The clinical significance of mitochondrial calcium uniporter in gastric cancer patients and its preliminary exploration of the impact on mitochondrial function and metabolism.

Objective: The objective of this study is to elucidate the influence of MCU on the clinical pathological features of GC patients, to investigate the function and mechanism of the mitochondrial calcium uptake transporter MCU in the initiation and progression of GC, and to explore its impact on the metabolic pathways and biosynthesis of mitochondria. The ultimate goal is to identify novel targets and strategies for the clinical management of GC patients. Methods: Tumor and adjacent tissue specimens were obtained from 205 patients with gastric cancer, and immunohistochemical tests were performed to assess the expression of MCU and its correlation with clinical pathological characteristics and prognosis. Data from TCGA, GTEx and GEO databases were retrieved for gastric cancer patients, and bioinformatics analysis was utilized to investigate the association between MCU expression and clinical pathological features. Furthermore, we conducted an in-depth analysis of the role of MCU in GC patients. We investigated the correlation between MCU expression in GC and its impact on mitochondrial function, metabolism, biosynthesis, and immune cells. Additionally, we studied the proteins or molecules that interact with MCU. Results: Our research revealed high expression of MCU in the GC tissues. This high expression was associated with poorer T and N staging, and indicated a worse disease-free survival period. MCU expression was positively correlated with mitochondrial function, mitochondrial metabolism, nucleotide, amino acid, and fatty acid synthesis metabolism, and negatively correlated with nicotinate and nicotinamide metabolism. Furthermore, the MCU also regulates the function of the mitochondrial oxidative respiratory chain. The MCU influences the immune cells of GC patients and regulates ROS generation, cell proliferation, apoptosis, and resistance to platinum-based drugs in gastric cancer cells. Conclusion: High expression of MCU in GC indicates poorer clinical outcomes. The expression of the MCU are affected through impacts the function of mitochondria, energy metabolism, and cellular biosynthesis in gastric cancer cells, thereby influencing the growth and metastasis of gastric cancer cells. Therefore, the mitochondrial changes regulated by MCU could be a new focus for research and treatment of GC.

Modulating peppermint oil flavor release properties of emulsion-filled protein gels: Impact of cross-linking method and matrix composition.

For some food applications, it is desirable to control the flavor release profiles of volatile flavor compounds. In this study, the effects of crosslinking method and protein composition on the flavor release properties of emulsion-filled protein hydrogels were explored, using peppermint essential oil as a model volatile compound. Emulsion-filled protein gels with different properties were prepared using different crosslinking methods and gelatin concentrations. Flavor release from the emulsion gels was then monitored using an electronic nose, gas chromatography-mass spectrometry (GC-MS), and sensory evaluation. Enzyme-crosslinked gels had greater hardness and storage modulus than heat-crosslinked ones. The hardness and storage modulus of the gels increased with increasing gelatin concentration. For similar gel compositions, flavor release and sensory perception were faster from the heat-crosslinked gels than the enzyme-crosslinked ones. For the same crosslinking method, flavor release and perception decreased with increasing gelatin concentration, which was attributed to retardation of flavor diffusion through the hydrogel matrix. Overall, this study shows that the release of hydrophobic aromatic substances can be modulated by controlling the composition and crosslinking of protein hydrogels, which may be useful for certain food applications.

Intravenous administration of IL-12 encoding self-replicating RNA-lipid nanoparticle complex leads to safe and effective antitumor responses.

Interleukin 12 (IL-12) is a potent immunostimulatory cytokine mainly produced by antigen-presenting cells (e.g., dendritic cells, macrophages) and plays an important role in innate and adaptive immunity against cancers. Therapies that can synergistically modulate innate immunity and stimulate adaptive anti-tumor responses are of great interest for cancer immunotherapy. Here we investigated the lipid nanoparticle-encapsulated self-replicating RNA (srRNA) encoding IL-12 (referred to as JCXH-211) for the treatment of cancers. Both local (intratumoral) and systemic (intravenous) administration of JCXH-211 in tumor-bearing mice induced a high-level expression of IL-12 in tumor tissues, leading to modulation of tumor microenvironment and systemic activation of antitumor immunity. Particularly, JCXH-211 can inhibit the tumor-infiltration of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs). When combined with anti-PD1 antibody, it was able to enhance the recruitment of T cells and NK cells into tumors. In multiple mouse solid tumor models, intravenous injection of JCXH-211 not only eradicated large preestablished tumors, but also induced protective immune memory that prevented the growth of rechallenged tumors. Finally, intravenous injection of JCXH-211 did not cause noticeable systemic toxicity in tumor-bearing mice and non-human primates. Thus, our study demonstrated the feasibility of intravenous administration of JCXH-211 for the treatment of advanced cancers.

Metaplastic regeneration in the mouse stomach requires a reactive oxygen species pathway.

In pyloric metaplasia, mature gastric chief cells reprogram via an evolutionarily conserved process termed paligenosis to re-enter the cell cycle and become spasmolytic polypeptide-expressing metaplasia (SPEM) cells. Here, we use single-cell RNA sequencing (scRNA-seq) following injury to the murine stomach to analyze mechanisms governing paligenosis at high resolution. Injury causes induced reactive oxygen species (ROS) with coordinated changes in mitochondrial activity and cellular metabolism, requiring the transcriptional mitochondrial regulator Ppargc1a (Pgc1α) and ROS regulator Nf2el2 (Nrf2). Loss of the ROS and mitochondrial control in Ppargc1a-/- mice causes the death of paligenotic cells through ferroptosis. Blocking the cystine transporter SLC7A11(xCT), which is critical in lipid radical detoxification through glutathione peroxidase 4 (GPX4), also increases ferroptosis. Finally, we show that PGC1α-mediated ROS and mitochondrial changes also underlie the paligenosis of pancreatic acinar cells. Altogether, the results detail how metabolic and mitochondrial changes are necessary for injury response, regeneration, and metaplasia in the stomach.

Unveiling the potential of HSPA4: a comprehensive pan-cancer analysis of HSPA4 in diagnosis, prognosis, and immunotherapy.

With the global rise in cancer incidence and mortality rates, research on the topic has become increasingly urgent. Among the significant players in this field are heat shock proteins (HSPs), particularly HSPA4 from the HSP70 subfamily, which has recently garnered considerable interest for its role in cancer progression. However, despite numerous studies on HSPA4 in specific cancer types, a comprehensive analysis across all cancer types is lacking. This study employs various bioinformatics techniques to delve into the role of HSPA4 in pan-cancer. Our objective is to assess its potential in clinical diagnosis, prognosis, and as a future molecular target for therapy. The research findings reveal significant differences in HSPA4 expression across different cancer types, suggesting its diagnostic value and close association with cancer staging and patient survival rates. Furthermore, genetic variations and methylation status of HSPA4 play critical roles in tumorigenesis. Lastly, the interaction of HSPA4 with immune cells is linked to the tumor microenvironment (TME) and immunotherapy. In summary, HSPA4 emerges as a promising cancer biomarker and a vital member of the HSPs family, holding potential applications in diagnosis, prognosis, and immunotherapy.

Differential airway resistome and its correlations with clinical characteristics in Haemophilus- or Pseudomonas-predominant microbial subtypes of bronchiectasis.

The prevalence and clinical correlates of antibiotic resistance genes (ARGs) in bronchiectasis are not entirely clear. We aimed to profile the ARGs in sputum from adults with bronchiectasis, and explore the association with airway microbiome and disease severity and subtypes. In this longitudinal study, we prospectively collected 118 sputum samples from stable and exacerbation visits of 82 bronchiectasis patients and 19 healthy subjects. We profiled ARGs with shotgun metagenomic sequencing, and linked these to sputum microbiome and clinical characteristics, followed by validation in an international cohort. We compared ARG profiles in bronchiectasis according to disease severity, blood and sputum inflammatory subtypes. Unsupervised clustering revealed a Pseudomonas predominant subgroup (n = 16), Haemophilus predominant subgroup (n = 48), and balanced microbiome subgroup (N = 54). ARGs of multi-drug resistance were over-dominant in the Pseudomonas-predominant subgroup, while ARGs of beta-lactam resistance were most abundant in the Haemophilus-predominant subgroup. Pseudomonas-predominant subgroup yielded the highest ARG diversity and total abundance, while Haemophilus-predominant subgroup and balanced microbiota subgroup were lowest in ARG diversity and total abundance. PBP-1A, ksgA and emrB (multidrug) were most significantly enriched in Haemophilus-predominant subtype. ARGs generally correlated positively with Bronchiectasis Severity Index, fluoroquinolone use, and modified Reiff score. 68.6% of the ARG-clinical correlations could be validated in an independent international cohort. In conclusion, ARGs are differentially associated with the dominant microbiome and clinical characteristics in bronchiectasis.

The airway microbiome mediates the interaction between environmental exposure and respiratory health in humans.

Exposure to environmental pollution influences respiratory health. The role of the airway microbial ecosystem underlying the interaction of exposure and respiratory health remains unclear. Here, through a province-wide chronic obstructive pulmonary disease surveillance program, we conducted a population-based survey of bacterial (n = 1,651) and fungal (n = 719) taxa and metagenomes (n = 1,128) from induced sputum of 1,651 household members in Guangdong, China. We found that cigarette smoking and higher PM2.5 concentration were associated with lung function impairment through the mediation of bacterial and fungal communities, respectively, and that exposure was associated with an enhanced inter-kingdom microbial interaction resembling the pattern seen in chronic obstructive pulmonary disease. Enrichment of Neisseria was associated with a 2.25-fold increased risk of high respiratory symptom burden, coupled with an elevation in Aspergillus, in association with occupational pollution. We developed an individualized microbiome-based health index, which covaried with exposure, respiratory symptoms and diseases, with potential generalizability to global datasets. Our results may inform environmental risk prevention and guide interventions that harness airway microbiome.

Gut Microbiome Signatures in the Progression of Hepatitis B Virus-Induced Liver Disease.

The gut microbiome is associated with hepatitis B virus (HBV)-induced liver disease, which progresses from chronic hepatitis B, to liver cirrhosis, and eventually to hepatocellular carcinoma. Studies have analyzed the gut microbiome at each stage of HBV-induced liver diseases, but a consensus has not been reached on the microbial signatures across these stages. Here, we conducted by a systematic meta-analysis of 486 fecal samples from publicly available 16S rRNA gene datasets across all disease stages, and validated the results by a gut microbiome characterization on an independent cohort of 15 controls, 23 chronic hepatitis B, 20 liver cirrhosis, and 22 hepatocellular carcinoma patients. The integrative analyses revealed 13 genera consistently altered at each of the disease stages both in public and validation datasets, suggesting highly robust microbiome signatures. Specifically, Colidextribacter and Monoglobus were enriched in healthy controls. An unclassified Lachnospiraceae genus was specifically elevated in chronic hepatitis B, whereas Bilophia was depleted. Prevotella and Oscillibacter were depleted in liver cirrhosis. And Coprococcus and Faecalibacterium were depleted in hepatocellular carcinoma. Classifiers established using these 13 genera showed diagnostic power across all disease stages in a cross-validation between public and validation datasets (AUC = 0.65-0.832). The identified microbial taxonomy serves as non-invasive biomarkers for monitoring the progression of HBV-induced liver disease, and may contribute to microbiome-based therapies.

Phospho-Ser727 triggers a multistep inactivation of STAT3 by rapid dissociation of pY705-SH2 through C-terminal tail modulation.

Signal transducer and activator of transcription 3 (STAT3) is involved in many biological processes, including immunity and cancer. STAT3 becomes phosphorylated at Tyr705 and Ser727 on IL-6 stimulation. Phospho-Tyr705 (pY705) stabilizes the STAT3 dimer with reciprocal interactions between pY705 and the SH2 of the other molecule and phospho-Ser727 (pS727) accelerates pY705 dephosphorylation. We study how pS727 regulates STAT3 in both structural and biological perspectives. Using STAT3 reconstituted in HepG2-stat3-knockout cells, we show that pS727, together with a handshake N-terminal domain (NTD) interaction, causes rapid inactivation of STAT3 for pY705 dephosphorylation and a chromosome region maintenance 1 (CRM1)-independent nuclear export, which is critical for faithful STAT3 response to the cellular signals. The various N-terminal tags, GFP-related Ruby and FLAG, rendered the export CRM1-dependent and especially FLAG-tag caused nuclear accumulation of STAT3, indicating the presence of conformational changes in inactivation. Impaired reactivation of STAT3 by S727A or FLAG-tag delayed or inhibited the IL-6-induced saa1 mRNA expression, respectively. The detailed analysis of the pY705-SH2 structure identified the C-terminal tail (CTT) from L706 to P715 as a key regulator of the CTT-CTT intermolecular and the CTT-SH2 intramolecular interactions that support pY705-SH2 association. The functional studies using multiple STAT3 mutants indicated that the degree of the two interactions determines the stability of pY705-SH2 interaction. Importantly, Pro715 was critical for the pS727's destabilizing activity and the known phosphorylation and acetylation at the CTT structurally inhibited the pY705-SH2 interaction. Thus, pS727 triggers pY705-SH2 dissociation by weakening the supportive interactions likely through CTT modulation, inducing rapid cycles of STAT3 activation-inactivation for proper function of STAT3.

RBM10 regulates centriole duplication in HepG2 cells by ectopically assembling PLK4-STIL complexes in the nucleus.

RNA-binding motif protein 10 (RBM10) primarily regulates alternative splicing of certain genes. Loss-of-function mutations in RBM10 have been frequently reported in patients with various cancers. However, how RBM10 levels affect cell proliferation and tumorigenesis remains unknown. To elucidate the role of RBM10 in cell proliferation, we established HepG2-RBM10 knockout cell lines and derivative doxycycline-inducible RBM10-expressing cells. RBM10 over-expression caused growth arrest in the M phase with a monopolar spindle because of impaired centriole duplication. Two RBM10 splicing mutants, one with F345A/F347A and the other with only the C-terminal half (401-930), were sufficient to cause growth arrest, whereas an RBM10 mutant with cytoplasmic localization forced by an NES did not show growth arrest. RBM10 over-expression induced the formation of many large nuclear domains containing RBM10, PLK4, STIL and SAS6, which are the regulatory proteins involved in centriole duplication. Consistently, the centrioles in the RBM10-over-expressing HepG2 cells lost PLK4 and STIL, accounting for the unsuccessful centriole duplication. In contrast, RBM10 depletion resulted in elevated levels of cytoplasmic PLK4 with a concomitant increase in the number of centrioles in HepG2 cells but not in A549 cells. Thus, nuclear RBM10 regulates normal chromosomal division in a cell-type-specific manner, independent of alternative RNA splicing.

RF1 attenuation enables efficient non-natural amino acid incorporation for production of homogeneous antibody drug conjugates.

Amber codon suppression for the insertion of non-natural amino acids (nnAAs) is limited by competition with release factor 1 (RF1). Here we describe the genome engineering of a RF1 mutant strain that enhances suppression efficiency during cell-free protein synthesis, without significantly impacting cell growth during biomass production. Specifically, an out membrane protease (OmpT) cleavage site was engineered into the switch loop of RF1, which enables its conditional inactivation during cell lysis. This facilitates extract production without additional processing steps, resulting in a scaleable extract production process. The RF1 mutant extract allows nnAA incorporation at previously intractable sites of an IgG1 and at multiple sites in the same polypeptide chain. Conjugation of cytotoxic agents to these nnAAs, yields homogeneous antibody drug conjugates (ADCs) that can be optimized for conjugation site, drug to antibody ratio (DAR) and linker-warheads designed for efficient tumor killing. This platform provides the means to generate therapeutic ADCs inaccessible by other methods that are efficient in their cytotoxin delivery to tumor with reduced dose-limiting toxicities and thus have the potential for better clinical impact.

Production of bispecific antibodies in "knobs-into-holes" using a cell-free expression system.

Bispecific antibodies have emerged in recent years as a promising field of research for therapies in oncology, inflammable diseases, and infectious diseases. Their capability of dual target recognition allows for novel therapeutic hypothesis to be tested, where traditional mono-specific antibodies would lack the needed mode of target engagement. Among extremely diverse architectures of bispecific antibodies, knobs-into-holes (KIHs) technology, which involves engineering CH3 domains to create either a "knob" or a "hole" in each heavy chain to promote heterodimerization, has been widely applied. Here, we describe the use of a cell-free expression system (Xpress CF) to produce KIH bispecific antibodies in multiple scaffolds, including 2-armed heterodimeric scFv-KIH and one-armed asymmetric BiTE-KIH with tandem scFv. Efficient KIH production can be achieved by manipulating the plasmid ratio between knob and hole, and further improved by addition of prefabricated knob or hole. These studies demonstrate the versatility of Xpress CF in KIH production and provide valuable insights into KIH construct design for better assembly and expression titer.

Microscale to manufacturing scale-up of cell-free cytokine production--a new approach for shortening protein production development timelines.

Engineering robust protein production and purification of correctly folded biotherapeutic proteins in cell-based systems is often challenging due to the requirements for maintaining complex cellular networks for cell viability and the need to develop associated downstream processes that reproducibly yield biopharmaceutical products with high product quality. Here, we present an alternative Escherichia coli-based open cell-free synthesis (OCFS) system that is optimized for predictable high-yield protein synthesis and folding at any scale with straightforward downstream purification processes. We describe how the linear scalability of OCFS allows rapid process optimization of parameters affecting extract activation, gene sequence optimization, and redox folding conditions for disulfide bond formation at microliter scales. Efficient and predictable high-level protein production can then be achieved using batch processes in standard bioreactors. We show how a fully bioactive protein produced by OCFS from optimized frozen extract can be purified directly using a streamlined purification process that yields a biologically active cytokine, human granulocyte-macrophage colony-stimulating factor, produced at titers of 700 mg/L in 10 h. These results represent a milestone for in vitro protein synthesis, with potential for the cGMP production of disulfide-bonded biotherapeutic proteins.

Cell-free production of scFv fusion proteins: an efficient approach for personalized lymphoma vaccines.

The unique immunoglobulin (Ig) idiotype on the surface of each B-cell lymphoma represents an ideal tumor-specific antigen for use as a therapeutic vaccine. We have used an Escherichia coli-based, cell-free protein-expression system to produce a vaccine within hours of cloning the Ig genes from a B-cell tumor. We demonstrated that a fusion protein consisting of an idiotypic single chain Fv antibody fragment (scFv) linked to a cytokine (GM-CSF) or to an immunostimulatory peptide was an effective lymphoma vaccine. These vaccines elicited humoral immune responses against the native Ig protein displayed on the surface of a tumor and protected mice against tumor challenge with efficacy equal to that of the conventional Ig produced in a mammalian cell and chemically coupled to keyhole limpet hemocyanin. The cell-free E coli system offers a platform for rapidly generating individualized vaccines, thereby allowing much more efficient application in the clinic.

Rapid expression of vaccine proteins for B-cell lymphoma in a cell-free system.

The idiotype (Id)-granulocyte-macrophage colony-stimulating factor (GM-CSF) fusion proteins are potential vaccines for immunotherapy of B-cell lymphoma. In this study, four vaccine candidates were constructed by fusing murine GM-CSF to the amino- or carboxy-terminus of the 38C13 murine B-lymphocyte Id scFv with two different arrangements of the variable regions of the heavy chain and light chain (VL-VH and VH-VL). scFv (VH-VL) and GM-CSF/scFv fusion proteins were expressed in an Escherichia coli cell-free protein synthesis system. In order to promote disulfide bond formation during cell-free expression, cell extract was pretreated with iodoacetamide (IAM), and a sulfhydryl redox buffer composed of oxidized and reduced glutathione was added. The E. coli periplasmic disulfide isomerase, DsbC, was also added to rearrange incorrectly formed disulfide linkages. The 38C13 B-lymphocyte Id scFv was expressed with 30% of its soluble yield in active form (43 microg/ml) when tested with an anti-idiotypic mAb, S1C5, as the capture antibody in radioimmunoassay. It was found that the amino-terminal GM-CSF fusion proteins, GM-VL-VH and GM-VH-VL, showed much higher activity than the carboxy-terminal GM-CSF fusion proteins, VL-VH-GM and VH-VL-GM, in stimulating the cell proliferation of a GM-CSF-dependent cell line, NFS-60. Between the two amino-terminal GM-CSF fusion proteins, GM-VL-VH showed a higher total and soluble yield than GM-VH-VL.

Effect of apolipoprotein A-I on ATP binding cassette transporter A1 degradation and cholesterol efflux in THP-1 macrophage-derived foam cells.

Cholesterol-loaded macrophage foam cells are a central component of atherosclerotic lesions. ATP binding cassette transporter A1 (ABCA1), the defective molecule in Tangier disease, mediates the efflux of phospholipid and cholesterol from cells to apolipoprotein A-I (apoA-I), reversing foam cell formation. This study investigated the effect of apoA-I on ABCA1 degradation and cholesterol efflux in THP-1 macrophage-derived foam cells. After exposure of the cultured THP-1 macrophage-derived foam cells to apoA-I for different time, cholesterol efflux, ABCA1 mRNA and protein levels were determined by FJ-2107P type liquid scintillator, RT-PCR and Western blot, respectively. The mean ABCA1 fluorescence intensity on THP-1 macrophage-derived foam cells was detected by flow cytometry. Results showed that apoA-I markedly increased ABCA1-mediated cholesterol efflux from THP-1 macrophage-derived foam cells. This was accompanied by an increase in the content of ABCA1. ApoA-I did not alter ABCA1 mRNA abundance. Significantly, thiol protease inhibitors increased the level of ABCA1 protein and slowed its decay in THP-1 macrophage-derived foam cells, whereas none of the proteosome-specific inhibitor lactacystin, other protease inhibitors, or the lysosomal inhibitor NH4Cl showed such effects. The apoA-I-mediated cellular cholesterol efflux was enhanced by thiol protease inhibitors. Our results suggested that thiol protease inhibitors might provide an alternative way to upregulate ABCA1 protein. This strategy is especially appealing since it may mimic the stabilizing effect of the natural ligands apoA-I.

Oxidized LDL upregulated ATP binding cassette transporter-1 in THP-1 macrophages.

AIM: To study the effect of oxidized low density lipoprotein (ox-LDL) on ATP binding cassette transporter A1 (ABCA1) in THP-1 macrophages. METHODS: After exposing the cultured THP-1 macrophages to ox-LDL for different periods, cholesterol efflux was determined by FJ-2107P type liquid scintillator. ABCA1 mRNA and protein level were determined by reverse trancriptase-polymerase chain reaction (RT-PCR) and Western blot, respectively. The cholesterol level in THP-1 macrophage foam cells was detected by high performance liquid chromatography. RESULTS: ox-LDL elevated ABCA1 in both protein and mRNA levels and increased apolipoprotein (apo) A-I-mediated cholesterol efflux in a time- and dose-dependent manner. 22(R)-hydroxycholesterol and 9-cis-retinoic acid did significantly increase cholesterol efflux in THP-1 macrophage foam cells (P<0.05), respectively. Both of them further promoted cholesterol efflux (P<0.01). As expected, liver X receptor (LXR) agonist decreased content of esterified cholesterol in the macrophage foam cells compared with control, whereas only a slight decrease of free cholesterol was observed. LXR activity was slightly increased by oxidized LDL by 12 % at 12 h compared with 6 h. However, LXR activity was increased about 1.8 times at 24 h, and oxidized LDL further increased LXR activity by about 2.6 times at 48 h. CONCLUSION: ABCA1 gene expression was markedly increased in cholesterol-loaded cells as a result of activation of LXR/RXR. ABCA1 plays an important role in the homeostasis of cholesterol in the macrophages.