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Wu, X.

Publications and source records attributed to Wu, X..

At least 217 records · Page 12Linked to original sources

Murine gut microbiota dysbiosis via enteric infection modulates the foreign body response to a distal biomaterial implant

The gut microbiota influences systemic immunity and the function of distal tissues, including the brain, liver, skin, lung, and muscle. However, the role of the gut microbiota in the foreign body response (FBR) and fibrosis around medical implants is largely unexplored. To investigate this connection, we perturbed the homeostasis of the murine gut microbiota via enterotoxigenic Bacteroides fragilis (ETBF) infection and implanted the synthetic polymer polycaprolactone (PCL) into a distal muscle injury. ETBF infection in mice led to increased neutrophil and {gamma}{delta} T cell infiltration into the PCL implant site. ETBF infection alone promoted systemic inflammation and increased levels of neutrophils in the blood, spleen, and bone marrow. At the PCL implant site, we found significant changes in the transcriptome of sorted fibroblasts but did not observe gross ETBF- induced differences in the fibrosis levels after 6 weeks. These results demonstrate the ability of the gut microbiota to mediate long-distance effects such as immune and stromal responses to a distal biomaterial implant. Significance StatementThe foreign body response to implants leads to chronic inflammation and fibrosis that can be highly variable in the general patient population. Here, we demonstrate that gut dysbiosis via enteric infection promoted systemic inflammation and increased immune cell recruitment to an anatomically distant implant site. These results implicate the gut microbiota as a potential source of variability in the clinical biomaterial response and illustrate that the local tissue environment can be influenced by host factors that modulate systemic interactions.

bioengineering↗

Dimerization and dynamics of angiotensin-I converting enzyme revealed by cryoEM and MD simulations

Angiotensin-I converting enzyme (ACE) regulates the levels of disparate bioactive peptides, notably converting angiotensin-I to angiotensin-II and degrading amyloid beta. ACE is a heavily glycosylated dimer, containing 4 analogous catalytic sites, and exists in membrane bound and soluble (sACE) forms. ACE inhibition is a frontline, FDA-approved, therapy for cardiovascular diseases yet is associated with significant side effects, including higher rates of lung cancer. To date, structural studies have been confined to individual domains or partially denatured cryo-EM structures. Here we report the cryo-EM structure of the glycosylated full sACE dimer. We resolved four structural states at 2.99 to 3.65 [A] resolution which are primarily differentiated by varying degrees of solvent accessibility to the active sites and reveal the full dimerization interface. We also employed all-atom molecular dynamics (MD) simulations and heterogeneity analysis in cryoSPARC, cryoDRGN, and RECOVAR to elucidate the conformational dynamics of sACE and identify key regions mediating conformational change. We identify differences in the mechanisms governing the conformational dynamics of individual domains that have implications for the design of domain-specific sACE modulators.

biophysics↗

Role of drug induced nuclear CTSL (nCTSL) in DNA damage response in cancer- therapeutic implications

ABSTRACT Background: Despite treatment advances, ovarian cancer (OC) remains the deadliest gynecological cancer, with a ~70% mortality rate. OCs initially respond to platinum chemotherapy but often develop resistance. While PARP inhibitors improve relapse-free survival in responsive cases, they do not extend overall survival. This underscores the need for effective combination therapies beyond BRCA status. The lysosomal protein cathepsin L (CTSL) can traffic to the nucleus, where it can regulate DNA repair and serve as a chromatin modifier. Here, we tested the hypothesis that nucleoside analog clofarabine (CLF) induced nuclear CTSL (nCTSL) serves as a biomarker of sensitivity to DNA-damaging agents in ovarian cancer. Methods: Role of nuclear CTSL in DNA damage, synergy with PARPis and CRM1 inhibitor, tumor regression was analyzed by 3D-spheroid culture, colony forming assay, subcellular fractions, comet assay, and IFC/ western-blotting using PARP inhibitor-sensitive and -resistant OC cell lines, ex vivo cultures of patient-derived OC ascites cells (OVA), primary OCs, and patient-derived xenografts (PDXs). Results: In OC cell lines, treatment with CLF induced nCTSL in a subset of models-designated CLF-responsive (CLF-r)-and sensitized them to the PARP inhibitors olaparib and rucaparib. In CLF-non-responsive (CLF-nr) models, nCTSL trafficking and drug synergy were achieved with the CLF+olaparib combination. Synergy was observed in 47% of CLF-r and 24% of CLF-nr OVA samples. Mechanistically, CLF monotherapy induced nuclear import of CTSL via KPNB1 in CLF-r cells, while combination treatment with CLF+olaparib was required in CLF-nr cells. CLF also downregulated the nuclear export protein CRM1 in both CLF-r and CLF-nr models. CTSL knockdown conferred resistance to CLF+olaparib in both cohorts, supporting a functional role for nCTSL in therapeutic response. Notably, in a subset of OVAs (29%) classified as CLF-resistant (CLF-Res), CLF failed to downregulate CRM1; however, pharmacological inhibition of CRM1 with KPT8602 restored sensitivity to CLF+PARP inhibitor treatment. In vivo, CLF+olaparib treatment in CLF-r and CLF-nr PDX models led to enhanced DNA damage, reduced tumor burden, and prolonged survival. Conclusion: Collectively, these findings identify nCTSL as a predictive marker of response to DNA-damaging therapies and support the CLF+olaparib combination as a promising strategy for overcoming drug resistance in OC via CTSL-mediated DNA damage.

cancer biology↗

A De Novo Design Strategy to Convert FAcD from Dimer to Active Monomer

Enzymes largely exist in various oligomeric states, but monomeric enzymes are more conducive to industrial applications. Converting an oligomeric enzyme into an active monomer is a significant challenge. In this study, we present a de novo design strategy to convert fluoroacetate dehalogenase (FAcD) from its native dimeric form to an active monomer. Using the AI-based method ProteinMPNN, we identified critical protein-protein interaction (PPI) sites at the dimer interface. ArDCA, another AI tool, was employed to pinpoint catalytic hotspots. Six mutants, Mu1-Mu6, were designed. Molecular dynamics (MD) simulations, coupled with mass spectrometry, confirmed that these mutants form stable monomers. The pre-reaction state (PRS) model predicted that three of these mutants exhibited catalytic activity. In particular, Mu5 with 11 mutations from the wild-type, was predicted to have high catalytic activity, and was subsequently confirmed by kinetics experiment, with a kcat of 672.2 min-1 and a T5030 > 100 {degrees}C, comparable to the wild-type enzyme (kcat = 676.3 min-1, T5030 = 84 {degrees}C). Notably, the Y149M mutation increased catalytic activity nearly forty-fold, demonstrating the effectiveness of our design strategy.

bioengineering↗

Collagen Hydrogel Tube Microbioreactors for Cell and Tissue Manufacturing

The production of mammalian cells in large quantities is essential for various applications. However, scaling up cell culture using existing bioreactors poses significant technical challenges and high costs. To address this, we previously developed an innovative 3D culture system, known as the AlgTube cell culture system, for high-density cell cultivation. This system involves processing cells into microscale alginate hydrogel tubes, which are suspended in the culture medium within a vessel. These hydrogel tubes shield cells from hydrodynamic stress and maintain the cell mass below 400 {micro}m in diameter, facilitating efficient mass transport and creating a favorable microenvironment for cell growth. Under optimized conditions, AlgTubes supported long-term culture with high cell viability, rapid expansion (1000-fold increase over 9 days per passage), and high yield (5x10 cells/mL), offering significant advantages over conventional methods. Despite these benefits, AlgTubes have critical drawbacks. They are mechanically fragile, with frequent breakage during culture leading to cell leakage and production failures. Additionally, many cell types exhibit poor growth due to the inability to adhere to the alginate surface, making alginate hydrogel microtubes unsuitable for industrial-scale cell production. To overcome these challenges, we developed a novel collagen hydrogel tube-based microbioreactor system in this work. This system provides enhanced robustness and adhesion, enabling scalable, cost-effective, and efficient cell production for a wide range of applications.

bioengineering↗

MiRNA Locker: A Modularized DNA Assembly As miRNA Inhibitors

MicroRNAs (miRNAs) are vital post-transcriptional regulators that govern key cellular processes such as proliferation, migration, and apoptosis. Current loss-of-function approaches, including chemically modified antisense oligonucleotides (ASOs), face significant challenges, including high costs, limited scalability, and off-target effects. To overcome these limitations, we developed "miRNA Locker", a novel miRNA inhibition platform created using the Overlapped Oligo Assembly (OOA) method. This innovative platform constructs highly stable dumbbell-shaped single-stranded DNA structures, offering improved target specificity, scalability, and cost-effectiveness. Using miR-214 as a proof-of-concept target, we demonstrated that miRNA Lockers effectively bind Argonaute-miRNA complexes, reduce miRNA levels, and induce downstream changes in gene expression and cellular phenotypes, surpassing the performance of commercial antagomirs. Furthermore, applying miRNA Lockers to miR-654 validated the regulatory role of miR-654 in modulating RNF8 expression and promoting epithelial-mesenchymal transition (EMT) in lung cancer cells. Our results highlight the potential of miRNA Lockers as a versatile tool for studying miRNA function and advancing miRNA-based therapies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/632138v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@c6ac1eorg.highwire.dtl.DTLVardef@14e2698org.highwire.dtl.DTLVardef@1c7d84aorg.highwire.dtl.DTLVardef@148f9c1_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

The miR156/SPL9 interaction mode regulates the anthocyanin accumulation in potato

Anthocyanins are an essential class of flavonoids that represent a large group of plant secondary metabolites. microRNAs (miRNAs) can target TFs related to anthocyanin synthesis and inhibit their expression, thereby affecting the expression level of key downstream structural genes and ultimately regulating the synthesis and accumulation of anthocyanins in plants. Nevertheless, the regulatory function of miR156a in anthocyanin synthesis, especially in potatoes, has yet to be investigated. In this study, the miR156a and its target gene StSPL9 were screened and analyzed from small RNA sequencing, degradome sequencing and transient expression assay. The function of miR156a in anthocyanin synthesis of potato tuber was investigated. Overexpression of miR156a (OE-miR156a) in potato tubers promoted anthocyanin synthesis while concurrently reducing flavonoid synthesis compared with wild-type (WT) potatoes. The miR156a-silenced tubers (STTM-miR156a) achieved using short tandem target mimics (STTM) contained significantly lower anthocyanin content and increased flavonoid accumulation compared to WT and OE-miR156a. Notably, overexpression of StSPL9 in potato yielded identical results. The relative expression levels of the anthocyanin-related structural genes PAL, 4CL, CHS, CHI, F3H, DFR, and UFGT in STTM-miR156a transgenic potato tubers showed the opposite trend to that observed in OE-miR156a potato tubers, as demonstrated by RNA sequencing and quantitative real-time PCR (qRT-PCR). The integrated analysis of the transcriptome and metabolome revealed that the accumulation of flavonoids and flavonols in STTM-miR156a was significantly increased than in the wild type, whereas the contents of anthocyanins and phenolic acids were notably reduced. Further analysis showed that the expression of CHS, FG2, BEATH, and CYP75A were up-regulated in STTM-miR156a, and HCT was downregulated, which resulted in a reduced accumulation of methyl 4-caffeoylquinate, a phenolic acid metabolite. Therefore, it is demonstrated that miR156a indirectly modulates the expression of CHS, BEATH, CYP75A, HCT, and FG2 by targeting and degrading its target gene StSPL9 and other SPL genes, thus influencing anthocyanin accumulation in potato tuber. This study provides valuable insights into the complex regulatory network governing anthocyanin biosynthesis in Solanum species.

molecular biology↗

Global expression profile of Enterohemorrhagic Escherichia coli O157:H7 in phagosome of murine macrophages

EHEC O157:H7 is responsible for Hemolytic Uremic Syndrome (HUS) outbreaks in humans and its virulence is associated with Shiga toxin (Stx). During the pathogenesis of HUS, EHEC O157:H7 is phagocytized by the intestinal host macrophage into the phagosome. According to our study, a low percentage of phagocytized bacteria survived in the murine macrophage phagosome. To understand the initials mechanisms involved in bacterial persistence, we performed a global profile of bacterial RNAseq analysis in the early phagosome of murine macrophage and in vitro assays with more extreme stress conditions. The defense strategy of an early murine phagosome consisted on producing DNA damage, membrane damage, acid pH and nitric oxide (NO) agents. These hostile conditions triggering the bacterial SOS response, lipid biosynthesis and NO detoxification enzymes in response to DNA damage, membrane damage and nitric oxide agents, respectively. In turn, to survive, the bacterium conserves energy by downregulating flagellar biosynthesis, T3SS and T6SS virulence mechanisms. In contrast, stx2a expression is upregulated. At the same time, it increases the ribosomal levels and amino acid synthesis to react more effectively against adverse conditions. Under more extreme stress conditions, EHEC O157:H7 expressed genes related to an acidic environment (pH 4.5) upregulating the acid stress response pathways AR2-5, whereas, upon high concentrations of hydrogen peroxide, it transcribed higher levels of genes of the OxyR pathway and subsequently hydrogen peroxide oxidative stress-related genes.

microbiology↗

ORP2 regulates free cholesterol accumulation in hepatocytes during MASH

BackgroundCholesterol crystals in hepatocytes are known to strongly associate with human Metabolic Dysfunction-Associated Steatohepatitis (MASH). However, it remains unclear which molecular pathway(s) regulates free cholesterol accumulation, and therefore the formation of cholesterol crystals in hepatocytes. In cultured cell lines, oxysterol-binding protein-related protein 2 (ORP2) functions to deliver cholesterol to the plasma membrane from internal organelles. MethodsHere, we generated liver-specific ORP2 knockout (ORP2-LKO) mice and characterized their metabolic phenotypes on chow and high fat diet. ResultsThe ORP2-LKO mice developed much more severe hepatic steatosis than wild type mice after high-fat diet feeding. They also demonstrated more severe liver inflammation and damage. Notably, free but not esterified cholesterol, as well as cholesterol crystals accumulated in ORP2-LKO liver. The expression of Cyp7a1 was significantly upregulated in ORP2-LKO liver, accompanied with accumulation of taurocholic acid. Our results thus unveil an important in vivo function of ORP2 in preventing free cholesterol from accumulating in mouse liver. Conclusionsour results suggest that impaired cholesterol trafficking may enhance the deposition of cholesterol crystals in hepatocytes, promoting the development of MASH.

cell biology↗

Structural basis for postfusion-specific binding to Respiratory Syncytial Virus F protein by the canonical antigenic site I antibody 131-2a

The Respiratory Syncytial Virus (RSV) Fusion (F) protein is a major target of antiviral antibodies following natural infection or vaccination and responsible for mediating fusion between the viral envelope and the host membrane. The fusion process is driven by a large-scale conformational change in F, switching irreversibly from the metastable prefusion state to the stable postfusion conformation. Previous research has identified six distinct antigenic sites in RSV-F, termed sites O, I, II, III, IV, and V. Of these, only antigenic site I is fully specific to the postfusion conformation of F. A monoclonal antibody 131-2a that specifically targets postfusion F has been widely used as a research tool to probe for postfusion F and to define antigenic site I in serological studies, yet its sequence and precise epitope have remained unknown. Here we use mass spectrometry-based de novo sequencing of 131-2a to reverse engineer a recombinant product and study the epitope to define antigenic site I with molecular detail, revealing the structural basis for the antibodys specificity towards postfusion RSV-F.

immunology↗

Recessive but damaging alleles of muscle-specific ribosomal protein gene RPL3L drive neonatal dilated cardiomyopathy

The heart employs a specialized ribosome in its muscle cells to translate genetic information into proteins, a fundamental adaptation with an elusive physiological role1-3. Its significance is underscored by the discovery of neonatal patients suffering from often fatal heart failure caused by severe dilated cardiomyopathy when both copies of the gene RPL3L are mutated4-9. RPL3L is a muscle-specific paralog1-3 of the ubiquitous ribosomal protein L3 (RPL3), which makes the closest contact of any protein to the ribosomes RNA-based catalytic center10. RPL3L-linked heart failure represents the only known human disease associated with tissue-specific ribosomes, yet the underlying pathogenetic mechanisms remain poorly understood. Intriguingly, disease is linked to a large number of mostly missense variants in RPL3L, and RPL3L-knockout resulted in no severe heart defect in either human or mice3, 11-13, challenging the prevailing view that autosomal recessive diseases are caused by loss-of-function mutations. Here, we report three new cases of RPL3L-linked severe neonatal heart failure and present a unifying pathogenetic mechanism by which a large number of variants in the muscle-specific ribosome led to disease. Specifically, affected families often carry one of two recurrent toxic gain-of-function variants alongside a family-specific putative loss-of-function variant. While the non-recurrent variants often trigger partial compensation of RPL3 similar to Rpl3l-knockout mice, both recurrent variants exhibit increased affinity for the RPL3/RPL3L chaperone GRWD114-16 and 60S biogenesis factors, sequester 28S rRNA in the nucleus, disrupt ribosome biogenesis, and trigger severe cellular toxicity that extends beyond the loss of ribosomes. These findings provide critical insights for genetic screening and therapeutic development of neonatal heart failure. Our results suggest that gain-of-toxicity mechanisms may be more prevalent in autosomal recessive diseases, and a combination of gain-of-toxicity and loss-of-function mechanisms could underlie many diseases involving genes with paralogs.

molecular biology↗

Dissection of the Global Responses of Mandarin Fish Pyloric Caecum to An Acute Ranavirus (MRV) Infection Reveals the Formation of Serositis and Then Ascites

Mandarin fish ranavirus (MRV), a variant of largemouth bass virus (LMBV), is a distinct member of the genus Ranavirus within the family Iridoviridae. Acute MRV infection predominantly affects the pyloric caecum, a critical visceral organ in mandarin fish, and is hypothesized to drive the characteristic external clinical sign of severe ascites. In this study, we reveal that acute MRV infection initially targets the serosal layer of the pyloric caecum of mandarin fish, leading to rapid progression into fibrinous serositis characterized by serosal hypertrophy, fibrosis, hyperemia, edema, and tissue adhesions. Using single-cell RNA sequencing, we dissect the cellular composition of epithelial, immune, and stromal populations, identifying significant enrichment of macrophages and granulocytes, alongside T and natural killer cells, as key mediators of acute cytokine and inflammatory responses. Then, robust experimental evidence demonstrates that MRV attacks specific immune cell subsets of T and B cells and stromal cells of fibroblasts, myofibroblasts, endothelial cells, and pericytes, resulting in upregulation of genes and pathways associated with extracellular matrix (ECM) formation, collagen biosynthesis, and vascular remodeling in the hyperplastic serosal zone. Additionally, both host-derived type V collagens and MRV-encoded collagens are implicated in ECM formation in the hypertrophic serosa. Collectively, this study provides a comprehensive single-cell resolution analysis of the pyloric caecums response to acute MRV infection and highlights virus- driven serositis as the underlying cause of severe ascites in mandarin fish. ImportanceThe pyloric caecum is a vital digestive and immune organ in many bony fish species, including the mandarin fish, a carnivorous species with an exceptionally developed pyloric caecum comprising 207-326 caeca per individual. While MRV/LMBV infects various fish species, severe ascites is uniquely observed in infected mandarin fish. This study demonstrates that acute MRV infection induces fibrinous serositis in the pyloric caecum, characterized by hyperemia, edema, and hyperplasia, ultimately resulting in ascites and mortality. Leveraging single-cell RNA sequencing, we provide a detailed landscape of the cell types affected or involved in the inflammatory response, revealing their roles in the pathogenesis of serositis. These findings advance our understanding of MRV-induced pathology and its species-specific manifestations.

microbiology↗

Gene Conversion Directed Successive Engineering of Modular Polyketide Synthases

Modular polyketide synthases (PKSs) can produce various secondary metabolites in a collinearity fashion. Although rational engineering of modular PKS can ultimately create a diverse array of novel compounds, de novo generation of defined structures usually results in the loss or remarkable decline of productivity due primarily to the incompatibility of different elements. Here, we present a modular PKS engineering strategy driven by an evolutionary event of gene conversion to accomplish successive engineering of the modular PKS in cinnamomycin biosynthetic gene cluster (cmm BGC). By simulating the gene conversion process, cmm BGC is consecutively reprogrammed to generate a novel macrolide with predicted structural features. Moreover, in contrast to previous notion, the intra-module KS domain is demonstrated to associate with the selectivity of extender units. Collectively, the coordination between evolutionary consequence and functional manipulation of assembly line may shed a new light on modular PKS engineering. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/630704v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@11bd249org.highwire.dtl.DTLVardef@18af1a9org.highwire.dtl.DTLVardef@ffa287org.highwire.dtl.DTLVardef@10756cf_HPS_FORMAT_FIGEXP M_FIG Gene conversion process, typically occurred in KS (purple squares) and AT (blue squares), plays an integral role of structural diversity of polyketides. In this study, an unusual gene conversion was observed in cmm BGC. Subsequently, a homologous BGC was obtained through genome mining by a gene conversion-associated KS domain. Under the direction of gene conversion, the modular PKS in cmm BGC was successively reprogrammed, resulting in de novo biosynthesis of a new-to-nature polyketide. C_FIG

bioengineering↗

Glycerol-3-phosphate activates ChREBP, FGF21 transcription and lipogenesis in Citrin Deficiency

Citrin Deficiency (CD) is caused by inactivation of SLC25A13, a mitochondrial membrane protein required to move electrons from cytosolic NADH to the mitochondrial matrix in hepatocytes. People with CD do not like sweets. We discovered that SLC25A13 loss causes accumulation of glycerol-3-phosphate (G3P), which activates carbohydrate response element binding protein (ChREBP) to transcribe FGF21, which acts in the brain to restrain intake of sweets and alcohol, and to transcribe key genes of de novo lipogenesis. Mouse and human data establish G3P-ChREBP as a new mechanistic component of the Randle Cycle that contributes to metabolic dysfunction-associated steatotic liver disease (MASLD) and forms part of a system that communicates metabolic states from liver to brain in a manner that alters food and alcohol choices. The data provide a framework for understanding FGF21 induction in varied conditions, suggest ways to develop FGF21-inducing drugs, and drug candidates for both lean MASLD and support of urea cycle function in CD.

molecular biology↗

The role of inflammation in a humanized mouse model of transthyretin cardiac amyloidosis

BackgroundSystemic amyloidosis represents a group of protein-misfolding diseases that confer significant morbidity and mortality for millions of patients worldwide. Transthyretin cardiac amyloidosis (ATTR) is a particularly devastating amyloid disease that affects middle-aged and elderly individuals and leads to cardiomyopathy (ATTR-CM), which has a median survival of 2.5 to 3.5 years [1, 2]. ATTR-CM can be hereditary, leading to a more aggressive disease course in younger patients. The most prevalent TTR variant in the United States is V122I, which is found in 3-4 % of African Americans [3]. Despite the significant healthcare burden, ATTR-CM remains underdiagnosed due to a lack of disease awareness and limited diagnostic techniques [4]. Informative in vivo models have proven elusive during the past decade [5]. Moreover, there is no available treatment to reverse cardiac dysfunction due to amyloid fibril deposition [1, 6, 7]. Therefore, a better understanding of the molecular mechanisms of ATTR-CM is imperative to developing novel, effective therapies. Method and ResultsTo explore the pathogenesis of ATTR, we created a murine TTR knockout (TTR-KO) model expressing the human V122I TTR variant. To study the gender differences, both male and female TTR-KO mice were utilized in this study. Significant elevations of human TTR were observed in both male and female ATTR murine plasma post-injection 3 months (human TTR level (ng/ml) Male ATTR: 109.9 {+/-} 5.568; Male control: 28.17 {+/-} 7.010; p=0.0008, N=3 mice/group; Female ATTR: 127.5 {+/-} 32.43; Female control: 20.08 {+/-} 8.351; p=0.0327, N=3 mice/group) with preserved cardiac function (FS% Male ATTR: 26.07 {+/-} 3.667; Male control: 22.69 {+/-} 1.585; p=0.3712, N=6-8 mice/group; Female ATTR: 26.62 {+/-} 1.980; Female control: 31.25 {+/-} 4.482;p=0.3397, N=5-6 mice/group). Notably, the mouse model exhibited cardiac amyloid deposits confirmed by amyloidotic-specific Congo Red staining and Thioflavin T Staining. Transmission electron microscopy revealed both immature and mature amyloid fibrils in the extracellular matrix. RNA-sequencing of the ATTR mouse heart identified distinct transcriptomic patterns and conserved inflammation pathways similar to those seen in a cohort of human ATTR heart samples, including leukocyte transendothelial migration, T-cell receptor signaling, and apoptosis, along with upregulation of inflammatory markers CXCL-1/2/3 and CCL20, were observed in ATTR murine hearts. At the posttranslational level, we confirmed an increased level of CCL5 (MFI ATTR: 801 {+/-} 105; Control: 426{+/-} 64; p=0.0061, N=3 mice/group) in murine plasma post-injection 3 months by a luminance-based immunoassay. The CXCL- and CCL-chemokines family are critical for directing leukocytes to inflammation sites. ConclusionIn this study, we developed a humanized V122I ATTR mouse model with elevated circulating human TTR level and Congophilic amyloid deposits in the murine heart and kidneys. Our transcriptomic study suggested that inflammation may contribute to the ATTR-CM pathogenesis. Further studies are needed to decipher the precise interactions between inflammation and ATTR-CM. Highlights/Whats new/Clinical relevanceO_LIWe developed a humanized mouse model to replicate the multisystem complexity and clinical diversity associated with V122I ATTR-CM. C_LIO_LIOur study unveiled the pathogenic molecular mechanisms of amyloid deposition in ATTR-CM via a novel mouse model. C_LIO_LIWe identified signature inflammatory pathways that uncover potential therapeutic targets for ATTR-CM. C_LIO_LIOur ATTR mouse model allows for preclinical pharmacogenomic assessments of novel therapeutics, which will undoubtedly improve outcomes for ATTR-CM patients. C_LI

pathology↗

Cardiomyocyte STIM1 downregulation exacerbates post-Myocardial Infarction remodeling by dysregulating mitochondrial ultrastructure and metabolic signaling

BackgroundStromal interaction molecule 1 (STIM1) is a key regulator of intracellular calcium (Ca{superscript 2}) homeostasis and mitochondrial function, two tightly coupled processes that critically determine electro-mechanical function and post-myocardial infarction (MI) remodeling. HypothesisWe hypothesized that cardiomyocyte STIM1 deficiency induced by AAV9-mediated knockdown triggers mitochondrial and metabolic remodeling that alters the electrophysiological (EP) substrate and exacerbates post-MI heart failure. MethodsEight-week-old mice received cardiotropic AAV9-mediated delivery of short hairpin RNA targeting STIM1 (AAV9-shSTIM1) or a control construct (AAV9-shLuc/PBS). Four weeks later, in vivo echocardiography followed by ex vivo optical action potential (AP) mapping were performed to determine the impact of myocardial STIM1 downregulation on mechanical and EP properties. Electron microscopy and western blotting were performed to assess mitochondrial network architecture, dynamics and related metabolic signaling. A separate cohort of mice underwent a 30-minute coronary occlusion followed by reperfusion to induce MI. One week post-MI, left ventricular (LV) function, structural remodeling, and EP properties were assessed in mice with and without myocardial STIM1 knockdown. ResultsAAV9-shSTIM1 delivery markedly reduced cardiomyocyte STIM1 expression. STIM1 downregulation induced extensive mitochondrial fragmentation, increased DRP1 phosphorylation at S616 (+55%, p=0.0057), reduced OPA1 expression, and altered AMPK-dependent signaling (reduced phospho-ACC and phospho-Raptor). These mitochondrial and metabolic abnormalities were accompanied by EP instability. In response to in vivo I/R injury, shSTIM1 mice exhibited significantly greater LV dysfunction (-44.3% vs. -12.2% fractional shortening, p<0.0001) and more pronounced structural and EP remodeling with formation of spatially discordant AP alternans 1-week following MI indicative of accelerated heart failure progression. ConclusionsAAV9-mediated STIM1 knockdown exacerbates post-MI cardiac dysfunction likely through impaired mitochondrial dynamics and disrupted metabolic signaling. Beyond worsening post-MI function, STIM1 deficiency alone induces profound EP remodeling, underscoring the pivotal role of cardiomyocyte STIM1 in coordinating mitochondrial homeostasis, electrical stability, and cardiac recovery after injury.

physiology↗

An immunomechanical checkpoint PYK2 governs monocyte-to-macrophage differentiation in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a fibrotic, stiff tumor microenvironment (TME), where tumor-associated macrophages (TAMs) drive ECM remodeling, progression, and immune evasion. The contribution of mechanical cues to monocyte differentiation into TAMs remains largely unexplored. Here we show that mechanical force is required for monocyte-to-macrophage differentiation. PYK2, as an innovative immunomechanical checkpoint, de facto governs this differentiation process. We demonstrated that PYK2 senses mechanical signals via Piezo1 and integrins, triggering F-actin polymerization and translocating to the nucleus to regulate mechanotransduction and differentiation genes (e.g., ACTR3, RELA). Targeted deletion of PYK2 impairs the differentiation and polarization of monocyte-derived macrophages, reshapes the PDAC microenvironment, and enhances the efficacy of anti-PD-1 immunotherapy. These findings underscore the critical role of mechanical cues in monocyte differentiation and suggest that targeting PYK2 is a promising strategy to modulate TAM function and improve immunotherapy outcomes in patients with PDAC. Statement of significanceThis study identifies PYK2 as an immunomechanical checkpoint that drives monocyte-to-macrophage differentiation in PDAC via Piezo1/integrin-mediated mechanical cues. Targeted deletion of PYK2 reshapes the PDAC microenvironment, and enhances the efficacy of anti-PD-1 immunotherapy, suggesting PYK2 as a promising therapeutic target to overcome immunotherapy resistance.

immunology↗

Beta-DIA: Integrating learning-based and function-based feature scores to optimize the proteome profiling of single-shot diaPASEF mass spectrometry data

We present a freely available diaPASEF data analysis software, Beta-DIA, that utilizes deep learning methods to score coelution consistency in retention time-ion mobility dimensions and spectrum similarity. Beta-DIA integrates these learning-based scores with traditional function-based scores, enhancing the qualitative analysis performance. In some low detection datasets, Beta-DIA identifies twice as many protein groups as DIA-NN. The success of Beta-DIA has paved another way for the application of deep learning in fundamental proteome profiling.

bioinformatics↗