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Abstract Background The role of diet choline in atherosclerotic cardiovascular disease (ASCVD) is uncertain. Findings from animal experiments are contradictory while there is a lack of clinical investigations. This study aimed to investigate the association between choline intake and ASCVD based on individuals from the National Health and Nutrition Examination Survey (NHANES) database. Methods This cross-sectional study was conducted in 5525 individuals from the NHANES between 2011 and 2018. Participants were categorized into the ASCVD (n = 5015) and non-ASCVD (n = 510) groups. Univariable and multivariable-adjusted regression analyses were employed to investigate the relationship between diet choline and pertinent covariates. Logistic regression analysis and restricted cubic spline analysis were used to evaluate the association between choline intake and ASCVD. Results ASCVD participants had higher choline intake compared to those without ASCVD. In the higher tertiles of choline intake, there was a greater proportion of males, married individuals, highly educated individuals, and those with increased physical activity, but a lower proportion of smokers and drinkers. In the higher tertiles of choline intake, a lower proportion of individuals had a history of congestive heart failure and stroke. After adjusting for age, gender, race, ethnicity, and physical activity, an inverse association between choline intake and heart disease, stroke, and ASCVD was found. A restricted cubic spline analysis showed a mirrored J-shaped relationship between choline and ASCVD, stroke and congestive heart failure in males. There was no association between dietary choline and metabolic syndrome. Conclusion An inverse association was observed between choline intake and ASVCD among U.S. adults. Further large longitudinal studies are needed to test the causal relationship of choline and ASVCD.

The endoplasmic reticulum (ER) plays a pivotal role in protein folding and secretion, Ca2+ storage, and lipid synthesis in eukaryotic cells. When the burden of protein synthesis and folding required to be handled exceeds the processing capacity of the ER, the accumulation of misfolded/unfolded proteins triggers ER stress. In response to short-term ER stress, the unfolded protein response (UPR) is activated to allow cells to survive. When ER stress is severe and sustained, it typically provokes cell death through multiple approaches. It is well documented that ER stress and metabolic deregulation are functionally intertwined, both are considered contributing factors to the pathogenesis of liver diseases, including non-alcoholic fatty liver disease (NAFLD), alcoholic liver disease (ALD), ischemia/reperfusion (I/R) injury, viral hepatitis, liver fibrosis, and hepatocellular carcinoma (HCC). Hepatocytes are rich in smooth and rough ER, which harbor metabolic enzymes that are capable of sensing alterations in various nutritional status and external stimuli. Extensive research has focused on the molecular mechanism linking ER stress with metabolic enzymes. The purpose of this review is to summarize the current knowledge regarding the effects of ER stress on metabolic enzymes in various liver diseases and to provide potential therapeutic strategies for chronic liver diseases via targeting UPR.

Background Human olfactory receptors (ORs) account for approximately 60% of all human G protein-coupled receptors. The functions of ORs extend beyond olfactory perception and have garnered significant attention in tumor biology. However, a comprehensive pan-cancer analysis of ORs in human cancers is lacking. Methods Using data from public databases, such as HPA, TCGA, GEO, GTEx, TIMER2, TISDB, UALCAN, GEPIA2, and GSCA, this study investigated the role of olfactory receptor family 7 subfamily A member 5 (OR7A5) in various cancers. Functional analysis of OR7A5 in LGG and GBM was performed using the CGGA database. Molecular and cellular experiments were performed to validate the expression and biological function of OR7A5 in gliomas. Results The results revealed heightened OR7A5 expression in certain tumors, correlating with the expression levels of immune checkpoints and immune infiltration. In patients with gliomas, the expression levels of OR7A5 were closely associated with adverse prognosis, 1p/19p co-deletion status, and wild-type IDH status. Finally, in vitro experiments confirmed the inhibitory effect of OR7A5 knockdown on the proliferative capacity of glioma cells and on the expression levels of proteins related to lipid metabolism. Conclusion This study establishes OR7A5 as a novel biomarker, potentially offering a novel therapeutic target for gliomas.

Shezhi Huangling Decoction (SHD) has been proven clinically effective in regulating metabolic and immune homeostasis in the treatment of glioma. The investigation aimed to deconstruct the active constituents and mechanisms of SHD. Effects of SHD on malignant characteristics of HS683 and KNS89 cells have been investigated by CCK-8, clone formation, flow cytometry, and Transwell assays. A mouse xenograft model was established to assess the effect of SHD or SHD + temozolomide (TMZ) in vivo. A total of 461 constituents were found from SHD in UPLC/Q-TOF-MS/MS analysis. Functional enrichment analysis showed that pathway in cancer, proteoglycans in cancer, regulation of epithelial cell proliferation, inflammation/immune, gliogenesis, brain development, cell adhesion, and autophagy could participate in the treatment of SHD. Additionally, 9 hub genes (AKT1, TP53, CTNNB1, STAT3, EGFR, VEGFA, PIK3CA, ERBB2, and HIF1A) were identified as hub genes. Moreover, we found that SHD may greatly reduce the migration and accelerate apoptosis of HS683 and KNS89 cells. Additionally, SHD coordinates TMZ to restrict tumor growth were found in the mice. Our results suggest that the malignant behaviors of glioma cells are suppressed by SHD and the mechanism may be closing on the inhibition of the PI3K/Akt-HIF1A axis. SHD may serve as a synergistic therapeutic choice for TMZ to suppress glioblastoma growth.

Abstract Brain cancer is regarded among the deadliest forms of cancer worldwide. The distinct tumor microenvironment and inherent characteristics of brain tumor cells virtually render them resistant to the majority of conventional and advanced therapies. Oxidative stress (OS) is a key disruptor of normal brain homeostasis and is involved in carcinogenesis of different forms of brain cancers. Thus, antioxidants may inhibit tumorigenesis by preventing OS induced by various oncogenic factors. Antioxidants are hypothesized to inhibit cancer initiation by endorsing DNA repair and suppressing cancer progression by creating an energy crisis for preneoplastic cells, resulting in antiproliferative effects. These effects are referred to as chemopreventive effects mediated by an antioxidant mechanism. In addition, antioxidants minimize chemotherapy-induced nonspecific organ toxicity and prolong survival. Antioxidants also support the prooxidant chemistry that demonstrate chemotherapeutic potential, particularly at high or pharmacological doses and trigger OS by promoting free radical production, which is essential for activating cell death pathways. A growing body of evidence also revealed the roles of exogenous antioxidants as adjuvants and their ability to reverse chemoresistance. In this review, we explain the influences of different exogenous and endogenous antioxidants on brain cancers with reference to their chemopreventive and chemotherapeutic roles. The role of antioxidants on metabolic reprogramming and their influence on downstream signaling events induced by tumor suppressor gene mutations are critically discussed. Finally, the review hypothesized that both pro- and antioxidant roles are involved in the anticancer mechanisms of the antioxidant molecules by killing neoplastic cells and inhibiting tumor recurrence followed by conventional cancer treatments. The requirements of pro- and antioxidant effects of exogenous antioxidants in brain tumor treatment under different conditions are critically discussed along with the reasons behind the conflicting outcomes in different reports. Finally, we also mention the influencing factors that regulate the pharmacology of the exogenous antioxidants in brain cancer treatment. In conclusion, to achieve consistent clinical outcomes with antioxidant treatments in brain cancers, rigorous mechanistic studies are required with respect to the types, forms, and stages of brain tumors. The concomitant treatment regimens also need adequate consideration.

The tumor microenvironment (TME) is a crucial driving factor for tumor progression and it can hinder the body’s immune response by altering the metabolic activity of immune cells. Both tumor and immune cells maintain their proliferative characteristics and physiological functions through transporter-mediated regulation of nutrient acquisition and metabolite efflux. Transporters also play an important role in modulating immune responses in the TME. In this review, we outline the metabolic characteristics of the TME and systematically elaborate on the effects of abundant metabolites on immune cell function and transporter expression. We also discuss the mechanism of tumor immune escape due to transporter dysfunction. Finally, we introduce some transporter-targeted antitumor therapeutic strategies, with the aim of providing new insights into the development of antitumor drugs and rational drug usage for clinical cancer therapy.

Although consistent evidence has revealed that cognitive impairment is a common sequela in patients with mild stroke, few studies have focused on it, nor the impact of lesion location on cognitive function. Evidence on the neural mechanisms underlying the effects of mild stroke and lesion location on cognitive function is limited. This prompted us to conduct a comprehensive and quantitative study of functional brain network properties in mild stroke patients with different lesion locations. Specifically, an empirical approach was introduced in the present work to explore the impact of mild stroke-induced cognitive alterations on functional brain network reorganization during cognitive tasks (i.e., visual and auditory oddball). Electroencephalogram functional connectivity was estimated from three groups (i.e., 40 patients with cortical infarctions, 48 patients with subcortical infarctions, and 50 healthy controls). Using graph theoretical analysis, we quantitatively investigated the topological reorganization of functional brain networks at both global and nodal levels. Results showed that both patient groups had significantly worse behavioral performance on both tasks, with significantly longer reaction times and reduced response accuracy. Furthermore, decreased global and local efficiency were found in both patient groups, indicating a mild stroke-related disruption in information processing efficiency that is independent of lesion location. Regarding the nodal level, both divergent and convergent node strength distribution patterns were revealed between both patient groups, implying that mild stroke with different lesion locations would lead to complex regional alterations during visual and auditory information processing, while certain robust cognitive processes were independent of lesion location. These findings provide some of the first quantitative insights into the complex neural mechanisms of mild stroke-induced cognitive impairment and extend our understanding of underlying alterations in cognition-related brain networks induced by different lesion locations, which may help to promote post-stroke management and rehabilitation.

Glioma is the most common tumor of the central nervous system (CNS), with a 5-year survival rate of

Severe acute pancreatitis (SAP) is a critical illness characterized by a severe systemic inflammatory response resulting in persistent multiple organ failure and sepsis. The intestinal microbiome is increasingly appreciated to play a crucial role in modulation of AP disease outcome, but limited information is available about the identity and mechanism of action for specific commensal bacteria involved in AP-associated inflammation. Here we show that Bifidobacteria, particularly B. animalis, can protect against AP by regulating pancreatic and systemic inflammation in germ-free (GF) and oral antibiotic-treated (Abx) mouse models. Colonization by B. animalis and administration of its metabolite lactate protected Abx and GF mice from AP by reducing serum amylase concentration, ameliorating pancreatic lesions and improving survival rate after retrograde injection of sodium taurocholate. B. animalis relieved macrophage-associated local and systemic inflammation of AP in a TLR4/MyD88- and NLRP3/Caspase1-dependent manner through its metabolite lactate. Supporting our findings from the mouse study, clinical AP patients exhibited a decreased fecal abundance of Bifidobacteria that was inversely correlated with the severity of systemic inflammatory responses. These results may shed light on the heterogeneity of clinical outcomes and drive the development of more efficacious therapeutic interventions for AP, and potentially for other inflammatory disorders.

Summary: Neurofibromatosis type 2 is an autosomal dominant multiple neoplasia syndrome and is usually caused by mutations in the neurofibromin 2 (NF2) gene, which encodes a tumor suppressor and initiates the Hippo pathway. However, the mechanism by which NF2 functions in the Hippo pathway isn’t fully understood. Here we identified a NF2 c.770-784del mutation from a neurofibromatosis type 2 family. MD simulations showed that this mutation significantly changed the structure of the F3 module of the NF2-FERM domain. Functional assays indicated that the NF2 c.770-784del variant formed LLPS in the cytoplasm with LATS to restrain LATS plasma membrane localization and inactivated the Hippo pathway. Besides, this deletion partly caused a skipping of exon 8 and reduced the protein level of NF2, collectively promoting proliferation and tumorigenesis of meningeal cells. We identified an unrecognized mechanism of LLPS and splicing skipping for the NF2-induced Hippo pathway, which provided new insight into the pathogenesis of neurofibromatosis type 2.

Generalized anxiety disorder (GAD) and depressive disorder (DD) are distinct mental disorders, which are characterized by complex and unique neuroelectrophysiological mechanisms in psychiatric neurosciences. The understanding of the brain functional differences between GAD and DD is crucial for the accurate diagnosis and clinical efficacy evaluation. The aim of this study was to reveal the differences in functional brain imaging between GAD and DD based on multidimensional electroencephalogram (EEG) characteristics. To this end, 10 min resting-state EEG signals were recorded from 38 GAD and 34 DD individuals. Multidimensional EEG features were subsequently extracted, which include power spectrum density (PSD), fuzzy entropy (FE), and phase lag index (PLI). Then, a direct statistical analysis (i.e., ANOVA) and three ensemble learning models (i.e., Random Forest (RF), Light Gradient Boosting Machine (LightGBM), eXtreme Gradient Boosting (XGBoost)) were used on these EEG features for the differential recognitions. Our results showed that DD has significantly higher PSD values in the alpha1 and beta band, and a higher FE in the beta band, in comparison with GAD, along with the aberrant functional connections in all four bands between GAD and DD. Moreover, machine learning analysis further revealed that the distinct features predominantly occurred in the beta band and functional connections. Here, we show that DD has higher power and more complex brain activity patterns in the beta band and reorganized brain functional network structures in all bands compared to GAD. In sum, these findings move towards the practical identification of brain functional differences between GAD and DD.

To deliver doxorubicin (DOX) with enhanced efficacy and safety in vivo, fullerenol-modified micelles were prepared with the amphiphilic polymer DSPE-PEG-C60 as a carrier, which was synthesized by linking C60(OH)22 with DSPE-PEG-NH2. Studies of its particle size, PDI, zeta potential, and encapsulation efficiency were performed. DOX was successfully loaded into the micelles, exhibiting a suitable particle size [97 nm, 211 nm, 260 nm, vector: DOX = 5:1, 10:1; 15:1 (W/W)], a negative zeta potential of around −30 mv, and an acceptable encapsulation efficiency [86.1, 95.4, 97.5%, vector: DOX = 5:1, 10:1; 15:1 (W/W)]. The release behaviors of DOX from DSPE-PEG-C60 micelles were consistent with the DSPE-PEG micelles, and it showed sustained release. There was lower cytotoxicity of DSPE-PEG-C60 micelles on normal cell lines (L02, H9c2, GES-1) than free DOX and DSPE-PEG micelles. We explored the protective role of DSPE-PEG-C60 on doxorubicin-induced cardiomyocyte damage in H9c2 cells, which were evaluated with a reactive oxygen species (ROS) assay kit, JC-1, and an FITC annexin V apoptosis detection kit for cellular oxidative stress, mitochondrial membrane potential, and apoptosis. The results showed that H9c2 cells exposed to DSPE-PEG-C60 micelles displayed decreased intracellular ROS, an increased ratio of red fluorescence (JC-1 aggregates) to green fluorescence (JC-1 monomers), and a lower apoptotic ratio than the control and DSPE-PEG micelle cells. In conclusion, the prepared DOX-loaded DSPE-PEG-C60 micelles have great promise for safe, effective tumor therapy.

To investigate the differences in functional brain network structures between patients with a high level of generalized anxiety disorder (HGAD) and those with a low level of generalized anxiety disorder (LGAD), a resting-state electroencephalogram (EEG) was recorded in 30 LGAD patients and 21 HGAD patients. Functional connectivity between all pairs of brain regions was determined by the Phase Lag Index (PLI) to construct a functional brain network. Then, the characteristic path length, clustering coefficient, and small world were calculated to estimate functional brain network structures. The results showed that the PLI values of HGAD were significantly increased in alpha2, and significantly decreased in the theta and alpha1 rhythms, and the small-world attributes for both HGAD patients and LGAD patients were less than one for all the rhythms. Moreover, the small-world values of HGAD were significantly lower than those of LGAD in the theta and alpha2 rhythms, which indicated that the brain functional network structure would deteriorate with the increase in generalized anxiety disorder (GAD) severity. Our findings may play a role in the development and understanding of LGAD and HGAD to determine whether interventions that target these brain changes may be effective in treating GAD.

Depression has become one of the most common mental illnesses, causing serious physical and mental harm. However, there remain unclear and uniform physiological indicators to support the diagnosis of clinical depression. This study aimed to use machine learning techniques to investigate the abnormal multidimensional EEG features in patients with depression. Resting-state EEG signals were recorded from 41 patients with depression and 34 healthy controls. Multiple dimensional characteristics were extracted, including power spectral density (PSD), fuzzy entropy (FE), and phase lag index (PLI). These three different dimensional characteristics with statistical differences between two groups were ranked by three machine learning algorithms. Then, the ranked characteristics were placed into the classifiers according to the importance of features to obtain the optimal feature subset with the highest classification accuracy. The results showed that the optimal feature subset contained 86 features with the highest classification accuracy of 98.54% ± 0.21%. According to the statistics of the optimal feature subset, PLI had the largest number of features among the three categories, and the number of beta features was bigger than other rhythms. Moreover, compared to the healthy controls, the PLI values in the depression group increased in theta and beta rhythms, but decreased in alpha1 and alpha2 rhythms. The PSD of theta and beta rhythms were significantly greater in depression group than that in healthy controls, and the FE of beta rhythm showed the same trend. These findings indicate that the distribution of abnormal multidimensional features is potentially useful for the diagnosis of depression and understanding of neural mechanisms.

Objective: To present our classification for peripheral nerve schwannomas as well as explore the surgical strategies and operative management of peripheral nerve schwannomas based on the intraoperative neurophysiological monitoring (INM) technique and to decrease the risk of postoperative neurological deficits in the management of these schwannomas. Materials and methods: A retrospective study was conducted on 92 cases of peripheral nerve microsurgery performed, using the INM technique. We also made the classification for peripheral nerve schwannomas into two types according to operative findings and proceeded corresponding surgical strategies. Results: All tumors were removed completely under microscopy and INM. Three patients developed residual neurological deficits at final follow-up. There were different results about temporary (18/92, 19.6%) and permanent (3/92, 3.3%) neurological deficits. The incidence of temporary and permanent neurological deficits in type II group was significantly higher than that in type I group (p

Background and AimsWomen with severe intrahepatic cholestasis of pregnancy (ICP) are at higher risks of fetal complications and without effective treatments. Changes in gut microbiota in pregnancy were found to be related to the altered intestinal bile acid composition, so we aimed to explore the alterations of microbiota in the gut of ICP patients.MethodsA total of 90 women were recruited, including 45 ICP patients and 45 healthy controls. The gut microbiota communities of ICP group were compared to control group through 16S ribosomal RNA gene sequencing. The results were then confirmed by real-time polymerase chain reaction (PCR) and generalized linear model (GLM). Furthermore, we analyzed the relationships between microbiota and the severity of ICP.ResultsA total of seven genera and nine taxa with differential abundances between the ICP patients and the controls were identified. All of the seven genera were verified through real-time PCR, and three key genera Parabacteroides, Flavonifractor, and Megamonas were confirmed by using the GLM model. Further analysis found that the genera Escherichia_Shigella, Olsenella, and Turicibacter were enriched in the severe ICP group, the microbial gene function related to biosynthesis of unsaturated fatty acids and propanoate metabolism were also increased in them.ConclusionsOverall, our study was the first in Asia to demonstrate an association between gut microbiota and ICP. Our findings would contribute to a better understanding of the occurrence of ICP.

BackgroundFamilial adenomatous polyposis (FAP) is an autosomal dominant disorder characterized primarily by the development of numerous adenomatous polyps in the colon and a high risk for colorectal cancer. FAP is caused by germline mutations of the adenomatous polyposis coli (APC) gene. The proband in this family was a 39-year-old female patient with the pathologic diagnosis of adenomatous polyps, and then a five-generation kindred with FAP was characterized in the following years. This article identified an APC mutation, and demonstrated the practical use of APC-linked STR markers, which could be used to reduce misdiagnosis of prenatal diagnosis or preimplantation genetic diagnosis resulted from contamination or allele drop-out.MethodsNext-generation sequencing (NGS) was used to identify the possible APC mutations in an affected individual from a family with autosomal dominant colon cancer. Targeted sequencing then used to identify additional related individuals with the mutation. Three short tandem repeat (STR) loci, D5S299, D5S134, and D5S346, were used for PCR-based microsatellite analysis of the APC gene in the extended family.ResultsWe identified an APC: p.W553X mutation. The STR haplotype at the APC locus, A1B4C1, was shared by all clinically affected individuals with the APC: p.W553X mutation. In addition, the APC: p.D1822V variant was observed in 40% affected individuals and in two unaffected individuals.ConclusionWe described a protein truncation mutation, APC: p.W553X; demonstrated the value of APC-linked STR markers (D5S299, D5S134, and D5S346) haplotypes; and suggested the potential role of these haplotypes in detecting loss of heterozygosity of the APC gene.

The phenotypic manifestation of mitochondrial DNA (mtDNA) mutations can be modulated by nuclear genes and environmental factors. However, neither the interaction among these factors nor their underlying mechanisms are well understood. The yeast Saccharomyces cerevisiae mtDNA 15S rRNA C1477G mutation (PR) corresponds to the human 12S rRNA A1555G mutation. Here we report that a nuclear modifier gene mss1 mutation suppresses the neomycin-sensitivity phenotype of a yeast C1477G mutant in fermentable YPD medium. Functional assays show that the mitochondrial function of the yeast C1477G mutant was impaired severely in YPD medium with neomycin. Moreover, the mss1 mutation led to a significant increase in the steady-state level of HAP5 (heme activated protein), which greatly up-regulated the expression of glycolytic transcription factors RAP1, GCR1, and GCR2 and thus stimulated glycolysis. Furthermore, the high expression of the key glycolytic enzyme genes HXK2, PFK1 and PYK1 indicated that enhanced glycolysis not only compensated for the ATP reduction from oxidative phosphorylation (OXPHOS) in mitochondria, but also ensured the growth of the mss1(PR) mutant in YPD medium with neomycin. This study advances our understanding of the phenotypic manifestation of mtDNA mutations.

Organised stroke care has become a keystone in delivering efficient and effective treatment to patients with stroke with improved outcomes. Delivering timely acute reperfusion therapy to those with acute ischaemic strokes is key to good recovery. Emergency stroke unit (ESU) is a novel organised stroke care system developed in China. It centralises all necessary procedures for the diagnosis and treatment of acute stroke into one unit that can perform clinical assessment, imaging examination and acute treatments. In ESU, artificial intelligence algorithms are used to aid in reading brain images and making clinical decisions. Therefore, ESU can significantly enhance the efficiency of emergent stroke care. In this guideline, we aim to clarify the concept, construction standards and personnel requirements of an ESU, standardise ESU-based acute stroke triage and treatment workflow, establish metrics of quality control, facilitate the construction and promotion of ESU and continue the improvement of the quality of stroke care.

BackgroundAlarmins mediate type 2 T helper cell (Th2) inflammation and serve as upstream signaling elements in allergic inflammation and autoimmune responses. The alarmin interleukin (IL)-25 binds to a multi-domain receptor consisting of IL-17RA and IL-17RB subunits, resulting in the release of Th2 cytokines IL-4, IL-5, IL-9 and IL-13 to drive an inflammatory response. Therefore, the blockage of IL-17RB via SM17, a novel humanized monoclonal antibody, offers an attractive therapeutic target for Th2-mediated diseases, such as asthma.MethodsWild-type mice were stimulated with house dust mite (HDM) extracts for evaluation of SM17’s pre-clinical efficacy in allergic asthma. The safety, pharmacokinectics (PK), pharmacodynamics (PD), and immunogenicity of intravenous (IV) doses of SM17 were assessed in a 2-part clinical study in healthy adult subjects. In Part A, 53 healthy participants were enrolled to receive a single IV dose of SM17 (2, 20, 70, 200, 400, 600, 1200 mg) or placebo. In Part B, 24 healthy subjects were enrolled to receive a single IV dose of SM17 every two weeks (Q2W; 200, 400, 600 mg) or placebo for a total of 3 doses.ResultsAnimal studies demonstrated that SM17 significantly suppressed Th2 inflammation in the bronchoalveolar lavage fluid and infiltration of immune cells into the lungs. In the Phase I clinical study, no drug-related serious adverse events were observed. Total SM17 exposure increased by approximately 60- to 188-fold with a 60-fold increase in dose from 20 to 1200 mg SM17. Upon administration of the third dose, mean accumulation ratios over 200-600 mg was 1.5 to 2.1, which confirms moderate accumulation of SM17. After Q2W dosing of SM17 over 4 weeks, total exposure increased in a dose-proportional manner from 200 mg to 600 mg SM17.ConclusionIn the pre-clinical studies, we demonstrated that SM17 is a potential therapeutic agent to treat allergic asthma. In the Phase 1 clinical trial, a single IV dose of SM17 up to 1200 mg and three Q2W doses up to 600 mg were well tolerated in healthy participants and demonstrated a favorable safety profile. The pre-clinical efficacy and clinical PK and immunogenicity results of SM17 support further clinical development.Clinical trial registrationhttps://clinicaltrials.gov/, identifier NCT05332834.

The metabolic pathway of 3-alkyl-2-methoxypyrazines (MPs) in grape remains largely unclear except for the final step. In this study, the 2,5-dicarbonyl-3-isobutyl-piperazine (DCIP), which is proposed as the key intermediate of 3-isobutyl-2-methoxypyrazine (IBMP) biosynthesis, was incorporated into Cabernet Sauvignon clusters in situ using a soaking method. The IBMP concentration of grape and the expression patterns of VvOMTs in berry skin were monitored over two consecutive years. The results showed that the IBMP concentration of grape treated with DCIP was significantly increased at maturity in both years. The relative expression levels of VvOMT1 and VvOMT3 in berry skin were positively correlated with the IBMP accumulation. After DCIP incorporation, the relative expression level of VvOMT1 and particularly that of VvOMT3 were obviously up-regulated and closely mirrored the IBMP accumulation pattern in two consecutive years. Therefore, we speculate that DCIP may be a key intermediate involved in the biosynthesis of IBMP and plays an important role in regulating IBMP accumulation.