Multiple and Optimal Screening Subset: a method selecting global characteristic congeners for robust foodomics analysis.

Metabolomics and foodomics shed light on the molecular processes within living organisms and the complex food composition by leveraging sophisticated analytical techniques to systematically analyze the vast array of molecular features. The traditional feature-picking method often results in arbitrary selections of the model, feature ranking, and cut-off, which may lead to suboptimal results. Thus, a Multiple and Optimal Screening Subset (MOSS) approach was developed in this study to achieve a balance between a minimal number of predictors and high predictive accuracy during statistical model setup. The MOSS approach compares five commonly used models in the context of food matrix analysis, specifically bourbons. These models include Student's t-test, receiver operating characteristic curve, partial least squares-discriminant analysis (PLS-DA), random forests, and support vector machines. The approach employs cross-validation to identify promising subset feature candidates that contribute to food characteristic classification. It then determines the optimal subset size by comparing it to the corresponding top-ranked features. Finally, it selects the optimal feature subset by traversing all possible feature candidate combinations. By utilizing MOSS approach to analyze 1406 mass spectral features from a collection of 122 bourbon samples, we were able to generate a subset of features for bourbon age prediction with 88% accuracy. Additionally, MOSS increased the area under the curve performance of sweetness prediction to 0.898 with only four predictors compared with the top-ranked four features at 0.681 based on the PLS-DA model. Overall, we demonstrated that MOSS provides an efficient and effective approach for selecting optimal features compared with other frequently utilized methods.

Arachidonic Acid Mobilization and Peroxidation Promote Microglial Dysfunction in Aß Pathology.

Aberrant increase of arachidonic acid (ARA) has long been implicated in the pathology of Alzheimer's disease (AD), while the underlying causal mechanism remains unclear. In this study, we revealed a link between ARA mobilization and microglial dysfunction in Aß pathology. Lipidomic analysis of primary microglia from AppNL-GF mice showed a marked increase in free ARA and lysophospholipids (LPLs) along with a decrease in ARA-containing phospholipids, suggesting increased ARA release from phospholipids (PLs). To manipulate ARA-containing PLs in microglia, we genetically deleted lysophosphatidylcholine acyltransferase 3 (Lpcat3), the main enzyme catalyzing the incorporation of ARA into PLs. Loss of microglial Lpcat3 reduced the levels of ARA-containing PLs, free ARA and LPLs, leading to a compensatory increase in monounsaturated fatty acid (MUFA)-containing PLs in both male and female App NL-GF mice. Notably, the reduction of ARA in microglia significantly ameliorated oxidative stress and inflammatory responses while enhancing the phagocytosis of Aß plaques and promoting the compaction of Aß deposits. Mechanistically, scRNA seq suggested that LPCAT3 deficiency facilitates phagocytosis by facilitating de novo lipid synthesis while protecting microglia from oxidative damage. Collectively, our study reveals a novel mechanistic link between ARA mobilization and microglial dysfunction in AD. Lowering brain ARA levels through pharmacological or dietary interventions may be a potential therapeutic strategy to slow down AD progression.

Chemical Proteomic Profiling of Protein Dopaminylation in Colorectal Cancer Cells.

Histone dopaminylation is a newly identified epigenetic mark that plays a role in the regulation of gene transcription, where an isopeptide bond is formed between the fifth amino acid of H3 (i.e., glutamine) and dopamine. Recently, we developed a chemical probe to specifically label and enrich histone dopaminylation via bioorthogonal chemistry. Given this powerful tool, we found that histone H3 glutamine 5 dopaminylation (H3Q5dop) was highly enriched in colorectal tumors, which could be attributed to the high expression level of its regulator, transglutaminase 2 (TGM2), in colon cancer cells. Due to the enzyme promiscuity of TGM2, nonhistone proteins have also been identified as dopaminylation targets; however, the dopaminylated proteome in cancer cells still remains elusive. Here, we utilized our chemical probe to enrich dopaminylated proteins from colorectal cancer cells in a bioorthogonal manner and performed the chemical proteomics analysis. Therefore, 425 dopaminylated proteins were identified, many of which are involved in nucleic acid metabolism and transcription pathways. More importantly, a number of dopaminylation sites were identified and attributed to the successful application of our chemical probe. Overall, these findings shed light on the significant association between cellular protein dopaminylation and cancer development, further suggesting that targeting these pathways may become a promising anticancer strategy.

pH-Controlled Chemoselective Rapid Azo-Coupling Reaction (CRACR) Enables Global Profiling of Serotonylation Proteome in Cancer Cells.

Serotonylation has been identified as a novel protein posttranslational modification for decades, where an isopeptide bond is formed between the glutamine residue and serotonin through transamination. Transglutaminase 2 (also known as TGM2 or TGase2) was proven to act as the main "writer" enzyme for this PTM, and a number of key regulatory proteins (including small GTPases, fibronectin, fibrinogen, serotonin transporter, and histone H3) have been characterized as the substrates of serotonylation. However, due to the lack of pan-specific antibodies for serotonylated glutamine, the precise enrichment and proteomic profiling of serotonylation still remain challenging. In our previous research, we developed an aryldiazonium probe to specifically label protein serotonylation in a bioorthogonal manner, which depended on a pH-controlled chemoselective rapid azo-coupling reaction. Here, we report the application of a photoactive aryldiazonium-biotin probe for the global profiling of serotonylation proteome in cancer cells. Thus, over 1,000 serotonylated proteins were identified from HCT 116 cells, many of which are highly related to carcinogenesis. Moreover, a number of modification sites of these serotonylated proteins were determined, attributed to the successful application of our chemical proteomic approach. Overall, these findings provided new insights into the significant association between cellular protein serotonylation and cancer development, further suggesting that to target TGM2-mediated monoaminylation may serve as a promising strategy for cancer therapeutics.

Effects of the FHL2 gene on the development of subcutaneous and intramuscular adipocytes in goats.

BACKGROUND: Adipose tissue affects not only the meat quality of domestic animals, but also human health. Adipocyte differentiation is regulated by a series of regulatory genes and cyclins. Four and half-LIM protein (FHL2) is positively correlated with the hypertrophy of adipocytes and can cause symptoms such as obesity and diabetes. RESULT: In the transcriptome sequencing analysis of intramuscular adipocytes after three days of differentiation, the differentially expressed gene FHL2 was found. To further explore the biological significance of the differentially expressed gene FHL2, which was downregulated in the mature adipocytes. We revealed the function of FHL2 in adipogenesis through the acquisition and loss of function of FHL2. The results showed that the overexpression of FHL2 significantly increased the expression of adipogenic genes (PPARγ, C/EBPß) and the differentiation of intramuscular and subcutaneous adipocytes. However, silencing FHL2 significantly inhibited adipocyte differentiation. The overexpression of FHL2 increased the number of adipocytes stained with crystal violet and increased the mRNA expression of proliferation marker genes such as CCNE, PCNA, CCND and CDK2. In addition, it significantly increased the rate of EdU positive cells. In terms of apoptosis, overexpression of FHL2 significantly inhibited the expression of P53 and BAX in both intramuscular and subcutaneous adipocytes, which are involved in cell apoptosis. However, overexpression of FHL2 promoted the expression of BCL, but was rescued by the silencing of FHL2. CONCLUSIONS: In summary, FHL2 may be a positive regulator of intramuscular and subcutaneous adipocyte differentiation and proliferation, and acts as a negative regulator of intramuscular and subcutaneous adipocyte apoptosis. These findings provide a theoretical basis for the subsequent elucidation of FHL2 in adipocytes.

Bioorthogonal Labeling and Enrichment of Histone Monoaminylation Reveal Its Accumulation and Regulatory Function in Cancer Cell Chromatin.

Histone monoaminylation (i.e., serotonylation and dopaminylation) is an emerging category of epigenetic mark occurring on the fifth glutamine (Q5) residue of H3 N-terminal tail, which plays significant roles in gene transcription. Current analysis of histone monoaminylation is mainly based on site-specific antibodies and mass spectrometry, which either lacks high resolution or is time-consuming. In this study, we report the development of chemical probes for bioorthogonal labeling and enrichment of histone serotonylation and dopaminylation. These probes were successfully applied for the monoaminylation analysis of in vitro biochemical assays, cells, and tissue samples. The enrichment of monoaminylated histones by the probes further confirmed the crosstalk between H3Q5 monoaminylation and H3K4 methylation. Finally, combining the ex vivo and in vitro analyses based on the developed probes, we have shown that both histone serotonylation and dopaminylation are highly enriched in tumor tissues that overexpress transglutaminase 2 (TGM2) and regulate the three-dimensional architecture of cellular chromatin.

Plasma and serum metabolic analysis of healthy adults shows characteristic profiles by subjects' sex and age.

INTRODUCTION: Pre-analytical factors like sex, age, and blood processing methods introduce variability and bias, compromising data integrity, and thus deserve close attention. OBJECTIVES: This study aimed to explore the influence of participant characteristics (age and sex) and blood processing methods on the metabolic profile. METHOD: A Thermo UPLC-TSQ-Quantiva-QQQ Mass Spectrometer was used to analyze 175 metabolites across 9 classes in 208 paired serum and lithium heparin plasma samples from 51 females and 53 males. RESULTS: Comparing paired serum and plasma samples from the same cohort, out of the 13 metabolites that showed significant changes, 4 compounds related to amino acids and derivatives had lower levels in plasma, and 5 other compounds had higher levels in plasma. Sex-based analysis revealed 12 significantly different metabolites, among which most amino acids and derivatives and nitrogen-containing compounds were higher in males, and other compounds were elevated in females. Interestingly, the volcano plot also confirms the similar patterns of amino acids and derivatives higher in males. The age-based analysis suggested that metabolites may undergo substantial alterations during the 25-35-year age range, indicating a potential metabolic turning point associated with the age group. Moreover, a more distinct difference between the 25-35 and above 35 age groups compared to the below 25 and 25-35 age groups was observed, with the most significant compound decreased in the above 35 age groups. CONCLUSION: These findings may contribute to the development of comprehensive metabolomics analyses with confounding factor-based adjustment and enhance the reliability and interpretability of future large-scale investigations.

Integrated analysis of differently expressed microRNAs and mRNAs at different postnatal stages reveals intramuscular fat deposition regulation in goats (Capra hircus).

Intramuscular fat refers to the adipose tissue distributed in the muscle. It is an important indicator that affects the quality of goat meat, and can directly affect the tenderness and flavor of goat meat. Our previous study revealed the mRNA that may be crucial for intramuscular fat deposition during goat growth; however, how the microRNAs (miRNAs) are involved in the process is largely unclear. In the present study, a total of 401 known miRNAs and 120 goat novel miRNAs, including 110 differentially expressed (DE) miRNAs, were identified among longissimus dorsi from three growth stages (2, 9, and 24 months) by miRNA sequencing. Combining analysis of the DE mRNAs and DE miRNAs was then performed by miRDB and miRwalk, and miR-145-5p and FOXO1, miR-487b-3p, and PPARG coactivator 1 α (PPARGC1A), miR-345-3p, and solute carrier family 2 member 4 (SLC2A4), etc. were shown to closely associate with lipid metabolism, which was then validated by a correlation analysis. The final DE mRNAs were significantly enriched in fatty acid transmembrane transport, fatty acid homeostasis, apelin signaling pathway, glucagon signaling pathway, insulin signaling pathway, and AMPK signaling pathway by gene ontology and Kyoto Encyclopedia of Genes and Genomes analysis. Besides, miR-145-5p showed a certain effect on goat intramuscular fat metabolism by acting on the possible target gene Forkhead Box O1 (FOXO1). These data provide some theoretical support for improving the quality of goat meat.

Meeting Report on the 3rd Chinese American Society for Mass Spectrometry Conference-Advancing Biological and Pharmaceutical Mass Spectrometry.

Following the highly successful Chinese American Society for Mass Spectrometry (CASMS) conferences in the previous 2 years, the 3rd CASMS Conference was held virtually on August 28-31, 2023, using the Gather.Town platform to bring together scientists in the MS field. The conference offered a 4-day agenda with a scientific program consisting of two plenary lectures, and 14 parallel symposia in which a total of 70 speakers presented technological innovations and their applications in proteomics and biological MS and metabo-lipidomics and pharmaceutical MS. In addition, 16 invited speakers/panelists presented at two research-focused and three career development workshops. Moreover, 86 posters, 12 lightning talks, 3 sponsored workshops, and 11 exhibitions were presented, from which 9 poster awards and 2 lightning talk awards were selected. Furthermore, the conference featured four young investigator awardees to highlight early-career achievements in MS from our society. The conference provided a unique scientific platform for young scientists (i.e. graduate students, postdocs, and junior faculty/investigators) to present their research, meet with prominent scientists, learn about career development, and job opportunities (http://casms.org).

Database-assisted, globally optimized targeted secondary electrospray ionization high resolution mass spectrometry (dGOT-SESI-HRMS) and spectral stitching enhanced volatilomics analysis of bacterial metabolites.

Secondary electrospray ionization high-resolution mass spectrometry (SESI-HRMS) is an innovative analytical technique for the rapid and non-invasive analysis of volatile organic compounds (VOCs). However, compound annotation and ion suppression in the SESI source has hindered feature detection, stability and reproducibility of SESI-HRMS in untargeted volatilomics. To address this, we have developed and optimized a novel pseudo-targeted approach, database-assisted globally optimized targeted (dGOT)-SESI-HRMS using the microbial-VOC (mVOC) database, and spectral stitching methods to enhance metabolite detection in headspace of anaerobic bacterial cultures. Headspace volatiles from representative bacteria strains were assessed using full scan with data dependent acquisition (DDA), conventional globally optimized targeted (GOT) method, and spectral stitching supported dGOT experiments based on a MS peaks list derived from mVOC. Our results indicate that spectral stitching supported dGOT-SESI-HRMS can proportionally fragment peaks with respect to different analysis windows, with a total of 109 VOCs fragmented from 306 targeted compounds. Of the collected spectra, 88 features were confirmed as culture derived volatiles with respect to media blanks. Annotation was also achieved with a total of 25 unique volatiles referenced to standard databases allowing for biological interpretation. Principal component analysis (PCA) summarizing the headspace volatile demonstrated improved separation of clusters when data was acquired using the dGOT method. Collectively, our dGOT-SESI-HRMS method afforded robust capability of capturing unique VOC profiles from different bacterial strains and culture conditions when compared to conventional GOT and DDA modes, suggesting the newly developed approach can serve as a more reliable analytical method for the sensitive monitoring of gut microbial metabolism.

Diacylglycerol acyltransferase 2 promotes the adipogenesis of intramuscular preadipocytes in goat.

Diacylglycerol acyltransferase 2 (DGAT2) is the key enzyme that catalyzes the last step of triglyceride synthesis. However, its role in intramuscular fat (IMF) deposition in goat remains unclear. The purpose of this study was to explore the role of DGAT2 in regulating goat IMF deposition. In the present study, the expression of DGAT2 was highest in goat triceps brachii, and highest on the first day after oleic acid induction in goat intramuscular preadipocytes. The overexpression of DGAT2 promoted the accumulation of lipid droplets and triglyceride synthesis, accompanied by the expression upregulation of DGAT1, TIP47, ACC and ACOX1 significantly, and expression downregulation of AGPAT6, LPIN1, LPL, HSL, ATGL and ADRP significantly. In contrast, the silencing of DGAT2 decreased the accumulation of lipid droplets, inhibited the expression of DGAT1, GPAM, ADRP, AGPAT6, LPL, HSL, ATGL, ACC, FASN, ACOX1 significantly, and enhanced that of TIP47 significantly. Overall, these data underscore DGAT2 may play a potentially important role in lipid droplets formation and triglyceride accumulation, so as to maintain intramuscular fat deposition, beyond triglyceride synthesis in goat.

PDZK1-interacting protein 1(PDZK1IP1) promotes subcutaneous preadipocyte proliferation in goats.

PDZK1-interacting protein 1(PDZK1IP1), also known as MAP17, is encoded by the PDZK1IP1 gene and is a membrane-associated protein. PDZK1IP1 have been proven to be a potent regulator of cancer cell proliferation. However, the role of PDZK1IP1 in regulating goat subcutaneous preadipocyte proliferation is unknown. Here, we cloned the full-length coding sequence of PDZK1IP1 gene, investigated the potential functional of PDZK1IP1 in goat subcutaneous preadipocyte proliferation by gaining or losing function in vitro. Our results indicated that goat PDZK1IP1 gene consists of 345 bp, encoding a protein of 114 amino acids containing a typical PDZK1IP1 (MAP17) super family domain. Overexpression of PDZK1IP1 significantly increased the number of EdU-positive cells and cell viability, and also upregulated mRNA expression of cell proliferation-associated genes including CCND1 and CDK2 in vitro cultured cells. Conversely, knockdown of PDZK1IP1 mediated by siRNA technique significantly inhibited subcutaneous preadipocyte proliferation and downregulated mRNA expression of cell proliferation-associated genes including CCNE1, CCND1 and CDK2. Collectively, these results suggested that PDZK1IP1 can promote proliferation of goat subcutaneous preadipocyte.

Knockdown of KLF7 inhibits the differentiation of both intramuscular and subcutaneous preadipocytes in goat.

KLF7 belongs to the Krüppel-like factors (KLFs) family, which function as transcriptional regulators controlling a number of basic cellular processes, involving proliferation, differentiation, and migration. Here, we reveal insights into the differentiated expression of KLF7 in different goat tissues and different stages of growth, and the inhibition role of KLF7 knockdown to differentiation by using goat intramuscular and subcutaneous preadipocytes. We demonstrate that KLF7 expression is obviously changed during the differentiation of preadipocytes into mature adipocytes. Knockdown of KLF7 inhibited lipid droplet accumulation, reduced the expression of adipogenic markers both in intramuscular and subcutaneous preadipocytes in goats, suggesting that KLF7 is a novel regulator of adipogenesis. KLF7 expression changed also up or down-regulation the other KLF family members, but there were differences between these two types of cells. Investigation into the mechanism that KLF7 regulates preadipocyte differentiation revealed that KLF family members KLF1, KLF5, KLF6, KLF8, KLF11, KLF12, KLF16, KLF17 and adipogenic markers C/EBPα and SREBP1 promoter region present KLF7 transcriptional binding sites. Altogether, the data here identify KLF7 as a novel regulator of adipogenesis.

FGF1 promotes the differentiation of goat intramuscular and subcutaneous preadipocytes.

Fibroblast growth factor 1(FGF1) has been proved to bind to specific signal molecules and activate intracellular signal transduction, leading to proliferation or differentiation of cells. However, the role of FGF1 in goat adipocytes is still unclear. Here, we investigated its role in lipogenesis of goats, which depends on the activation of FGFRs. In goat intramuscular and subcutaneous adipocytes, we observed that adipocytes accumulation was inhibited by interfering of FGF1, the expression of C/EBPα, C/EBPß, LPL, Pref-1, PPARγ, AP2, KLF4, KLF6, KLF10 and KLF17 were significantly down-regulated (p < 0.05). When the FGF1 was up-regulated, the opposite result was found, while the expression of C/EBPß, LPL, PPARγ, SREBP1, AP2, KLF4, KLF7, KLF15, KLF16 and KLF17 were increased significantly (p < 0.05) in goat intramuscular and subcutaneous adipocytes. The expression level of FGFR1 was significantly and decreased with the interference of FGF1, and increased with the overexpression of FGF1. But in goat subcutaneous adipocytes, only the expression of FGFR2 was consistent with the expression of FGF1. Interference methods confirmed that FGFR1 or FGFR2 and FGF1 have the similarly promoting function in adipocytes differentiation. With the co-transfection technology, we confirmed that FGF1 promoted the differentiation of intramuscular and subcutaneous adipocytes might via FGFR1 or FGFR2, respectively.

Expression Variation of CPT1A Induces Lipid Reconstruction in Goat Intramuscular Precursor Adipocytes.

Intramuscular fat (IMF) deposition is one of the most important factors affecting meat quality and is closely associated with the expression of carnitine palmitoyl transferase 1A (CPT1A) which facilitates the transfer of long-chain fatty acids (LCFAs) into the mitochondria. However, the role of how CPT1A regulates the IMF formation remains unclear. Herein, we established the temporal expression profile of CPT1A during the differentiation of goat intramuscular precursor adipocytes. Functionally, the knockdown of CPT1A by siRNA treatment significantly increased the mRNA expression of adipogenic genes and promoted lipid deposition in goat intramuscular precursor adipocytes. Meanwhile, a CPT1A deficiency inhibited cell proliferation and promoted cell apoptosis significantly. CPT1A was then supported by the overexpression of CPT1A which significantly suppressed the cellular triglyceride deposition and promoted cell proliferation although the cell apoptosis also was increased. For RNA sequencing, a total of 167 differential expression genes (DEGs), including 125 upregulated DEGs and 42 downregulated DEGs, were observed after the RNA silencing of CPT1A compared to the control, and were predicted to enrich in the focal adhesion pathway, cell cycle, apoptosis and the MAPK signaling pathway by KEGG analysis. Specifically, blocking the MAPK signaling pathway by a specific inhibitor (PD169316) rescued the promotion of cell proliferation in CPT1A overexpression adipocytes. In conclusion, the expression variation of CPT1A may reconstruct the lipid distribution between cellular triglyceride deposition and cell proliferation in goat intramuscular precursor adipocyte. Furthermore, we demonstrate that CPT1A promotes the proliferation of goat adipocytes through the MAPK signaling pathway. This work widened the genetic regulator networks of IMF formation and delivered theoretical support for improving meat quality from the aspect of IMF deposition.

Adaptive Metabolism of Staphylococcus aureus Revealed by Untargeted Metabolomics.

Staphylococcus aureus (SA) is an opportunistic pathogen that can cause a wide spectrum of infections, from superficial skin inflammation to severe and potentially fatal and invasive diseases. Due to the many potential routes of infection, host-derived environmental signals (oxygen availability, nutrients, etc.) are vital for host colonization and thus contribute to SA's pathogenesis. To uncover the direct effects of environmental factors on SA metabolism, we performed a series of experiments in diverse culture environments and correlated our findings of SA's metabolic adaptation to some of the pathogen's known virulence factors. Untargeted metabolomics was conducted on a Thermo Q-Exactive high-resolution mass spectrometer. We detected 260 intracellular polar metabolites from our bacteria cultured under both aerobic and anaerobic conditions and in glucose- and dextrin-supplemented cultures. These metabolites were mapped to relevant metabolic pathways to elucidate the adaptive metabolic processes of both methicillin-sensitive SA (MSSA) and methicillin-resistant SA (MRSA). We also detected an increased expression of virulence genes agr-I and sea of MRSA supplemented with both glucose and dextrin by qPCR. With the metabolic data collected that may be associated with the adaptive growth and virulence of SA, our study could set up the foundations for future work to identify metabolic inhibitors/modulators to mitigate SA infections in different growth environments.

More than Meets the Eye: Untargeted Metabolomics and Lipidomics Reveal Complex Pathways Spurred by Activation of Acid Resistance Mechanisms in Escherichia coli.

Escherichia coli is a ubiquitous group of bacteria that can be either commensal gut microbes or enterohemorrhagic food-borne pathogens. Regardless, both forms must survive acidic environments in the stomach and intestines to reach and colonize the gut, a process that partially relies on amino acid-dependent acid resistance (AR) mechanisms and modifications to membrane phospholipids. However, only the basic tenets of these mechanisms have been elucidated. In this paper, we aim to conduct a full-scale metabolic and lipidomic characterization of E. coli's adaptations to acid stress. We hypothesized that the use of untargeted metabolomics and lipidomics would reveal mechanisms downstream of AR processes that provide novel contributions to acid stress survival. We detected significant differences in the extracellular metabolome and the lipidome induced by amino acid supplementation (glutamine, arginine, or lysine) and contextualized these results using real-time quantitative polymerase chain reaction (RT-qPCR). We additionally identified several metabolic pathways as well as a significant alteration in phospholipid synthetic pathways induced by differential amino acid supplementation. These results demonstrate that AR may extend beyond canonical mechanisms to a coordinated metabolic phenotype. Future studies may benefit from our analysis to further elucidate distinct targets for prebiotic supplements to cultivate commensal strains or therapies to combat pathogenic ones.

HABP4 overexpression promotes apoptosis in goat turbinate bone cells.

Hyaluronic acid-binding protein (HABP4) plays important roles in regulating cell cycle and apoptosis. However, its functions in regulating cell apoptosis remain unclear. To reveal the effects of HABP4 on cell proliferation, cell cycle and apoptosis, the HABP4 sequence was cloned, and we investigated the gain and loss functions of HABP4 in goat turbinate bone cells. Our results showed that a 1,496-bp HABP4 sequence was cloned successfully. The interference effect of siRNA1 on HABP4 was the strongest, reducing its mRNA expression level by 83%, decreasing the cells in the G0/G1 and S phases of the cell cycle and inhibiting cell growth and apoptosis. The overexpression of HABP4 produced contrasting results. Furthermore, an HABP4 knockdown caused the up-regulated expression of genes associated with apoptosis, including Bcl-2 and BCL2L11, but the down-regulation of Caspase3, Caspase7, Bax, PARP1, SOCS2 and P53 mRNA levels. Additionally, HABP4 overexpression significantly up-regulated the expression levels of Bax, Caspase3, Caspase7, BCL2L11, P53, SOCS2 and PARP1. However, the expression of Bcl-2 was down-regulated. These data provide an important foundation for further in-depth studies of HABP4 functions.

Angiopoietin-like protein 8, molecular cloning and regulating lipid accumulation in goat intramuscular preadipocytes.

This study aimed to clone the full-length open reading frame (ORF) of goat ANGPTL8 gene sequence, reveal its molecular and expression characteristics, and explore its effect on the differentiation of goat intramuscular preadipocytes. The full-length ORF sequence of goat ANGPTL8 gene was cloned by RT-PCR technology, and bioinformatics analysis was performed by related biological software. RT-qPCR was used to detect the expression of ANGPTL8 mRNA in goat tissues. Further use of RNA interference to study the effect of ANGPTL8 on the differentiation of goat intramuscular preadipocytes. The total length of the ANGPTL8 gene nucleotide sequence is 717 bp, including 597 bp of ORF, encoding 198 amino acids. Goat ANGPTL8 has the closest relationship with sheep, it was widely expressed in different tissues, and relatively enriched in liver. The silence of ANGPTL8 inhibited the accumulation of lipid droplets by 5.76% in goat intramuscular preadipocytes (p > 0.05) and significantly suppressed the expression of the genes related to preadipocytes differentiation, fatty acid synthesis and transport (p＜0.05 or p＜0.01). These data illuminate the speculation that ANGPTL8 may involve in the lipid accumulation regulation via the control of PPARγ and C/EBPß in goat adipocytes.

MiR-421 regulates goat intramuscular preadipocytes differentiation via targeting FGF13.

As a member of the MicroRNA s (miRNAs) family, miR-421 has been widely studied in regulating the proliferation and apoptosis of cancer cells a. However, there are still no reports on miR-421 in regulating adipocyte differentiation and its related mechanisms. Accordingly, this study aimed to investigate the potential involvement of miR-421 in goat intramuscular preadipocytes (P_IMA). The expression level of miR-421 was measured via quantitative real-time PCR during goat P_IMA differentiation. And the effects of miR-421 on goat P_IMA differentiation were studied by liposome transfection, Oil red O staining and qRT-PCR. Furthermore, the miR-421 target was searched and the underlying mechanism was clarified by luciferase reporter assay and rescue experiment. Our results showed that inhibition of miR-421 could accumulation of lipid droplets by upregulation the expression level of AP2, LPL, C/EBPα and SREBP1. Further studies showed that fibroblast growth factor 13 (FGF13) was the direct target of miR-421. Knocking down of FGF13 expression could inhibit lipid droplet formation and down-regulated the expression of key adipogenic regulatory genes. In addition, the rescue experiment revealed that FGF13 is involved in miR-421-induced differentiation of goat P_IMA as a key factor. Overall, these findings indicate that miR-421 is a negative regulator in the progression of differentiation of goat P_IMA by inhibiting the expression of FGF13.