Embracing the era of antimicrobial peptides with marine organisms.

Covering: 2018 to Jun of 2023The efficiency of traditional antibiotics has been undermined by the proliferation of antibiotic-resistant pathogenic microorganisms, necessitating the pursuit of innovative therapeutic agents. Antimicrobial peptides (AMPs), which are part of host defence peptides found ubiquitously in nature, exhibiting a wide range of activity towards bacteria, fungi, and viruses, offer a highly promising candidate solution. The efficacy of AMPs can frequently be augmented via alterations to their amino acid sequences or structural adjustments. Given the vast reservoir of marine life forms and their distinctive ecosystems, marine AMPs stand as a burgeoning focal point in the quest for alternative peptide templates extracted from natural sources. Advances in identification and characterization techniques have accelerated the discoveries of marine AMPs, thereby stimulating AMP customization, optimization, and synthesis research endeavours. This review presents an overview of recent discoveries related to the intriguing qualities of marine AMPs. Emphasis will be placed upon post-translational modifications (PTMs) of marine AMPs and how they may impact functionality and potency. Additionally, this review considers ways in which marine PTM might support larger-scale, heterologous AMP manufacturing initiatives, providing insights into translational applications of these important biomolecules.

Oncolytic vaccinia virus harboring aphrocallistes vastus lectin exerts anti-tumor effects by directly oncolysis and inducing immune response through enhancing ROS in human ovarian cancer.

Aphrocallistes vastus lectin (AVL) is a Ca2+ dependent C-type lectin produced by sponges. Previous studies have demonstrated that oncolytic vaccinia virus harboring AVL (oncoVV-AVL) effectively triggers cell death in various tumors. However, the effects of oncoVV-AVL on human ovarian cancer (OV) remain unknown. This study aims to investigate the mechanism-of-action of oncoVV-AVL in human OV cell lines and in tumor-bearing nude mice. We found that oncoVV-AVL could directly induce apoptosis and autophagy in ovarian cancer cells. Additionally, our results showed that oncoVV-AVL increased the serum levels of mouse IFN-γ (mIFN-γ), leading to the activation of M1-polarized macrophages. Conversely, NADPH, a reducing agent by providing reducing equivalents, reduced the production of mIFN-γ, and suppressed M1-polarization of macrophage. Based on these findings, we propose that oncoVV-AVL not only contributes to direct cytolysis, but also enhances host immune response by promoting ROS levels.

Tolerance enhancement of Dendrobium officinale by salicylic acid family-related metabolic pathways under unfavorable temperature.

BACKGROUND: Unfavorable temperatures significantly constrain the quality formation of Dendrobium officinale, severely limiting its food demand. Salicylic acid (SA) enhances the resistance of D. officinale to stress and possesses various analogs. The impact and mechanism of the SA family on improving the quality of D. officinale under adverse temperature conditions remains unclear. RESULTS: Combined with molecular docking analysis, chlorophyll fluorescence and metabolic analysis after treatments with SA analogues or extreme temperatures are performed in this study. The results demonstrate that both heat and cold treatments impede several main parameters of chlorophyll fluorescence of D. officinale, including the ΦPSII parameter, a sensitive growth indicator. However, this inhibition is mitigated by SA or its chemically similar compounds. Comprehensive branch imaging of ΦPSII values revealed position-dependent improvement of tolerance. Molecular docking analysis using a crystal structure model of NPR4 protein reveals that the therapeutic effects of SA analogs are determined by their binding energy and the contact of certain residues. Metabolome analysis identifies 17 compounds are considered participating in the temperature-related SA signaling pathway. Moreover, several natural SA analogs such as 2-hydroxycinnamic acid, benzamide, 2-(formylamino) benzoic acid and 3-o-methylgallic acid, are further found to have high binding ability to NPR4 protein and probably enhance the tolerance of D. officinale against unfavorable temperatures through flavone and guanosine monophosphate degradation pathways. CONCLUSIONS: These results reveal that the SA family with a high binding capability of NPR4 could improve the tolerance of D. officinale upon extreme temperature challenges. This study also highlights the collaborative role of SA-related natural compounds present in D. officinale in the mechanism of temperature resistance and offers a potential way to develop protective agents for the cultivation of D. officinale.

Mendelian randomization in hepatology: A review of principles, opportunities, and challenges.

Mendelian randomization has become a popular tool to assess causal relationships using existing observational data. While randomized controlled trials are considered the gold standard for establishing causality between exposures and outcomes, it is not always feasible to conduct a trial. Mendelian randomization is a causal inference method that uses observational data to infer causal relationships by using genetic variation as a surrogate for the exposure of interest. Publications using the approach have increased dramatically in recent years, including in the field of hepatology. In this concise review, we describe the concepts, assumptions, and interpretation of Mendelian randomization as related to studies in hepatology. We focus on the strengths and weaknesses of the approach for a non-statistical audience, using an illustrative example to assess the causal relationship between body mass index and NAFLD.

Spatiotemporal dual-periodic soliton pulsation in a multimode fiber laser.

Spatiotemporal mode-locked (STML) fiber lasers have become a new platform for investigating nonlinear phenomena. In this work, spatiotemporal dual-periodic soliton pulsation (SDSP) is firstly observed in an STML fiber laser. It is found that in the SDSP, the long-period pulsations (LPPs) of different transverse modes are synchronous, while the short-period pulsations (SPPs) exhibit asynchronous modulations. The numerical simulation confirms the experimental results and further reveals that the proportion of transverse mode components can manipulate the periods of the LPP and SPP but does not affect the synchronous and asynchronous pulsations of different transverse modes. The obtained results bring the study of spatiotemporal dissipative soliton pulsation into the multi-period modulation stage, which helps to understand the complex spatiotemporal dynamics in STML fiber lasers and discover new dynamics in high-dimensional nonlinear systems.

Behavioral carry-over effect and power consideration in crossover trials.

A crossover trial is an efficient trial design when there is no carry-over effect. To reduce the impact of the biological carry-over effect, a washout period is often designed. However, the carry-over effect remains an outstanding concern when a washout period is unethical or cannot sufficiently diminish the impact of the carry-over effect. The latter can occur in comparative effectiveness research, where the carry-over effect is often non-biological but behavioral. In this paper, we investigate the crossover design under a potential outcomes framework with and without the carry-over effect. We find that when the carry-over effect exists and satisfies a sign condition, the basic estimator underestimates the treatment effect, which does not inflate the type I error of one-sided tests but negatively impacts the power. This leads to a power trade-off between the crossover design and the parallel-group design, and we derive the condition under which the crossover design does not lead to type I error inflation and is still more powerful than the parallel-group design. We also develop covariate adjustment methods for crossover trials. We evaluate the performance of cross-over design and covariate adjustment using data from the MTN-034/REACH study.

Adjusting for incomplete baseline covariates in randomized controlled trials: a cross-world imputation framework.

In randomized controlled trials, adjusting for baseline covariates is commonly used to improve the precision of treatment effect estimation. However, covariates often have missing values. Recently, Zhao and Ding studied two simple strategies, the single imputation method and missingness-indicator method (MIM), to handle missing covariates and showed that both methods can provide an efficiency gain compared to not adjusting for covariates. To better understand and compare these two strategies, we propose and investigate a novel theoretical imputation framework termed cross-world imputation (CWI). This framework includes both single imputation and MIM as special cases, facilitating the comparison of their efficiency. Through the lens of CWI, we show that MIM implicitly searches for the optimal CWI values and thus achieves optimal efficiency. We also derive conditions under which the single imputation method, by searching for the optimal single imputation values, can achieve the same efficiency as the MIM. We illustrate our findings through simulation studies and a real data analysis based on the Childhood Adenotonsillectomy Trial. We conclude by discussing the practical implications of our findings.

GENIUS-MAWII: for robust Mendelian randomization with many weak invalid instruments.

Mendelian randomization (MR) addresses causal questions using genetic variants as instrumental variables. We propose a new MR method, G-Estimation under No Interaction with Unmeasured Selection (GENIUS)-MAny Weak Invalid IV, which simultaneously addresses the 2 salient challenges in MR: many weak instruments and widespread horizontal pleiotropy. Similar to MR-GENIUS, we use heteroscedasticity of the exposure to identify the treatment effect. We derive influence functions of the treatment effect, and then we construct a continuous updating estimator and establish its asymptotic properties under a many weak invalid instruments asymptotic regime by developing novel semiparametric theory. We also provide a measure of weak identification, an overidentification test, and a graphical diagnostic tool.

Possibility of PD-1/PD-L1 Inhibitors for the Treatment of Patients with Chronic Hepatitis B Infection.

BACKGROUND: Chronic hepatitis B (CHB) infection is still a major global public health problem, with nearly two billion patients. Although current antiviral drugs can inhibit viral replication and reduce hepatitis B virus (HBV) related complications, it is difficult to achieve clinical endpoints due to drug resistance. SUMMARY: Immune checkpoint inhibitors (ICIs) are an important strategy to reverse T-cell exhaustion, and rebuilding an effective functional T-cell response is a promising immunomodulatory approach for CHB patients. However, ICIs may lead to viral reactivation or immune-related adverse effects. There are still many controversies in the application of ICIs in treating patients with CHB. KEY MESSAGES: This article reviews the research progress of ICIs in CHB infection and related issues. The goal of this paper was to summarize the possible impact of new therapies for CHB with the aim of reducing potential clinical risks.

Self-supported electrochemical sensor based on uniform palladium nanoparticles functionalized porous graphene film for monitoring H2O2 released from living cells.

Graphene film has been considered a promising material for the construction of self-supported electrodes due to its favorable flexibility and high conductivity. However, the film fabricated from pristine graphene or conventional graphene sheet reduced graphene oxide processes limited electrocatalytic performance. Decorating active metal species or incorporating heteroatoms into the graphene framework have been proved to be effective methods to enhance the electrocatalytic efficiency of graphene film-based self-supported electrodes. Herein, we present a freestanding electrode composed of uniform Pd nanoparticles decorating N,S co-doped porous graphene film (Pd/NSPGF) and explore its practical application in differentiating various human colon cell types by in situ tracking the amount of H2O2 secreted from live cells. Our findings reveal that, on the one hand, the NSPGF has abundant surface and inner pores, which promote active site exposure, and mass diffusion during electrochemical reactions; on the other hand, the substitutional doping of the graphene framework with heteroatoms (e.g., N or S) can tailor its electronic and chemical properties, and facilitate the uniform loading of high-density Pd nanoparticles. Moreover, the intrinsic activity of Pd/NSPGF is regulated by the interaction of Pd nanoparticles with the NSPGF support. Taking the advantages of morphology and composition, the self-supported Pd/NSPGF electrode displays remarkable electrochemical performance with a wide linear range up to 2.0 mM, low detection limit of 0.1 µM (S/N = 3), high sensitivity of 665 µA cm-2 mM-1, and good selectivity. When applied in real-time tracking of the H2O2 released from normal human colon epithelial cells and human colorectal cancer cells, the Pd/NSPGF-based electrochemical sensing system can distinguish the cell types by testing the number of extracellular H2O2 molecules released per cell, which holds considerable potential for early detection and monitoring of disease-related clinical specimens.

Chronic kidney disease and cognitive performance: NHANES 2011-2014.

PURPOSE: Previous studies suggest an association between chronic kidney disease (CKD) and cognitive impairment. The purpose of this study was to explore the association between the diverse stages of CKD and the cognitive performance of elderly American adults. METHODS: Data from the National Health and Nutrition Examination Survey (NHANES) 2011-2014 were used. Multivariate adjusted logistic regression, subgroup analysis, and the restricted cubic spline model were used to assess the associations of CKD stage and estimated glomerular filtration rate (eGFR) with cognitive performance. The measures used to evaluate cognitive function included the Consortium to Establish a Registry for Alzheimer's Disease (CERAD) test, the Animal Fluency test, and the Digit Symbol Substitution test (DSST). RESULTS: This study included 2234 participants aged ≥ 60 years. According to the fully adjusted model, stages 3-5 CKD were significantly associated with the CERAD test score (OR = 0.70, 95% CI [0.51, 0.97], p = 0.033), the Animal Fluency test score (OR = 0.64, 95% CI [0.48, 0.85], p = 0.005), and the DSST score (OR = 0.60, 95% CI [0.41, 0.88], p = 0.013). In addition, the incidence of poor cognitive function increased with decreasing eGFR, especially for individuals with low and moderate eGFRs. Both the DSST score (p nonlinearity < 0.0001) and the Animal Fluency test score (p nonlinearity = 0.0001) had nonlinear dose-response relationships with the eGFR. However, a linear relationship was shown between the eGFR and CERAD test score (p nonlinearity = 0.073). CONCLUSIONS: CKD, especially stages3-5 CKD, was significantly associated with poor cognitive performance in terms of executive function, learning, processing speed, concentration, and working memory ability. All adults with CKD should be screened for cognitive impairment.

Network pharmacology-based approach to understand the effect and mechanism of chrysophanol against cognitive impairment in Wilson disease.

Wilson disease (WD) is a rare hereditary copper metabolism disorder, wherein cognitive impairment is a common clinical symptom. Chrysophanol (CHR) is an active compound with neuroprotective effects. The study aims to investigate the neuroprotective effect of CHR in WD and attempted to understand the potential mechanisms. Network pharmacology analysis was applied to predict the core target genes of CHR against cognitive impairment in WD. The rats fed with copper-laden diet for 12 weeks, and the effect of CHR on the copper content in liver and 24-h urine, the learning and memory ability, the morphological changes and the apoptosis level of neurons in hippocampal CA1 region, the expression level of Bax, Bcl-2, Cleaved Caspase-3, p-PI3K, PI3K, p-AKT, and AKT proteins were detected. Network pharmacology analysis showed that cell apoptosis and PI3K-AKT signaling pathway might be the main participants in CHR against cognitive impairment in WD. The experiments showed that CHR could reduce the copper content in liver, increase the copper content in 24-h urine, improve the ability of the learning and memory, alleviate the damage and apoptosis level of hippocampal neurons, down-regulate the expression of Bax, Cleaved Caspase-3, and up-regulate the expressions of Bcl-2, p-PI3K/PI3K, p-AKT/AKT. These results suggested that CHR could alleviate cognitive impairment in WD by inhibiting cell apoptosis and triggering the PI3K-AKT signaling pathway.

ROS Induced by Aphrocallistes vastus Lectin Enhance Oncolytic Vaccinia Virus Replication and Induce Apoptosis in Hepatocellular Carcinoma Cells.

Oncolytic virotherapy is expected to provide a new treatment strategy for cancer. Aphrocallistes vastus lectin (AVL) is a Ca2+-dependent lectin receptor containing the conserved domain of C-type lectin and the hydrophobic N-terminal region, which can bind to the bird's nest glycoprotein and D-galactose. Our previous studies suggested that the oncolytic vaccinia virus (oncoVV) armed with the AVL gene exerted remarkable replication and antitumor effects in vitro and in vivo. In this study, we found that oncoVV-AVL may reprogram the metabolism of hepatocellular carcinoma cells to promote ROS, and elevated ROS subsequently promoted viral replication and induced apoptosis. This study will provide a new theoretical basis for the application of oncoVV-AVL in liver cancer.

Cognitive and emotional impairment in stroke survivors: insights from a multi-center study on inpatient rehabilitation therapy.

BACKGROUND: Individuals recovering from stroke often experience cognitive and emotional impairments, but rehab programs tend to focus on motor skills. The aim of this investigation is to systematically assess the change of magnitude of cognitive and emotional function subsequent to a conventional rehabilitative protocol administered to stroke survivors within a defined locale in China. METHODS: This is a multicenter study; a total of 1884 stroke survivors who received in-hospital rehabilitation therapy were assessed on admission (T0) and discharge (T1). The tool of InterRAI was used to assess cognitive, emotional, and behavioral abnormality. RESULTS: The patients aged >60 years, with a history of hypertension, and long stroke onset duration were more exposed to functional impairment (all p < 0.05). Both cognitive and emotional sections were significantly improved at T1 compared to T0 (p < 0.001). Initially, 64.97% and 46.55% of patients had cognitive or emotional impairment at T0, respectively; this percentage was 58.55% and 37.15% at T1. CONCLUSION: Many stroke survivors have ongoing cognitive and emotional problems that require attention. It is essential to focus on rehabilitating these areas during the hospital stay, especially for older patients, those with a longer recovery, and those with hypertension history.

Metabonomics and Transcriptomics Analyses Reveal the Development Process of the Auditory System in the Embryonic Development Period of the Small Yellow Croaker under Background Noise.

Underwater noise pollution has become a potential threat to aquatic animals in the natural environment. The main causes of such pollution are frequent human activities creating underwater environmental noise, including commercial shipping, offshore energy platforms, scientific exploration activities, etc. However, in aquaculture environments, underwater noise pollution has also become an unavoidable problem due to background noise created by aquaculture equipment. Some research has shown that certain fish show adaptability to noise over a period of time. This could be due to fish's special auditory organ, i.e., their "inner ear"; meanwhile, otoliths and sensory hair cells are the important components of the inner ear and are also essential for the function of the auditory system. Recently, research in respect of underwater noise pollution has mainly focused on adult fish, and there is a lack of the research on the effects of underwater noise pollution on the development process of the auditory system in the embryonic development period. Thus, in this study, we collected embryo-larval samples of the small yellow croaker (Larimichthys polyactis) in four important stages of otic vesicle development through artificial breeding. Then, we used metabonomics and transcriptomics analyses to reveal the development process of the auditory system in the embryonic development period under background noise (indoor and underwater environment sound). Finally, we identified 4026 differentially expressed genes (DEGs) and 672 differential metabolites (DMs), including 37 DEGs associated with the auditory system, and many differences mainly existed in the neurula stage (20 h of post-fertilization/20 HPF). We also inferred the regulatory mode and process of some important DEGs (Dnmt1, CPS1, and endothelin-1) in the early development of the auditory system. In conclusion, we suggest that the auditory system development of L. polyactis begins at least in the neurula stage or earlier; the other three stages (tail bud stage, caudal fin fold stage, and heart pulsation stage, 28-35 HPF) mark the rapid development period. We speculate that the effect of underwater noise pollution on the embryo-larval stage probably begins even earlier.

QTL Mapping-Based Identification of Visceral White-Nodules Disease Resistance Genes in Larimichthys polyactis.

Disease outbreaks in aquaculture have recently intensified. In particular, visceral white-nodules disease, caused by Pseudomonas plecoglossicida, has severely hindered the small yellow croaker (Larimichthys polyactis) aquaculture industry. However, research on this disease is limited. To address this gap, the present study employed a 100K SNP chip to genotype individuals from an F1 full-sib family, identify single nucleotide polymorphisms (SNPs), and construct a genetic linkage map for this species. A high-density genetic linkage map spanning a total length of 1395.72 cM with an average interval of 0.08 cM distributed across 24 linkage groups was obtained. Employing post-infection survival time as an indicator of disease resistance, 13 disease resistance-related quantitative trait loci (QTLs) were detected, and these regions included 169 genes. Functional enrichment analyses pinpointed 11 candidate disease resistance-related genes. RT-qPCR analysis revealed that the genes of chmp1a and arg1 are significantly differentially expressed in response to P. plecoglossicida infection in spleen and liver tissues, indicating their pivotal functions in disease resistance. In summary, in addition to successfully constructing a high-density genetic linkage map, this study reports the first QTL mapping for visceral white-nodules disease resistance. These results provide insight into the intricate molecular mechanisms underlying disease resistance in the small yellow croaker.

[Mechanism of ganoderic acid X in treating hepatoblastoma based on proteomics].

This research explored the mechanism of ganoderic acid X(GAX) on human hepatocellular carcinoma cell models(HepG2, HuH6) and nonobese diabetic-severe combined immune deficient(NOD-SCID) mouse subcutaneous tumor models using proteomics, aiming to provide a basis for the clinical application of GAX. CCK-8 assay was employed to evaluate the effect of GAX on the viability of HepG2 and HuH6 cells. EdU assay was used to assess the effect of GAX on cell proliferation. Scratch assay was used to examine the effect of GAX on cell migration ability. Hoechst 33258 staining was used to investigate the effect of GAX on cell apoptosis. Moreover, a NOD-SCID mouse subcutaneous tumor model was established to analyze the tumor volume and weight in control group and GAX low-, medium-, and high-dose groups(5, 10, and 20 mg·kg~(-1)). HE staining was conducted to evaluate the drug toxicity of GAX. Additionally, HepG2 cells in the control group and the GAX high-dose group were subjected to label-free proteomics analysis to identify differential proteins and enrich relevant signaling pathways. CYTO-ID® staining was performed to detect autophagy, and Western blot was conducted to measure the expression levels of relevant proteins. In vitro results demonstrated that GAX dose-depen-dently inhibited proliferation, migration, and induced apoptosis in HepG2 and HuH6 cells. In vivo studies showed that GAX significantly inhibited tumor volume and weight without causing significant damage to major organs(heart, liver, spleen, lung, and kidney) in mice. Label-free proteomics analysis revealed that GAX participated in multiple signaling pathways during the treatment of hepatocellular carcinoma, with a high enrichment in the autophagy pathway. CYTO-ID® staining and Western blot results showed that GAX induced autophagy, upregulated the expression of Beclin-1, ATG5, and LC3-Ⅱ proteins, and downregulated the expression of p62 protein. This study suggests that GAX inhibits the proliferation, migration, and induces apoptosis of hepatocellular carcinoma cells by inducing autophagy, thereby significantly inhibiting tumor growth. GAX represents a promising adjuvant therapy for cancer treatment.

Formulation design and characterization of silymarin liposomes for enhanced antitumor activity.

Liposomes, a nanoscale carrier, plays an important role in the delivery of drug, affects the in vivo efficacy of drugs. In this paper, silymarin(SM)-loaded liposomes was optimized using the response surface method (RSM), with entrapment efficiency (EE%) as an index. The formulation was optimized as follow: lecithin (7.8mg/mL), SM/lecithin (1/26) and lecithin/cholesterol (10/1). The optimized SM liposomes had a high EE (96.58 ±3.06%), with a particle size of 290.3 ±10.5nm and a zeta potential of +22.98 ±1.73mV. In vitro release tests revealed that SM was released in a sustained-release manner, primarily via diffusion mechanism. In vitro cytotoxicity studies demonstrated that the prepared SM liposomes had stronger inhibitory effects than the model drug. Overall, these results indicate that this liposome system is suitable for intravenous delivery to enhance the antitumor effects of SM.

Esophageal cancer in China: Practice and research in the new era.

China, as the one of the largest developing countries in the world and with about one-fifth of the global population, is bearing an increasing burden on health from cancer. In the area of esophageal cancer (EC), China accounts for more than 50% of the global cases, with this disease being a particularly worse for those in disadvantaged populations. Along with China's socioeconomic condition, the epidemiology, diagnosis, therapeutics and research of EC have developed throughout the 21st century. In the current review, existing control measures for EC in China are outlined, including the incidence, mortality, screening, clinical diagnosis, multidisciplinary treatment and research landscape. EC in China are very different from those in some other parts of the world, especially in Western countries. Core measures that could contribute to the prevention of EC and improve clinical outcomes in patients of less developed countries and beyond are recommended. International cooperation among academia, government and industry is especially warranted in global EC control.

Cancer-associated fibroblasts contribute to the immunosuppressive landscape and influence the efficacy of the combination therapy of PD-1 inhibitors and antiangiogenic agents in hepatocellular carcinoma.

BACKGROUND: Chronic inflammation is considered the most critical predisposing factor of hepatocellular carcinoma (HCC), with inflammatory cell heterogeneity, hepatic fibrosis accumulation, and abnormal vascular proliferation as prominent features of the HCC tumor microenvironment (TME). Cancer-associated fibroblasts (CAFs) play a key role in HCC TME remodeling. Therefore, the level of abundance of CAFs may significantly affect the prognosis and outcome in HCC patients. METHODS: Unsupervised clustering was performed based on 39 genes related to CAFs in HCC identified by single-cell RNA sequencing data. Patients of bulk RNA were grouped into CAF low abundance cluster and high abundance clusters. Subsequently, prognosis, immune infiltration landscape, metabolism, and treatment response between the two clusters were investigated and validated by immunohistochemistry. RESULTS: Patients in the CAF high cluster had a higher level of inflammatory cell infiltration, a more significant immunosuppressive microenvironment, and a significantly worse prognosis than those in the low cluster. At the metabolic level, the CAF high cluster had lower levels of aerobic oxidation and higher angiogenic scores. Drug treatment response prediction indicated that the CAF high cluster could have a better response to PD-1 inhibitors and conventional chemotherapeutic agents for HCC such as anti-angiogenic drugs, whereas CAF low cluster may be more sensitive to transarterial chemoembolization treatment. CONCLUSIONS: This study not only revealed the TME characteristics of HCC with the difference in CAF abundance but also further confirmed that the combination therapy of PD-1 inhibitors and anti-angiogenic drugs may be more valuable for patients with high CAF abundance.