tRNA-derived fragments in T lymphocyte-beta cell crosstalk and in type 1 diabetes pathogenesis in NOD mice.

AIMS/HYPOTHESIS: tRNAs play a central role in protein synthesis. Besides this canonical function, they were recently found to generate non-coding RNA fragments (tRFs) regulating different cellular activities. The aim of this study was to assess the involvement of tRFs in the crosstalk between immune cells and beta cells and to investigate their contribution to the development of type 1 diabetes. METHODS: Global profiling of the tRFs present in pancreatic islets of 4- and 8-week-old NOD mice and in extracellular vesicles released by activated CD4+ T lymphocytes was performed by small RNA-seq. Changes in the level of specific fragments were confirmed by quantitative PCR. The transfer of tRFs from immune cells to beta cells occurring during insulitis was assessed using an RNA-tagging approach. The functional role of tRFs increasing in beta cells during the initial phases of type 1 diabetes was determined by overexpressing them in dissociated islet cells and by determining the impact on gene expression and beta cell apoptosis. RESULTS: We found that the tRF pool was altered in the islets of NOD mice during the initial phases of type 1 diabetes. Part of these changes were triggered by prolonged exposure of beta cells to proinflammatory cytokines (IL-1ß, TNF-α and IFN-γ) while others resulted from the delivery of tRFs produced by CD4+ T lymphocytes infiltrating the islets. Indeed, we identified several tRFs that were enriched in extracellular vesicles from CD4+/CD25- T cells and were transferred to beta cells upon adoptive transfer of these immune cells in NOD.SCID mice. The tRFs delivered to beta cells during the autoimmune reaction triggered gene expression changes that affected the immune regulatory capacity of insulin-secreting cells and rendered the cells more prone to apoptosis. CONCLUSIONS/INTERPRETATION: Our data point to tRFs as novel players in the crosstalk between the immune system and insulin-secreting cells and suggest a potential involvement of this novel class of non-coding RNAs in type 1 diabetes pathogenesis. DATA AVAILABILITY: Sequences are available from the Gene Expression Omnibus (GEO) with accession numbers GSE242568 and GSE256343.

Comparative study on seasonal hair follicle cycling by analysis of the transcriptomes from cashmere and milk goats.

Guard hair and cashmere undercoat are developed from primary and secondary hair follicle, respectively. Little is known about the gene expression differences between primary and secondary hair follicle cycling. In this study, we obtained RNA-seq data from cashmere and milk goats grown at four different seasons. We studied the differentially expressed genes (DEGs) during the yearly hair follicle cycling, and between cashmere and milk goats. WNT, NOTCH, MAPK, BMP, TGFß and Hedgehog signaling pathways were involved in hair follicle cycling in both cashmere and milk goat. However, Milk goat DEGs between different months were significantly more than cashmere goat DEGs, with the largest difference being identified in December. Some expression dynamics were confirmed by quantitative PCR and western blot, and immunohistochemistry. This study offers new information sources related to hair follicle cycling in milk and cashmere goats, which could be applicable to improve the wool production and quality.

DBU-Glycerol Solution: A CO2 Absorbent with High Desorption Ratio and Low Regeneration Energy.

1,8-Diazabicyclo[5.4.0]-undec-7-ene (DBU)-glycerol solution is employed as a promising CO2 absorbent. The regeneration of the CO2-loaded solution is of vital importance for its commercialization. It was investigated and compared with 30 wt % monoethanolamine (MEA). Variables affecting the absorption and desorption processes were studied, including the solvent composition, reaction temperature, and agitation. It shows that the absorption capacity for DBU-glycerol is comparable with 30 wt % MEA, and the desorption ratio for CO2-loaded DBU-glycerol mixture can reach as high as 95% in 60 min, 363 K at the 3:1 molar ratio of DBU to glycerol, while it is only 68% even after 165 min for CO2-saturated 30 wt % MEA. DBU-glycerol solution has higher cycling CO2 loading than 30 wt % MEA. Its cyclic capacity could keep above 90% after 10 cycles of absorption-desorption experiments. The desorption reaction is irreversible at the initial stage, and the reaction rate is expected as a first-order reaction from 349 to 377 K, and the apparent activation energy is 68.94 kJ/mol. Moreover, the heat duty of the reboiler during regeneration is estimated to be reduced by about 27% when compared with 30 wt % MEA.

The dynamics of FTO binding and demethylation from the m6A motifs.

N6-methyladenosine (m6A) is considered as a reversible RNA modification occurring more frequently on the GAC than AAC context in vivo, which regulates post-transcriptional gene expression in mammalian cells. m6A 'writers' METTL3 and METTL14 demonstrate a strong preference for binding AC-containing motifs in living cells. However, this evidence is currently lacking for m6A erasers, leaving the dynamics of the internal m6A modification under debate recently. We analysed three recently published FTO CLIP-seq data sets and two generated in this study, one of the two known m6A 'erasers'. FTO binding peaks from all cell lines contain RRACH motifs. Only those from K562, 3T3-L1and HeLa cells were enriched in AC-containing motifs, while those from HEK293 were not. The exogenously overexpressed FTO effectively binds to m6A motif-containing RNA sites. FTO overexpression specifically removed m6A modification from GGACU and RRACU motifs in a concentration-dependent manner. These findings underline the dynamics of FTO in target selection, which is predicted to contribute to both the m6A dynamics and the FTO plasticity in biological functions and diseases.

Association of Toll-like receptor 7 and 8 gene polymorphisms with Graves' disease in Chinese Cantonese population.

Graves' disease (GD) is a common polygenic multifactorial autoimmune disease. Toll-like receptors (TLRs) play critical roles in the activation of innate and adaptive immune responses. This study investigated the association of TLR7 and TLR8 gene polymorphisms with susceptibility of GD. Five single nucleotide polymorphisms (SNPs), namely, rs179019, rs179010 and rs3853839 in TLR7 and rs3764880 and rs5744088 in TLR8, were evaluated in 332 GD patients and 351 controls using High-Resolution Melting analysis. After adjusting for age, SNP rs179010 was found to decrease the risk of GD in females (OR(T vs C) = 0.64, P = 0.004). In the additive model, the risk of GD decreased significantly as the number of T alleles increased in females [odds ratio (OR) = 0.67 (0.50-0.90), P = 0.007]. The multivariate logistic regression analysis confirmed the independent contribution of rs179010 to the protective effect against GD. This study indicates that rs179010 in TLR7 may be associated with the decreased susceptibility to GD in Chinese Cantonese.

Plastron effect enhanced electrochemical CO2 reduction activity over hydrophobic three-dimensional nanoporous silver.

The electrochemical reduction of CO2 on catalyst surfaces is hindered by the inefficient mass transfer of CO2 in aqueous solutions. In this study, we employed an electrochemical reduction approach to fabricate a hydrophobic three-dimensional nanoporous silver catalyst with a plastron effect, aiming to enhance the CO2 diffusion. The resulting catalyst exhibited an exceptional performance with the FECO peaking at 95% at -0.65 V (vs. RHE) and demonstrated remarkable stability during continuous electrolysis for 48 hours. Control experiments, together with Tafel analysis, EIS measurements, and contact angle results, confirmed that the notable enhancement of performance was attributed to the hydrophobic porous structure that facilitated efficient storage and rapid mass transfer of low-solubility CO2 gas reactants.

RNA interactome of hypervirulent Klebsiella pneumoniae reveals a small RNA inhibitor of capsular mucoviscosity and virulence.

Hypervirulent Klebsiella pneumoniae (HvKP) is an emerging bacterial pathogen causing invasive infection in immune-competent humans. The hypervirulence is strongly linked to the overproduction of hypermucoviscous capsule, but the underlying regulatory mechanisms of hypermucoviscosity (HMV) have been elusive, especially at the post-transcriptional level mediated by small noncoding RNAs (sRNAs). Using a recently developed RNA interactome profiling approach iRIL-seq, we interrogate the Hfq-associated sRNA regulatory network and establish an intracellular RNA-RNA interactome in HvKP. Our data reveal numerous interactions between sRNAs and HMV-related mRNAs, and identify a plethora of sRNAs that repress or promote HMV. One of the strongest HMV repressors is ArcZ, which is activated by the catabolite regulator CRP and targets many HMV-related genes including mlaA and fbp. We discover that MlaA and its function in phospholipid transport is crucial for capsule retention and HMV, inactivation of which abolishes Klebsiella virulence in mice. ArcZ overexpression drastically reduces bacterial burden in mice and reduces HMV in multiple hypervirulent and carbapenem-resistant clinical isolates, indicating ArcZ is a potent RNA inhibitor of bacterial pneumonia with therapeutic potential. Our work unravels a novel CRP-ArcZ-MlaA regulatory circuit of HMV and provides mechanistic insights into the posttranscriptional virulence control in a superbug of global concern.

The mitochondrial tRNA-derived fragment, mt-tRF-LeuTAA, couples mitochondrial metabolism to insulin secretion.

OBJECTIVE: The contribution of the mitochondrial electron transfer system to insulin secretion involves more than just energy provision. We identified a small RNA fragment (mt-tRF-LeuTAA) derived from the cleavage of a mitochondrially-encoded tRNA that is conserved between mice and humans. The role of mitochondrially-encoded tRNA-derived fragments remains unknown. This study aimed to characterize the impact of mt-tRF-LeuTAA, on mitochondrial metabolism and pancreatic islet functions. METHODS: We used antisense oligonucleotides to reduce mt-tRF-LeuTAA levels in primary rat and human islet cells, as well as in insulin-secreting cell lines. We performed a joint transcriptome and proteome analysis upon mt-tRF-LeuTAA inhibition. Additionally, we employed pull-down assays followed by mass spectrometry to identify direct interactors of the fragment. Finally, we characterized the impact of mt-tRF-LeuTAA silencing on the coupling between mitochondrial metabolism and insulin secretion using high-resolution respirometry and insulin secretion assays. RESULTS: Our study unveils a modulation of mt-tRF-LeuTAA levels in pancreatic islets in different Type 2 diabetes models and in response to changes in nutritional status. The level of the fragment is finely tuned by the mechanistic target of rapamycin complex 1. Located within mitochondria, mt-tRF-LeuTAA interacts with core subunits and assembly factors of respiratory complexes of the electron transfer system. Silencing of mt-tRF-LeuTAA in islet cells limits the inner mitochondrial membrane potential and impairs mitochondrial oxidative phosphorylation, predominantly by affecting the Succinate (via Complex II)-linked electron transfer pathway. Lowering mt-tRF-LeuTAA impairs insulin secretion of rat and human pancreatic ß-cells. CONCLUSIONS: Our findings indicate that mt-tRF-LeuTAA interacts with electron transfer system complexes and is a pivotal regulator of mitochondrial oxidative phosphorylation and its coupling to insulin secretion.

Association mapping combined with linkage analysis for aluminum tolerance among soybean cultivars released in Yellow and Changjiang River Valleys in China.

Association mapping (AM) combined with linkage mapping (LM) was executed to identify molecular markers and QTL regions associated with aluminum (Al) tolerance using relative root elongation (RRE) in hydroponics as an indicator. A set of 188 soybean cultivars released in Yellow and Changjiang River Valleys and 184 recombinant inbred lines (RIL) derived from a cross KF No. 1 (tolerant) × NN1138-2 (susceptible) was used in the study. Inheritance analysis of the RIL population suggested four major genes and polygenes controlled Al-tolerance. Further, LM indicated four additive and four epistatic QTL pairs plus a collective unmapped minor QTL were responsible for Al-tolerance and explained 29.39, 18.75 and 43.07 % of the phenotypic variation (PV), respectively. In the set of released cultivars, AM identified 11 markers significant at P < 0.03 that explained 85.2 % of PV with six of which at P < 0.01 accounted for 57.9 % of PV. Ten of these eleven AM marker-QTL were mapped within range of ~2.0 cM to ~43.0 cM outside confidence interval of respective Al-tolerance QTL in previous studies. Five markers, Satt209, Sat_364, Sat_240, Sct_190 and Satt284, were located near Al-tolerance QTL regions in this and previous LM studies. Thus, the two methods confirmed these markers as being the most likely candidate regions for Al-tolerance. Allele effects relative to the population mean for the 11 QTL were estimated, and the allele A210 of Satt209 showed greatest phenotypic effect on Al-tolerance. The two most favorable alleles from each of the 11 marker loci and their carriers were identified, and accordingly the genetic constitution of Al-tolerance for the 188 cultivars was dissected as a QTL-allele matrix. Therefore, marker-assisted pairing of crosses and marker-assisted selection of progenies can be carried out to pyramid favorable alleles of all the 11 loci. This marker-assisted breeding procedure was designated as breeding by design using a QTL-allele matrix.

In vivo RNA interactome profiling reveals 3'UTR-processed small RNA targeting a central regulatory hub.

Small noncoding RNAs (sRNAs) are crucial regulators of gene expression in bacteria. Acting in concert with major RNA chaperones such as Hfq or ProQ, sRNAs base-pair with multiple target mRNAs and form large RNA-RNA interaction networks. To systematically investigate the RNA-RNA interactome in living cells, we have developed a streamlined in vivo approach iRIL-seq (intracellular RIL-seq). This generic approach is highly robust, illustrating the dynamic sRNA interactomes in Salmonella enterica across multiple stages of growth. We have identified the OmpD porin mRNA as a central regulatory hub that is targeted by a dozen sRNAs, including FadZ cleaved from the conserved 3'UTR of fadBA mRNA. Both ompD and FadZ are activated by CRP, constituting a type I incoherent feed-forward loop in the fatty acid metabolism pathway. Altogether, we have established an approach to profile RNA-RNA interactomes in live cells, highlighting the complexity of RNA regulatory hubs and RNA networks.

The structure and properties of lignin isolated from various lignocellulosic biomass by different treatment processes.

The structure and properties of lignin can vary depending on the type of lignocellulosic biomass it comes from and the separation techniques used, and also affects its suitability for different applications. In this work, the structure and properties of lignin isolated from moso bamboo, wheat straw, and poplar wood by different treatment processes were compared. Results show that deep eutectic solvent (DES) extracted lignin exhibits well-preserved structures (including ß-O-4, ß-ß, and ß-5 linkages), a low molecular weight (Mn = 2300-3200 g/mol), and relatively homogeneous lignin fragments (1.93 < PDI < 2.33) compared to dealkaline lignin (DL) and milled wood lignin (MWL). Besides, lignin samples extracted by DES have a regular nanostructure, higher carbon residue content (>40 %), and excellent antioxidant properties (the free radical scavenging index >20). Among the three types of biomass, the structural destruction of lignin in straw is the most obvious, which is due to the degradation of ß-O-4 and ß-ß linkages during DES treatment. These findings can contribute to a better understanding of the structural changes that occur in various treatment processes from different lignocellulosic biomass, and help maximize the targeted development of their applications based on the characteristics of lignin.

Multistage treatment of bamboo powder waste biomass: Highly efficient and selective isolation of lignin components.

Bamboo pulp and papermaking produce a lot of bamboo powder waste, and its resource utilization is of great significance for biomass refining and environmental protection. Here, we propose an integrated approach involving mechanical activation, hydrothermal extraction, and deep eutectic solvents (DESs) multiple delignification for the efficient separation of bamboo powder. Among seven carboxylic acids based DESs, choline chloride (ChCl)-lactic acid (La) DES (1:1) is the most effective, with over 78.0% lignin removal and 88.9% cellulose retained after mechanical-hydrothermal (180 °C, 5 h)-DES (110 °C, 12 h) treatment. Notably, 84.7% of delignification is achieved after three times of ChCl-La DES treatment at 70, 90, and 110 °C respectively. The delignification rate is negatively correlated with the amount of carboxyl group in the DESs. The lower the pKa value, the higher the delignification rate. Additionally, the selectivity for lignin is improved with decreasing solvent polarity. DES treatment effectively degrades the guaiacyl unit lignin fractions and disrupts several ß-aryl-ether bonds (e.g., ß-O-4, ß-ß, and ß-5). Furthermore, DESs exhibit good recyclability, with less than 10% reduction in delignification after three cycles. Theory calculations confirm that ChCl-carboxylic acid DESs could compete with lignin to break hydrogen bonds in lignocellulosic biomass by providing their chloride, hydroxyl, and carboxyl groups. Overall, this study demonstrates the practical significance of multistage treatment for the effective fractionation of biomass into its three components.

Heterogeneous evolutionary rates of Pi2/9 homologs in rice.

BACKGROUND: The Pi2/9 locus contains multiple nucleotide binding site-leucine-rich repeat (NBS-LRR) genes in the rice genome. Although three functional R-genes have been cloned from this locus, little is known about the origin and evolutionary history of these genes. Herein, an extensive genome-wide survey of Pi2/9 homologs in rice, sorghum, Brachypodium and Arabidopsis, was conducted to explore this theme. RESULTS: In our study, 1, 1, 5 and 156 Pi2/9 homologs were detected in Arabidopsis, Brachypodium, sorghum and rice genomes, respectively. Two distinct evolutionary patterns of Pi2/9 homologs, Type I and Type II, were observed in rice lines. Type I Pi2/9 homologs showed evidence of rapid gene diversification, including substantial copy number variations, obscured orthologous relationships, high levels of nucleotide diversity or/and divergence, frequent sequence exchanges and strong positive selection, whereas Type II Pi2/9 homologs exhibited a fairly slow evolutionary rate. Interestingly, the three cloned R-genes from the Pi2/9 locus all belonged to the Type I genes. CONCLUSIONS: Our data show that the Pi2/9 locus had an ancient origin predating the common ancestor of gramineous species. The existence of two types of Pi2/9 homologs suggest that diversifying evolution should be an important strategy of rice to cope with different types of pathogens. The relationship of cloned Pi2/9 genes and Type I genes also suggests that rapid gene diversification might facilitate rice to adapt quickly to the changing spectrum of the fungal pathogen M. grisea. Based on these criteria, other potential candidate genes that might confer novel resistance specificities to rice blast could be predicted.

An Alternative Class of Targets for microRNAs Containing CG Dinucleotide.

MicroRNAs (miRNAs) are endogenous ~23 nt RNAs which regulate message RNA (mRNA) targets mainly through perfect pairing with their seed region (positions 2-7). Several instances of UTR sequence with an additional nucleotide that might form a bulge within the pairing region, can also be recognized by miRNA as their target (bugle-target). But the prevalence of such imperfect base pairings in human and their roles in the evolution are incompletely understood. We found that human miRNAs with the CG dinucleotides (CG dimer) in their seed region have a significant low mutation rate than their putative binding sites in mRNA targets. Interspecific comparation shows that these miRNAs had very few conservative targets with the perfect seed-pairing, while potentially having a subclass of bulge-targets. Compared with the canonical target (perfect seed-pairing), these bulge-targets had a lower negative correlation with the miRNA expression, and either were down-regulated in the miRNA overexpression experiment or up-regulated in the miRNA knock-down experiment. Our results show that the bulge-targets are widespread in the miRNAs with CG dinucleotide within their seed regions, which could in part explain the rare conserved targets of these miRNAs based on seed rule. Incorporating these bulge-targets, together with conservation information, could more accurately predict the entire targets of these miRNAs.

Small RNAs derived from tRNA fragmentation regulate the functional maturation of neonatal ß cells.

tRNA-derived fragments (tRFs) are an emerging class of small non-coding RNAs with distinct cellular functions. Here, we studied the contribution of tRFs to the regulation of postnatal ß cell maturation, a critical process that may lead to diabetes susceptibility in adulthood. We identified three tRFs abundant in neonatal rat islets originating from 5' halves (tiRNA-5s) of histidine and glutamate tRNAs. Their inhibition in these islets reduced ß cell proliferation and insulin secretion. Mitochondrial respiration was also perturbed, fitting with the mitochondrial enrichment of nuclear-encoded tiRNA-5HisGTG and tiRNA-5GluCTC. Notably, tiRNA-5 inhibition reduced Mpc1, a mitochondrial pyruvate carrier whose knock down largely phenocopied tiRNA-5 inhibition. tiRNA-5HisGTG interactome revealed binding to Musashi-1, which was essential for the mitochondrial enrichment of tiRNA-5HisGTG. Finally, tiRNA-5s were dysregulated in the islets of diabetic and diabetes-prone animals. Altogether, tiRNA-5s represent a class of regulators of ß cell maturation, and their deregulation in neonatal islets may lead to diabetes susceptibility in adulthood.

Insights into the relationships between physicochemical properties, solvent performance, and applications of deep eutectic solvents.

Deep eutectic solvent (DES) is regarded as a new generation of green solvent due to its distinctive and tailorable physicochemical properties, such as low volatility, strong solubility, biodegradability, low-cost, environment-friendly, and feasibility of the structural design. As an alternative to traditional organic solvents and ionic liquids (ILs), DESs have been widely applied in many fields, such as organic chemical synthesis, electrochemical deposition, material preparation, biomass catalytic conversion, extraction and separation, detection and analysis, nanotechnology, gas absorption, and drug delivery. In this paper, through in-depth discussion on factors influencing the physicochemical properties of DESs, we summarized the relations between their composition, structure, and performance. Focusing on their solvent performance, we analyzed the latest research results of DESs with different physicochemical properties in various fields. It should be pointed out that designing and synthesizing DESs from the molecular structure aspect to regulate their physicochemical properties is the direction of accurately developing new functional applications of DESs.

The role of adsorbed oleylamine on gold catalysts during synthesis for highly selective electrocatalytic reduction of CO2 to CO.

The low-coordinated sites of electrocatalysts favour hydrogen evolution, while the edge sites are active for CO2 reduction. Oleylamine is used to stabilize nanoparticles by adsorbing on the low-coordinated sites. The hydrogen evolution reaction was dramatically suppressed and the FECO remained >93% from -0.4 to -0.8 V (vs. RHE) when oleylamine ligands existed on the surface of a gold catalyst. More H+ and electrons were involved in the CO evolution reaction, which changed the rate-limiting step from single-electron transfer to the chemical reaction step. The results establish that the surface-adsorbed surfactants during catalyst synthesis have an important effect on CO2 electrocatalytic reduction.

Roles of Noncoding RNAs in Islet Biology.

The discovery that most mammalian genome sequences are transcribed to ribonucleic acids (RNA) has revolutionized our understanding of the mechanisms governing key cellular processes and of the causes of human diseases, including diabetes mellitus. Pancreatic islet cells were found to contain thousands of noncoding RNAs (ncRNAs), including micro-RNAs (miRNAs), PIWI-associated RNAs, small nucleolar RNAs, tRNA-derived fragments, long non-coding RNAs, and circular RNAs. While the involvement of miRNAs in islet function and in the etiology of diabetes is now well documented, there is emerging evidence indicating that other classes of ncRNAs are also participating in different aspects of islet physiology. The aim of this article will be to provide a comprehensive and updated view of the studies carried out in human samples and rodent models over the past 15 years on the role of ncRNAs in the control of α- and ß-cell development and function and to highlight the recent discoveries in the field. We not only describe the role of ncRNAs in the control of insulin and glucagon secretion but also address the contribution of these regulatory molecules in the proliferation and survival of islet cells under physiological and pathological conditions. It is now well established that most cells release part of their ncRNAs inside small extracellular vesicles, allowing the delivery of genetic material to neighboring or distantly located target cells. The role of these secreted RNAs in cell-to-cell communication between ß-cells and other metabolic tissues as well as their potential use as diabetes biomarkers will be discussed. © 2020 American Physiological Society. Compr Physiol 10:893-932, 2020.

ZrMn Oxides for Aqueous-Phase Ketonization of Acetic Acid: Effect of Crystal and Porosity.

Aqueous-phase ketonization of bio-based acetic acid is important to improve the conversion efficiency of biomass resources. In this study, ZrMn mixed oxides (ZrMnOx ) with high aqueous-phase ketonization activity are synthetized through a carbonization/oxidation method (COM) and solvothermal method (STM). The results show that ZrMnOx prepared by COM possesses tetragonal ZrO2 , and hausmannite Mn3 O4 is observed only at a high oxidation temperature of 750 °C. Low-temperature and long oxidation results in decreased crystallinity and crystallite size, which is related to highly dispersed Mnn+ species. The catalysts with improved acid sites possess high ketonization activity. Surface areas and pore size of ZrMnOx synthetized by STM are controlled by the solvents for thermal treatment. Compared with water as solvent, ethanol increases the surface area and pore size, resulting in high ketonization activity.

Enhanced hydrolysis of mechanically pretreated cellulose in water/CO2 system.

The aim of this work was to study promotion of ball milling and CO2 assistance on cellulose hydrolysis kinetics in water medium. Kinetic behaviors were analyzed based on first-order and shrinking core models. The results showed that cellulose hydrolysis is enhanced by ball milling and CO2 assistance. Ball milling reduced crystallinity and particle size of cellulose, resulting in high cellulose conversion, while hydrolysis promoted by CO2 assistance was weaker. Double-layer hydrolysis was observed for ball-milled cellulose, and rate constant in active layer is higher. Based on double-layer shrinking core model (DL-SCM), activation energy of cellulose conversion decreased from 73.6 to 39.8 kJ/mol when ball milling and CO2 assistance were applied. Hydrolysis active layer was about 0.9 µm, representing activated thickness of ball-milled cellulose. Hydrolysis promotion by crystallinity and particle size reduction was distinguished via DL-SCM, and crystal evolution possesses greater improvement than particle size decrease on hydrolysis of ball-milled cellulose.