resolved X-ray absorption near edge structure (XANES) spectroscopy that enables faster data acquisition and requires smaller sample quantities for high-quality data, thus allowing the analysis of more samples in a shorter time is introduced. The method uses large bandwidth free electron laser pulses to measure laser-excited XANES spectra in transmission mode. A beam-splitting grating configuration allows simultaneous measurements of the spectra of the incoming X-ray Free Electron Laser (XFEL) pulses and transmission XANES, which is crucial for compensating the pulse-dependent intensity and spectrum fluctuations due to the self-amplified spontaneous emission operation. The implementation of this new methodology is applied on a liquid solution of ammonium iron(III) oxalate jet and is compared to previous results, showing great improvements in the speed of acquisition and spectral resolution, and the ability to measure a large 2-D spectral-time map quickly.

The RNA binding proteins LARP4A and LARP4B promote sarcoma and carcinoma growth and metastasis.

RNA-binding proteins (RBPs) are emerging as important regulators of cancer pathogenesis. We reveal that the RBPs LARP4A and LARP4B are differentially overexpressed in osteosarcoma and osteosarcoma lung metastases, as well as in prostate cancer. Depletion of LARP4A and LARP4B reduced tumor growth and metastatic spread in xenografts, as well as inhibiting cell proliferation, motility, and migration. Transcriptomic profiling and high-content multiparametric analyses unveiled a central role for LARP4B, but not LARP4A, in regulating cell cycle progression in osteosarcoma and prostate cancer cells, potentially through modulating key cell cycle proteins such as Cyclins B1 and E2, Aurora B, and E2F1. This first systematic comparison between LARP4A and LARP4B assigns new pro-tumorigenic functions to LARP4A and LARP4B in bone and prostate cancer, highlighting their similarities while also indicating distinct functional differences. Uncovering clear biological roles for these paralogous proteins provides new avenues for identifying tissue-specific targets and potential druggable intervention.

Serologic Detection of Hepatocellular Carcinoma: Application of Machine Learning and Implications for Diagnostic Models.

PURPOSE: The gender, age, lens culinaris agglutinin-reactive fraction of alphafetoprotein, alphafetoprotein, des-gamma-carboxyprothrombin (GALAD) score is a biomarker-based statistical model for the serologic diagnosis of hepatocellular carcinoma (HCC) that has been developed and validated using the case-control approach with a view to early detection. Performance has, however, been suboptimal in the first prospective studies which better reflect the real-world situation. In this article, we report the application of machine learning to a large, prospectively accrued, HCC surveillance data set. PATIENTS AND METHODS: Models were built on a cohort of 3,473 patients with chronic liver disease within a rigorous surveillance program between 1998 and 2014, during which 459 patients with HCC were detected. Two random forest (RF) models were trained. The first RF model uses the same variables as the original GALAD model (GALAD-RF); the second is based on routinely available clinical and laboratory features (RF-practical). For comparison, we evaluated a logistic regression GALAD model trained on this longitudinal prospective data set (termed GALAD-Ogaki). RESULTS: Models were evaluated using a repetitive cross-validation approach with the metrics averaged over 100 independent runs. As judged by area under the receiver operator curve (AUROC) and F1 score, the GALAD RF model significantly outperformed the original GALAD model. The RF-practical model also outperformed the original GALAD model in terms of both AUROC and F1 score, and both models outperformed the individual biomarkers. An online web application that implemented the GALAD-RF and RF-practical models is presented. CONCLUSION: RF-based models improve on the diagnostic performance of the original GALAD model in the setting of a standard HCC surveillance program. Further prospective validation studies are warranted using these models and could be expanded to offer prediction of risk of HCC development over defined periods of time.

Accessing metal-specific orbital interactions in C-H activation with resonant inelastic X-ray scattering.

Photochemically prepared transition-metal complexes are known to be effective at cleaving the strong C-H bonds of organic molecules in room temperature solutions. There is also ample theoretical evidence that the two-way, metal to ligand (MLCT) and ligand to metal (LMCT), charge-transfer between an incoming alkane C-H group and the transition metal is the decisive interaction in the C-H activation reaction. What is missing, however, are experimental methods to directly probe these interactions in order to reveal what determines reactivity of intermediates and the rate of the reaction. Here, using quantum chemical simulations we predict and propose future time-resolved valence-to-core resonant inelastic X-ray scattering (VtC-RIXS) experiments at the transition metal L-edge as a method to provide a full account of the evolution of metal-alkane interactions during transition-metal mediated C-H activation reactions. For the model system cyclopentadienyl rhodium dicarbonyl (CpRh(CO)2), we demonstrate, by simulating the VtC-RIXS signatures of key intermediates in the C-H activation pathway, how the Rh-centered valence-excited states accessible through VtC-RIXS directly reflect changes in donation and back-donation between the alkane C-H group and the transition metal as the reaction proceeds via those intermediates. We benchmark and validate our quantum chemical simulations against experimental steady-state measurements of CpRh(CO)2 and Rh(acac)(CO)2 (where acac is acetylacetonate). Our study constitutes the first step towards establishing VtC-RIXS as a new experimental observable for probing reactivity of C-H activation reactions. More generally, the study further motivates the use of time-resolved VtC-RIXS to follow the valence electronic structure evolution along photochemical, photoinitiated and photocatalytic reactions with transition metal complexes.

Influence of pump laser fluence on ultrafast myoglobin structural dynamics.

High-intensity femtosecond pulses from an X-ray free-electron laser enable pump-probe experiments for the investigation of electronic and nuclear changes during light-induced reactions. On timescales ranging from femtoseconds to milliseconds and for a variety of biological systems, time-resolved serial femtosecond crystallography (TR-SFX) has provided detailed structural data for light-induced isomerization, breakage or formation of chemical bonds and electron transfer1,2. However, all ultrafast TR-SFX studies to date have employed such high pump laser energies that nominally several photons were absorbed per chromophore3-17. As multiphoton absorption may force the protein response into non-physiological pathways, it is of great concern18,19 whether this experimental approach20 allows valid conclusions to be drawn vis-à-vis biologically relevant single-photon-induced reactions18,19. Here we describe ultrafast pump-probe SFX experiments on the photodissociation of carboxymyoglobin, showing that different pump laser fluences yield markedly different results. In particular, the dynamics of structural changes and observed indicators of the mechanistically important coherent oscillations of the Fe-CO bond distance (predicted by recent quantum wavepacket dynamics21) are seen to depend strongly on pump laser energy, in line with quantum chemical analysis. Our results confirm both the feasibility and necessity of performing ultrafast TR-SFX pump-probe experiments in the linear photoexcitation regime. We consider this to be a starting point for reassessing both the design and the interpretation of ultrafast TR-SFX pump-probe experiments20 such that mechanistically relevant insight emerges.

Identification of a novel perifornical-hypothalamic-area-projecting serotonergic system that inhibits innate panic and conditioned fear responses.

The serotonin (5-HT) system is heavily implicated in the regulation of anxiety and trauma-related disorders such as panic disorder and post-traumatic stress disorder, respectively. However, the neural mechanisms of how serotonergic neurotransmission regulates innate panic and fear brain networks are poorly understood. Our earlier studies have identified that orexin (OX)/glutamate neurons within the perifornical hypothalamic area (PFA) play a critical role in adaptive and pathological panic and fear. While site-specific and electrophysiological studies have shown that intracranial injection and bath application of 5-HT inhibits PFA neurons via 5-HT1a receptors, they largely ignore circuit-specific neurotransmission and its physiological properties that occur in vivo. Here, we investigate the role of raphe nuclei 5-HT inputs into the PFA in panic and fear behaviors. We initially confirmed that photostimulation of glutamatergic neurons in the PFA of rats produces robust cardioexcitation and flight/aversive behaviors resembling panic-like responses. Using the retrograde tracer cholera toxin B, we determined that the PFA receives discrete innervation of serotonergic neurons clustered in the lateral wings of the dorsal (lwDRN) and in the median (MRN) raphe nuclei. Selective lesions of these serotonergic projections with saporin toxin resulted in similar panic-like responses during the suffocation-related CO2 challenge and increased freezing to fear-conditioning paradigm. Conversely, selective stimulation of serotonergic fibers in the PFA attenuated both flight/escape behaviors and cardioexcitation responses elicited by the CO2 challenge and induced conditioned place preference. The data here support the hypothesis that PFA projecting 5-HT neurons in the lwDRN/MRN represents a panic/fear-off circuit and may also play a role in reward behavior.

Correlation of refractive index based and THz streaking arrival time tools for a hard X-ray free-electron laser.

To fully exploit ultra-short X-ray pulse durations routinely available at X-ray free-electron lasers to follow out-of-equilibrium dynamics, inherent arrival time fluctuations of the X-ray pulse with an external perturbing laser pulse need to be measured. In this work, two methods of arrival time measurement were compared to measure the arrival time jitter of hard X-ray pulses. The methods were photoelectron streaking by a THz field and a transient refractive index change of a semiconductor. The methods were validated by shot-to-shot correction of a pump-probe transient reflectivity measurement. An ultimate shot-to-shot full width at half-maximum error between the devices of 19.2 ± 0.1 fs was measured.

Directed ultrafast conformational changes accompany electron transfer in a photolyase as resolved by serial crystallography.

Charge-transfer reactions in proteins are important for life, such as in photolyases which repair DNA, but the role of structural dynamics remains unclear. Here, using femtosecond X-ray crystallography, we report the structural changes that take place while electrons transfer along a chain of four conserved tryptophans in the Drosophila melanogaster (6-4) photolyase. At femto- and picosecond delays, photoreduction of the flavin by the first tryptophan causes directed structural responses at a key asparagine, at a conserved salt bridge, and by rearrangements of nearby water molecules. We detect charge-induced structural changes close to the second tryptophan from 1 ps to 20 ps, identifying a nearby methionine as an active participant in the redox chain, and from 20 ps around the fourth tryptophan. The photolyase undergoes highly directed and carefully timed adaptations of its structure. This questions the validity of the linear solvent response approximation in Marcus theory and indicates that evolution has optimized fast protein fluctuations for optimal charge transfer.

Soft robotics for farm to fork: applications in agriculture & farming.

Agricultural tasks and environments range from harsh field conditions with semi-structured produce or animals, through to post-processing tasks in food-processing environments. From farm to fork, the development and application of soft robotics offers a plethora of potential uses. Robust yet compliant interactions between farm produce and machines will enable new capabilities and optimize existing processes. There is also an opportunity to explore how modeling tools used in soft robotics can be applied to improve our representation and understanding of the soft and compliant structures common in agriculture. In this review, we seek to highlight the potential for soft robotics technologies within the food system, and also the unique challenges that must be addressed when developing soft robotics systems for this problem domain. We conclude with an outlook on potential directions for meaningful and sustainable impact, and also how our outlook on both soft robotics and agriculture must evolve in order to achieve the required paradigm shift.

Regional Differences in Clinical Presentation and Prognosis of Patients With Post-Sustained Virologic Response Hepatocellular Carcinoma.

BACKGROUND & AIMS: Widespread use of direct-acting antivirals for hepatitis C virus infection has been paralleled with increased numbers of patients with hepatocellular carcinoma (HCC) after achieving sustained virologic response (post-SVR HCC) worldwide. Few data compare regional differences in the presentation and prognosis of patients with post-SVR HCC. METHODS: We identified patients with advanced fibrosis (F3/F4) who developed incident post-SVR HCC between March 2015 and October 2021 from 30 sites in Europe, North America, South America, the Middle East, South Asia, East Asia, and Southeast Asia. We compared patient demographics, liver dysfunction, and tumor burden by region. We compared overall survival by region using Kaplan-Meier analysis and identified factors associated with survival using multivariable Cox regression analysis. RESULTS: Among 8796 patients with advanced fibrosis or cirrhosis who achieved SVR, 583 (6.6%) developed incident HCC. There was marked regional variation in the proportion of patients detected by surveillance (range: 59.5%-100%), median maximum tumor diameter (range, 1.8-5.0 cm), and the proportion with multinodular HCC (range, 15.4%-60.8%). The prognosis of patients highly varied by region (hazard ratio range, 1.82-9.92), with the highest survival rates in East Asia, North America, and South America, and the lowest survival rates in the Middle East and South Asia. After adjusting for geographic region, HCC surveillance was associated with early stage detection (Barcelona Clinic Liver Cancer stage 0/A, 71.0% vs 21.3%; P < .0001) and lower mortality rates (adjusted hazard ratio, 0.29; 95% CI, 0.18-0.46). CONCLUSIONS: Clinical characteristics, including early stage detection, and prognosis of post-SVR HCC differed significantly across geographic regions. Surveillance utilization appears to be a high-yield intervention target to improve prognosis among patients with post-SVR HCC globally.

A multi-reservoir extruder for time-resolved serial protein crystallography and compound screening at X-ray free-electron lasers.

Serial crystallography at X-ray free-electron lasers (XFELs) permits the determination of radiation-damage free static as well as time-resolved protein structures at room temperature. Efficient sample delivery is a key factor for such experiments. Here, we describe a multi-reservoir, high viscosity extruder as a step towards automation of sample delivery at XFELs. Compared to a standard single extruder, sample exchange time was halved and the workload of users was greatly reduced. In-built temperature control of samples facilitated optimal extrusion and supported sample stability. After commissioning the device with lysozyme crystals, we collected time-resolved data using crystals of a membrane-bound, light-driven sodium pump. Static data were also collected from the soluble protein tubulin that was soaked with a series of small molecule drugs. Using these data, we identify low occupancy (as little as 30%) ligands using a minimal amount of data from a serial crystallography experiment, a result that could be exploited for structure-based drug design.

Unlocking Pb2+ Sensing Potential in a DNA G-Quadruplex via Loop Modification with Fluorescent Chalcone Surrogates.

The ability of guanine (G)-rich DNA to bind toxic lead (Pb2+) ions within a G-quadruplex (GQ) motif is a leading DNA biosensor strategy. A major analytical hurdle for GQ detection of Pb2+ is competitive GQ templating by potassium (K+) ions. We employ the on-strand DNA synthesis of internal fluorescent chalcone surrogates within the 15-mer thrombin binding aptamer (TBA15) to address this challenge. Replacement of thymidine at the 3-position (T3) within TBA15 with an indole-4-hydroxy-indanone (Ind4HI) chalcone strongly decreases K+-GQ stability while enhancing Pb2+-GQ stability to increase Pb2+ binding specificity. The new T3-Ind4HI probe exhibits a 15-fold increase in fluorescence intensity upon binding of Pb2+ by the modified TBA15 and can detect 6.4 nM Pb2+ in the presence of 10 mM K+. Thus, replacement of the T3 residue of TBA15 with the new Ind4HI probe modulates metal ion affinity by native TBA15 to solve the analytical challenge posed by K+ in real water samples for detecting Pb2+ to meet regulatory guidelines by using a GQ biosensor.

Visualizing the DNA repair process by a photolyase at atomic resolution.

Photolyases, a ubiquitous class of flavoproteins, use blue light to repair DNA photolesions. In this work, we determined the structural mechanism of the photolyase-catalyzed repair of a cyclobutane pyrimidine dimer (CPD) lesion using time-resolved serial femtosecond crystallography (TR-SFX). We obtained 18 snapshots that show time-dependent changes in four reaction loci. We used these results to create a movie that depicts the repair of CPD lesions in the picosecond-to-nanosecond range, followed by the recovery of the enzymatic moieties involved in catalysis, completing the formation of the fully reduced enzyme-product complex at 500 nanoseconds. Finally, back-flip intermediates of the thymine bases to reanneal the DNA were captured at 25 to 200 microseconds. Our data cover the complete molecular mechanism of a photolyase and, importantly, its chemistry and enzymatic catalysis at work across a wide timescale and at atomic resolution.

Time-resolved crystallography captures light-driven DNA repair.

Photolyase is an enzyme that uses light to catalyze DNA repair. To capture the reaction intermediates involved in the enzyme's catalytic cycle, we conducted a time-resolved crystallography experiment. We found that photolyase traps the excited state of the active cofactor, flavin adenine dinucleotide (FAD), in a highly bent geometry. This excited state performs electron transfer to damaged DNA, inducing repair. We show that the repair reaction, which involves the lysis of two covalent bonds, occurs through a single-bond intermediate. The transformation of the substrate into product crowds the active site and disrupts hydrogen bonds with the enzyme, resulting in stepwise product release, with the 3' thymine ejected first, followed by the 5' base.

Predictive and Prognostic Potential of Liver Function Assessment in Patients with Advanced Hepatocellular Carcinoma: A Systematic Literature Review.

Introduction: We conducted a systematic literature review to assess the utility of liver function assessments for predicting disease prognosis and response to systemic anticancer therapy in patients with advanced hepatocellular carcinoma (aHCC). Methods: This was a PRISMA-standard review and was registered with PROSPERO (CRD42021244588). MEDLINE and Embase were systematically searched (March 24, 2021) to identify publications reporting the efficacy and/or safety of systemic anticancer therapy (vs. any/no comparator) in liver-function-defined subgroups in phase 2 or 3 aHCC trials. Screening was completed by a single reviewer, with uncertainties resolved by a second reviewer and/or the authors. English-language full-text articles and congress abstracts were eligible for inclusion. Included publications were described and assessed for risk of bias using the GRADE methodology. Results: Twenty (of 2,579) screened publications were eligible; seven categorized liver function using the albumin-bilirubin system, nine using the Child-Pugh system, four using both. GRADE assessment classified ten, nine, and one publication(s) as reporting moderate-quality, low-quality, and very-low-quality evidence, respectively. Analyses of cross-trial trends of within-exposure arm analyses (active and control) reported a positive relationship between baseline liver function and overall survival and progression-free survival, supporting liver function as a prognostic marker in aHCC. There were also signals for a modest relationship between more preserved baseline liver function and extent of systemic treatment benefit, and with more preserved liver function and lower incidence of safety events. Conclusion: This review supports liver function as a prognostic variable in aHCC and highlights the value of a priori stratification of patients by baseline liver function in aHCC trials. The predictive value of liver function warrants further study. Findings were limited by the quality of available data.

European' health care indicators and pancreatic cancer incidence and mortality: A mediation analysis of Eurostat data and Global Burden of Disease Study 2019.

AIM: To highlight correlations existing between incidence and mortality of pancreatic cancer, and health care indicators in 36 European countries. METHODS: The Global Burden of Disease (GBD) and Eurostat databases were queried between 2004 and 2019. Incidence and mortality were age-standardized. From Eurostat, indicators regarding expenditure, hospital beds, medical technology, health personnel, physicians by medical specialty and unmet needs for medical examination were extracted. Correlations between GBD and Eurostat data were analysed through mediation analysis applying clustering for countries. RESULTS: Incidence increased by +0.6% per year (p = 0.001) and mortality by +0.3% (p = 0.001), being increasing for most of the European countries considered. Incidence and mortality were strongly positively correlated (p = 0.001). Higher current health expenditure, expenditure in inpatient curative care, the number of available beds, the number of computed tomography scan, magnetic resonance units, practising medical doctors were all related to higher incidence (p < 0.05), whereas the unmet need for medical examinations was related to lower incidence. When the mediator' effect of incidence was handled, these indicators, together with expenditure on outpatient curative cares, the number of pet scanners and of radiation therapy equipment, were related to lower mortality (p < 0.05). CONCLUSIONS: Health care environment correlates with reported incidence and mortality of pancreatic cancer. This highlights both that ameliorated socio-economic societies suffer from higher incidence but lower mortality, as well as the epidemiological bias originating from countries' diagnostic ability.

Effect of Sugar Beet Pulp on the Composition and Predicted Function of Equine Fecal Microbiota.

The purpose of this study is to determine the effect of the partial replacement of dietary hay with sugar beet pulp (SBP) on the composition and predicted function of the fecal microbiota of healthy adult horses. Fecal samples were collected daily for 12 days from six adult horses after removal from pasture, including a five-day acclimation period, and a seven-day period following the introduction of SBP into their diet, and compared to six untreated horses over a comparable period. Fecal DNA was subjected to 16S rRNA amplicon sequencing and a longitudinal analysis was performed comparing the composition and predicted function. While no significant treatment-associated changes in the richness, alpha diversity, or beta diversity were detected, random forest regression identified several high-importance taxonomic features associated with change over time in horses receiving SBP. A similar analysis of the predicted functional pathways identified several high-importance pathways, including those involved in the production of L-methionine and butyrate. These data suggest that feeding SBP to healthy adult horses acutely increases the relative abundance of several Gram-positive taxa, including Cellulosilyticum sp., Moryella sp., and Weissella sp., and mitigates the predicted functional changes associated with removal from pasture. Large-scale studies are needed to assess the protective effect of SBP on the incidence of the gastrointestinal conditions of horses.

Surveillance and treatment of primary hepatocellular carcinoma (aka. STOP HCC): protocol for a prospective cohort study of high-risk patients for HCC using GALAD-score.

BACKGROUND: Vietnam and Saudi Arabia have high disease burden of primary hepatocellular carcinoma (HCC). Early detection in asymptomatic patients at risk for HCC is a strategy to improve survival outcomes in HCC management. GALAD score, a serum-based panel, has demonstrated promising clinical utility in HCC management. However, in order to ascertain its potential role in the surveillance of the early detection of HCC, GALAD needs to be validated prospectively for clinical surveillance of HCC (i.e., phase IV biomarker validation study). Thus, we propose to conduct a phase IV biomarker validation study to prospectively survey a cohort of patients with advanced fibrosis or compensated cirrhosis, irrespective of etiologies, using semi-annual abdominal ultrasound and GALAD score for five years. METHODS: We plan to recruit a cohort of 1,600 patients, male or female, with advanced fibrosis or cirrhosis (i.e., F3 or F4) and MELD ≤ 15, in Vietnam and Saudi Arabia (n = 800 each). Individuals with a liver mass ≥ 1 cm in diameter, elevated alpha-fetoprotein (AFP) (≥ 9 ng/mL), and/or elevated GALAD score (≥ -0.63) will be scanned with dynamic contrast-enhanced magnetic resonance imaging (MRI), and a diagnosis of HCC will be made by Liver Imaging Reporting and Data System (LiRADS) assessment (LiRADS-5). Additionally, those who do not exhibit abnormal imaging findings, elevated AFP titer, and/or elevated GALAD score will obtain a dynamic contrast-enhanced MRI annually for five years to assess for HCC. Only MRI nearest to the time of GALAD score measurement, ultrasound and/or AFP evaluation will be included in the diagnostic validation analysis. MRI will be replaced with an abdominal computed tomography scan when MRI results are poor due to patient conditions such as movement etc. Gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid-enhanced MRI will not be carried out in study sites in both countries. Bootstrap resampling technique will be used to account for repeated measures to estimate standard errors and confidence intervals. Additionally, we will use the Cox proportional hazards regression model with covariates tailored to the hypothesis under investigation for time-to-HCC data as predicted by time-varying biomarker data. DISCUSSION: The present work will evaluate the performance of GALAD score in early detection of liver cancer. Furthermore, by leveraging the prospective cohort, we will establish a biorepository of longitudinally collected biospecimens from patients with advanced fibrosis or cirrhosis to be used as a reference set for future research in early detection of HCC in the two countries. TRIAL REGISTRATION: Name of the registry: ClinicalTrials.gov Registration date: 22 April 2022 Trial registration number: NCT05342350 URL of trial registry record.