Common carotid artery diameter and the risk of cardiovascular disease mortality: a prospective cohort study in northeast China.

BACKGROUND: The association between the common carotid artery (CCA) diameter and cardiovascular disease (CVD) is recognized, but the precise nature of this link remains elusive. This study aimed to investigate the potential relationship between CCA diameter and the risk of CVD mortality in a large population in northeast China. METHODS: The current study included 5668 participants (mean age 58.9 ± 10.1 years) from a population-based study conducted in rural areas of northeast China between September 2017 and May 2018. Information on death was collected from baseline until July 31, 2022. The CCA inter-adventitial diameter was measured using ultrasound. Cox proportional-hazard models were employed to explore the relationship between the common carotid artery diameter and cardiovascular mortality. RESULTS: At baseline, the mean CCA diameter (mm) of subjects was 7.30 ± 0.99 and increased significantly with age, ranging from 6.65 ± 0.71 among people 40-49 years to 7.99 ± 1.04 among people ≥ 80 years. CCA diameter was significantly larger in males compared to females (7.51 ± 1.03 versus vs. 7.16 ± 0.94; P < 0.001). A total of 185 participants died of CVD during a median follow-up of 4.48 years. CCA diameters were divided into quartiles, and the highest quartile of carotid diameter (≥ 8.06 mm) had a 2.29 (95% confidence interval [CI]: 1.24, 4.22) times higher risk of CVD mortality than the lowest quartile (≤ 6.65 mm) (P < 0.01) in the fully adjusted model. Each increase in the diameter of the common carotid artery (per SD) raised the risk of cardiovascular death by 36% (hazard ratio [HR]: 1.36; 95% CI: 1.18, 1.57). The subgroup analysis results demonstrated that a per SD increase was associated with a 42% increased risk of CVD mortality in participants aged ≥ 64 years in the fully adjusted model (HR: 1.42; 95%CI: 1.21, 1.66). CONCLUSIONS: Our study indicates the possible incremental value of CCA diameter in optimizing the risk stratification of cardiovascular disease and provides essential insights into reducing the burden of cardiovascular disease.

Receptor modulators associated with the hypothalamus -pituitary-thyroid axis.

The hypothalamus-pituitary-thyroid (HPT) axis maintains normal metabolic balance and homeostasis in the human body through positive and negative feedback regulation. Its main regulatory mode is the secretion of thyrotropin (TSH), thyroid hormones (TH), and thyrotropin-releasing hormone (TRH). By binding to their corresponding receptors, they are involved in the development and progression of several systemic diseases, including digestive, cardiovascular, and central nervous system diseases. The HPT axis-related receptors include thyrotropin receptor (TSHR), thyroid hormone receptor (TR), and thyrotropin-releasing hormone receptor (TRHR). Recently, research on regulators has become popular in the field of biology. Several HPT axis-related receptor modulators have been used for clinical treatment. This study reviews the developments and recent findings on HPT axis-related receptor modulators. This will provide a theoretical basis for the development and utilisation of new modulators of the HPT axis receptors.

Insight into the Inhibitory Mechanism of Embryonic Ectoderm Development Subunit by Triazolopyrimidine Derivatives as Inhibitors through Molecular Dynamics Simulation.

Inhibition of the Embryonic Ectoderm Development (EED) subunit in Polycomb Repressive Complex 2 (PRC2) can inhibit tumor growth. In this paper, we selected six experimentally designed EED competitive Inhibitors of the triazolopyrimidine derivatives class. We investigated the difference in the binding mode of the natural substrate to the Inhibitors and the effects of differences in the parent nuclei, heads, and tails of the Inhibitors on the inhibitory capacity. The results showed that the binding free energy of this class of Inhibitors was close to or lower compared to the natural substrate, providing an energetic basis for competitive inhibition. For the Inhibitors, the presence of a strong negatively charged group at the 6-position of the parent nucleus or the 8'-position of the head would make the hydrogen atom on the head imino group prone to flip, resulting in the vertical movement of the parent nucleus, which significantly decreased the inhibitory ability. When the 6-position of the parent nucleus was a nonpolar group, the parent nucleus would move horizontally, slightly decreasing the inhibitory ability. When the 8'-position of the head was methylene, it formed an intramolecular hydrophobic interaction with the benzene ring on the tail, resulting in a significant increase in inhibition ability.

Solvent-controlled formation of alkali and alkali-earth-secured cucurbituril/guest trimers.

Cucurbit[7]uril (CB[7]) encapsulates adamantyl and trimethylsilyl substituents of positively charged guests in dimethyl sulfoxide (DMSO). Unlike in water or deuterium oxide, addition of a selection of alkali and alkali-earth cations with van der Waals radii between 1.0 and 1.4 Å (Na+, K+, Ca2+, Sr2+, Ba2+ and Eu3+) to the CB[7]/guest complexes triggers their cation-mediated trimerization, a process that is very slow on the nuclear magnetic resonance (NMR) time scale. Smaller (Li+, Mg2+) or larger cations (Rb+, Cs+ or NH4+) are inert. The trimers display extensive CH-O interactions between the equatorial and pseudo-equatorial hydrogens of CB[7] and the carbonyl rim of the neighboring CB[7] unit in the trimer, and a deeply nested cation between the three interacting carbonylated CB[7] rims; a counteranion is likely perched in the shallow cavity formed by the three outer walls of CB[7] in the trimer. Remarkably, a guest must occupy the cavity of CB[7] for trimerization to take place. Using a combination of semi-empirical and density functional theory techniques in conjunction with continuum solvation models, we showed that trimerization is favored in DMSO, and not in water, because the penalty for the partial desolvation of three of the six CB[7] portals upon aggregation into a trimer is less unfavorable in DMSO compared to water.

Mechanism Exploration of Amyloid-ß-42 Disaggregation by Single-Chain Variable Fragments of Alzheimer's Disease Therapeutic Antibodies.

Alzheimer's disease (AD) is a specific neurodegenerative disease. This study adopts single-chain variable fragments (scFvs) as a potential immunotherapeutic precursor for AD. According to the remarkable effects of monoclonal antibodies, such as the depolymerization or promotion of Aß42 efflux by Crenezumab, Solanezumab, and 12B4, it is attractive to prepare corresponding scFvs targeting amyloid-ß-42 protein (Aß42) and investigate their biological activities. Crenezumab-like scFv (scFv-C), Solanezumab-like scFv (scFv-S), and 12B4-like scFv (scFv-12B4) were designed and constructed. The thermal stabilities and binding ability to Aß42 of scFv-C, scFv-S, and scFv-12B4 were evaluated using unfolding profile and enzyme-linked immunosorbent assay. As the results indicated that scFv-C could recognize Aß42 monomer/oligomer and promote the disaggregation of Aß42 fiber as determined by the Thioflavin-T assay, the potential mechanism of its interaction with Aß42 was investigated using molecular dynamics analysis. Interactions involving hydrogen bonds and salt bonds were predicted between scFv-C and Aß42 pentamer, suggesting the possibility of inhibiting further aggregation of Aß42. The successfully prepared scFvs, especially scFv-C, with favorable biological activity targeting Aß42, might be developed for a potentially efficacious clinical application for AD.

Insight into the Inhibitory Mechanism of Aryl Formyl Piperidine Derivatives on Monoacylglycerol Lipase through Molecular Dynamics Simulations.

Monoacylglycerol lipase (MAGL) can regulate the endocannabinoid system and thus becomes a target of antidepressant drugs. In this paper, molecular docking and molecular dynamics simulations, combined with binding free energy calculation, were employed to investigate the inhibitory mechanism and binding modes of four aryl formyl piperidine derivative inhibitors with different 1-substituents to MAGL. The results showed that in the four systems, the main four regions where the enzyme bound to the inhibitor included around the head aromatic ring, the head carbonyl oxygen, the tail amide bond, and the tail benzene ring. The significant conformational changes in the more flexible lid domain of the enzyme were caused by 1-substituted group differences of inhibitors and resulted in different degrees of flipping in the tail of the inhibitor. The flipping led to a different direction of the tail amide bond and made a greater variation in its interaction with some of the charged residues in the enzyme, which further contributed to a different swing of the tail benzene ring. If the swing is large enough, it can weaken the binding strength of the head carbonyl oxygen to its nearby residues, and even the whole inhibitor with the enzyme so that the inhibition decreases.

Protonated α-N-Acetyl Galactose Glycopeptide Dissociation Chemistry.

We recently provided mass spectrometric, H/D labeling, and computational evidence of pyranose to furanose N-acetylated ion isomerization reactions that occurred prior to glycosidic bond cleavage in both O- and N-linked glycosylated amino acid model systems (Guan et al. Phys. Chem. Chem. Phys., 2021, 23, 23256-23266). These reactions occurred irrespective of the glycosidic linkage stereochemistry (α or ß) and the N-acetylated hexose structure (GlcNAc or GalNAc). In the present article, we test the generality of the preceding findings by examining threonyl α-GalNAc-glycosylated peptides. We utilize computational chemistry to compare the various dissociation and isomerization pathways accessible with collisional activation. We then interrogate the structure(s) of the resulting charged glycan and peptide fragments with infrared "action" spectroscopy. Isomerization of the original pyranose, the protonated glycopeptide [AT(GalNAc)A+H]+, is predicted to be facile compared to direct dissociation, as is the glycosidic bond cleavage of the newly formed furanose form, i.e., furanose oxazolinium ion structures are predicted to predominate. IR action spectra for the m/z 204, C8H14N1O5+, glycan fragment population support this prediction. The IR action spectra of the complementary m/z 262 peptide fragment were assigned as a mixture of the lowest-energy structures of [ATA+H]+ consistent with the literature. If general, the change to a furanose m/z 204 product ion structure fundamentally alters the ion population available for MS3 dissociation and glycopeptide sequence identification.

Polymer-solvent interaction and conformational changes at a molecular level: Implication to solvent-assisted deformation and aggregation at the polymer surface.

HYPOTHESIS: We hypothesize that varying the chemical structure of the monomeric unit in a polymer will affect the surface structure and interfacial molecular group orientations of the polymer film leveraging its response to solvents of different chemical affinities. EXPERIMENTS: Poly (2-methoxy ethyl methacrylate) and poly (2-tertbutoxy ethyl methacrylate) thin films exposed to either deuterated water (D2O) or deuterated chloroform (CDCl3) were studied by sum frequency generation (SFG) spectroscopy, contact angle goniometry, and atomic force microscopy (AFM) at the polymer-solvent interface, supported with molecular simulation studies. FINDINGS: SFG spectral analysis of the polymer thin films corroborated molecular re-organization at the surface when exposed to different chemical environments. The AFM height images of the polymer surfaces were homogeneously flat under CDCl3 and showed swollen regions under D2O. Following the removal of D2O, the exposed areas have imprinted, recessed locations and exposure to CDCl3 resulted in the formation of aggregates. The chemical affinity and characteristics of the solvents played a role in conformational change at the polymer surface. It had direct implications to interfacial processes involving adsorption, permeation which eventually leads to swelling, deformation or aggregation, and possibly dissolution.

Hemagglutinin-based DNA vaccines containing trimeric self-assembling nanoparticles confer protection against influenza.

Influenza viruses continue to threaten public health, and currently available vaccines provide insufficient immunity against seasonal and pandemic influenza. The use of recombinant trimeric hemagglutinin (HA) as an Ag provides an attractive alternative to current influenza vaccines. Aiming to develop an effective vaccine with rapid production, robust immunogenicity, and high protective efficiency, a DNA vaccine was designed by fusing influenza virus HA with self-assembled ferritin nanoparticles, denoted as HA-F. This candidate vaccine was prepared and purified in a 293-6E cell eukaryotic expression system. After BALB/c mice were immunized with 100 µg of HA-F DNA 3 times, HA-F elicited significant HA-specific humoral immunity and T cell immune responses. The HA-F DNA vaccine also conferred protection in mice against a lethal infection of homologous A/17/California/2009/38 (H1N1) virus. These results suggest that the HA-F DNA vaccine is a competitive vaccine candidate and presents a promising vaccination approach against influenza viruses.

Evidence of gas-phase pyranose-to-furanose isomerization in protonated peptidoglycans.

Peptidoglycans are diverse co- and post-translational modifications of key importance in myriad biological processes. Mass spectrometry is employed to infer their biomolecular sequences and stereochemisties, but little is known about the critical gas-phase dissociation processes involved. Here, using tandem mass spectrometry (MS/MS and MSn), isotopic labelling and high-level simulations, we identify and characterize a facile isomerization reaction that produces furanose N-acetylated ions. This reaction occurs for both O- and N-linked peptidoglycans irrespective of glycosidic linkage stereochemistry (α/ß). Dissociation of the glycosidic and other bonds thus occur from the furanose isomer critically altering the reaction feasibility and product ion structures.

Size Dependent Fragmentation Chemistry of Short Doubly Protonated Tryptic Peptides.

Tandem mass spectrometry of electrospray ionized multiply charged peptide ions is commonly used to identify the sequence of peptide(s) and infer the identity of source protein(s). Doubly protonated peptide ions are consistently the most efficiently sequenced ions following collision-induced dissociation of peptides generated by tryptic digestion. While the broad characteristics of longer (N ≥ 8 residue) doubly protonated peptides have been investigated, there is comparatively little data on shorter systems where charge repulsion should exhibit the greatest influence on the dissociation chemistry. To address this gap and further understand the chemistry underlying collisional-dissociation of doubly charged tryptic peptides, two series of analytes ([GxR+2H]2+ and [AxR+2H]2+, x = 2-5) were investigated experimentally and with theory. We find distinct differences in the preference of bond cleavage sites for these peptides as a function of size and to a lesser extent composition. Density functional calculations at two levels of theory predict that the threshold relative energies required for bond cleavages at the same site for peptides of different size are quite similar (for example, b2-yN-2). In isolation, this finding is inconsistent with experiment. However, the predicted extent of entropy change of these reactions is size dependent. Subsequent RRKM rate constant calculations provide a far clearer picture of the kinetics of the competing bond cleavage reactions enabling rationalization of experimental findings. The M06-2X data were substantially more consistent with experiment than were the B3LYP data.

Gas-Phase Dissociation Chemistry of Deprotonated RGD.

We investigate the structure and dissociation pathways of the deprotonated amphoteric peptide arginylglycylasparic acid, [RGD-H]-. We model the pertinent gas-phase structures and fragmentation chemistry of the precursor anions and predominant sequence-informative bond cleavages (b2+H2O, c2, and z1 peaks) and compare these predictions to our tandem mass spectra and infrared spectroscopy experiments. Formation of the b2+H2O anions requires rate-limiting intramolecular back biting to cleave the second amide bond and generate an anhydride structure. Facile cleavage of the newly formed ester bond with concerted expulsion of a cyclic anhydride neutral generates the product structure. IR spectroscopy supports this b2+H2O anion having structures that are essentially identical to C-terminally deprotonated arginylglycine, [RG-H]-. Formation of the c2 anion is predicted to require concerted expulsion of CO2 from the aspartyl side chain carboxylate and cleavage of the N-Calpha bond to produce a proton-bound dimer of arginylglycinamide and acrylate. Proton transfers within the dimer then enable predominant detection of a c2 anion with the negative charge nominally on the central, glycine nitrogen (amidate structure) as the proton affinity of this structure is predicted to be lower than acrylate by ∼27 kJ mol-1. Alternate means of cleaving the same N-Calpha bond produce deprotonated cis-1,4-dibut-2-enoic acid z1 anion structures. These lowest energy processes involve C-H proton mobilization from the aspartyl side chain prior to N-Calpha bond cleavage consistent with proposals from the literature.

Isomeric Differentiation and Acidic Metabolite Identification by Piperidine-Based Tagging, LC-MS/MS, and Understanding of the Dissociation Chemistries.

We demonstrate a method for facile differentiation of acidic, isomeric metabolites by attaching high proton affinity, piperidine-based chemical tags to each carboxylic acid group. These tags attach with high efficiency to the analytes, increase the signal, and result in the formation of multiply-charged cations. We illustrate the present approach with citrate and isocitrate, which are isomeric metabolites each containing three carboxylic acid groups. We observe a 20-fold increase in signal-to-noise for citrate and an 8-fold increase for isocitrate as compared to detection of the untagged analytes in negative mode. Collision-induced dissociation of the triply tagged, triply charged analytes results in distinct tandem mass spectra. The phenylene spacer groups limit proton mobility and enable access to structurally informative C-C bond cleavage reactions. Modeling of the gas-phase structures and dissociation chemistry of these triply charged analyte ions highlights the importance of hydroxyl proton mobilization in this low proton mobility environment. Tandem mass spectrometric analyses of deuterated congeners and MS3 spectra are consistent with the proposed fragment ion structures and mechanisms of formation. Direct evidence that these chemistries are more generally applicable is provided by subsequent analyses of doubly tagged, doubly charged malate ions. Future work will focus on applying these methods to identify new metabolites and development of general rules for structural determination of tagged metabolites with multiple charges.

How Different Substitution Positions of F, Cl Atoms in Benzene Ring of 5-Methylpyrimidine Pyridine Derivatives Affect the Inhibition Ability of EGFRL858R/T790M/C797S Inhibitors: A Molecular Dynamics Simulation Study.

Lung cancer is the most frequent cause of cancer-related deaths worldwide, and mutations in the kinase domain of the epidermal growth factor receptor (EGFR) are a common cause of non-small-cell lung cancers, which is a major subtype of lung cancers. Recently, a series of 5-methylpyrimidine-pyridinone derivatives have been designed and synthesized as novel selective inhibitors of EGFR and EGFR mutants. However, the binding-based inhibition mechanism has not yet been determined. In this study, we carried out molecular dynamic simulations and free-energy calculations for EGFR derivatives to fill this gap. Based on the investigation, the three factors that influence the inhibitory effect of inhibitors are as follows: (1) The substitution site of the Cl atom is the main factor influencing the activity through steric effect; (2) The secondary factors are repulsion between the F atom (present in the inhibitor) and Glu762, and the blocking effect of Lys745 on the phenyl ring of the inhibitor. (3) The two factors function synergistically to influence the inhibitory capacity of the inhibitor. The theoretical results of this study can provide further insights that will aid the design of oncogenic EGFR inhibitors with high selectivity.

Exploration of Binding Mechanism of a Potential Streptococcus pneumoniae Neuraminidase Inhibitor from Herbaceous Plants by Molecular Simulation.

Streptococcus pneumoniae can cause diseases such as pneumonia. Broad-spectrum antibiotic therapy for Streptococcus pneumoniae is increasingly limited due to the emergence of drug-resistant strains. The development of novel drugs is still currently of focus. Abundant polyphenols have been demonstrated to have antivirus and antibacterial ability. Chlorogenic acid is one of the representatives that has been proven to have the potential to inhibit both the influenza virus and Streptococcus pneumoniae. However, for such a potential neuraminidase inhibitor, the interaction mechanism studies between chlorogenic acid and Streptococcus pneumoniae neuraminidase are rare. In the current study, the binding mechanism of chlorogenic acid and Streptococcus pneumoniae neuraminidase were investigated by molecular simulation. The results indicated that chlorogenic acid might establish the interaction with Streptococcus pneumoniae neuraminidase via hydrogen bonds, salt bridge, and cation-π. The vital residues involved Arg347, Ile348, Lys440, Asp372, Asp417, and Glu768. The side chain of Arg347 might form a cap-like structure to lock the chlorogenic acid to the active site. The results from binding energy calculation indicated that chlorogenic acid had strong binding potential with neuraminidase. The results predicted a detailed binding mechanism of a potential Streptococcus pneumoniae neuraminidase inhibitor, which will be provide a theoretical basis for the mechanism of new inhibitors.

Structural basis of fullerene derivatives as novel potent inhibitors of protein acetylcholinesterase without catalytic active site interaction: insight into the inhibitory mechanism through molecular modeling studies.

Acetylcholinesterase (AChE) is an important kind of esterase that plays a key biological role in the central and peripheral nervous systems. Recent research has demonstrated that some fullerene derivatives serve as a new nanoscale class of potent inhibitors of AChE, but the specific inhibition mechanism remains unclear. In the present article, several molecular modeling methods, such as molecular docking, molecular dynamics simulations and molecular mechanics/generalized Born surface area calculations, were used for the investigation of the binding mode and inhibition mechanism of fullerene inhibitions for AChE. Results revealed that fullerene inhibitors block the entrance of substrates by binding with the peripheral anionic site (PAS) region. Thus, fullerene derivatives might mainly serve as competitive inhibitors. The interactions of a fullerene backbone with AChE are at the same level in different single side chain systems and seem to be related to the length or aromaticity of the side chain. The inhibitor with multihydroxyl side chains shows an obviously large electrostatic interaction as it forms additional hydrogen bonds with AChE. Moreover, fullerene derivatives might exhibit noncompetitive inhibition partly by affecting some secondary structures around them. Thus, the destructions of these secondary structures can lead to conformational changes in some important regions, such as the catalytic triad and acyl pocket. The investigation is of great importance to the discovery of good fullerene inhibitors.Communicated by Ramaswamy H. Sarma.

Effects of Se substitution and transition metal doping on the electronic and magnetic properties of a MoSxSe2-x/h-BN heterostructure.

The van der Waals heterostructures created by stacking two monolayer semiconductors have been rapidly developed experimentally and exhibit various unique physical properties. In this work, we investigate the effects of Se atom substitution and 3d-TM atom doping on the structural, electronic, and magnetic properties of the MoSe2/h-BN heterostructure, by using first-principles calculations based on density functional theory (DFT). It is found that Se atom substitution could considerably enhance the band gaps of MoSe2/h-BN heterostructures. With an increase in the substitution concentration, the energy band changes from an indirect to a direct band gap when the substitution concentration exceeds a critical value. For 3d-TM atom doping, it is shown that V-, Mn-, Fe-, and Co-doped systems exhibit a half-metallic state and magnetic behavior, while there is no spin polarization in the Ni-doped case. The results provide a theoretical basis for the development of diluted magnetic semiconductors and spin devices based on the MoSxSe2-x/h-BN heterostructure.

Leaving Group Effects in a Series of Electrosprayed CcHhN1 Anthracene Derivatives.

We investigate the gas-phase structures and fragmentation pathways of model compounds of anthracene derivatives of the general formula CcHhN1 utilizing tandem mass spectrometry and computational methods. We vary the substituent alkyl chain length, composition, and degree of branching. We find substantial experimental and theoretical differences between the linear and branched congeners in terms of fragmentation thresholds, available pathways, and distribution of products. Our calculations predict that the linear substituents initially isomerize to form lower energy branched isomers prior to loss of the alkyl substituents as alkenes. The rate-determining chemistry underlying these related processes is dominated by the ability to stabilize the alkene loss transition structures. This task is more effectively undertaken by branched substituents. Consequently, analyte lability systematically increased with degree of branching (linear < secondary < tertiary). The resulting anthracen-9-ylmethaniminium ion generated from these alkene loss reactions undergoes rate-limiting proton transfer to enable expulsion of either hydrogen cyanide or CNH. The combination of the differences in primary fragmentation thresholds and degree of radical-based fragmentation processes provide a potential means of distinguishing compounds that contain branched alkyl chain substituents from those with linear ones.

Effect of Chemical Structure on the Performance of Sulfonated Poly(aryl ether sulfone) Composite Nanofiltration Membranes.

This paper discusses the effect of the chemical structure of sulfonated poly(aryl ether sulfone) on the performance of composite nanofiltration membranes. The composite nanofiltration membranes were fabricated by coating sulfonated poly(aryl ether sulfone) solution onto the top surface of poly(phthalazinone ether sulfone ketone) support membranes. Three kinds of sulfonated poly(aryl ether sulfone)s with different amounts of phthalazinone moieties, namely, sulfonated poly(phthalazinone ether sulfone) (SPPES), sulfonated poly(phthalazinone biphenyl ether sulfone) (SPPBES), and sulfonated poly(phthalazinone hydroquinone ether sulfone)s (SPPHES), were used as coating materials. The solvents used in preparing the coating solution were investigated and optimized. The separation properties, thermal stability, and chlorine resistance of composite membranes were determined. The structures and morphologies of membranes were characterized with FTIR and SEM, respectively. The membrane prepared from SPPES with more phthalazinone moiety groups showed high water flux and salt rejection. The salt rejection of composite membranes followed the order SPPES > SPPHES > SPPBES. The rejection of the three composite membranes decreased slightly with the solution temperature rising from 20 to 90 °C, while the composite membrane with SPPES as the active layer showed a higher increase in flux than others. The results indicate that SPPES composite membranes show better thermal stability than others.

Insight into the process of product expulsion in cellobiohydrolase Cel6A from Trichoderma reesei by computational modeling.

Glycoside hydrolase cellulase family 6 from Trichoderma reesei (TrCel6A) is an important cellobiohydrolase to hydrolyze cellooligosaccharide into cellobiose. The knowledge of enzymatic mechanisms is critical for improving the conversion efficiency of cellulose into ethanol or other chemicals. However, the process of product expulsion, a key component of enzymatic depolymerization, from TrCel6A has not yet been described in detail. Here, conventional molecular dynamics and steered molecular dynamics (SMD) were applied to study product expulsion from TrCel6A. Tyr103 may be a crucial residue in product expulsion given that it exhibits two different posthydrolytic conformations. In one conformation, Tyr103 rotates to open the -3 subsite. However, Tyr103 does not rotate in the other conformation. Three different routes for product expulsion were proposed on the basis of the two different conformations. The total energy barriers of the three routes were calculated through SMD simulations. The total energy barrier of product expulsion through Route 1, in which Tyr103 does not rotate, was 22.2 kcal·mol-1. The total energy barriers of product expulsion through Routes 2 and 3, in which Tyr103 rotates to open the -3 subsite, were 10.3 and 14.4 kcal·mol-1, respectively. Therefore, Routes 2 and 3 have lower energy barriers than Route 1, and Route 2 is the thermodynamically optimal route for product expulsion. Consequently, the rotation of Tyr103 may be crucial for product release from TrCel6A. Results of this work have potential applications in cellulase engineering.