Endogenous Pathways Research Articles

Divergent C. elegans toxin alleles are suppressed by distinct mechanisms.

Toxin-antidote elements (TAs) are selfish DNA sequences that bias their transmission to the next generation. TAs typically consist of two linked genes: a toxin and an antidote. The toxin kills progeny that do not inherit the TA, while the antidote counteracts the toxin in progeny that inherit the TA. We previously discovered two TAs in C. elegans that follow the canonical TA model of two linked genes: peel-1/zeel-1 and sup-35/pha-1. Here, we report a new TA that exists in three distinct states across the C. elegans population. The canonical TA, which is found in isolates from the Hawaiian islands, consists of two genes that encode a maternally deposited toxin (MLL-1) and a zygotically expressed antidote (SMLL-1). The toxin induces larval lethality in embryos that do not inherit the antidote gene. A second version of the TA has lost the toxin gene but retains a partially functional antidote. Most C. elegans isolates, including the standard laboratory strain N2, carry a highly divergent allele of the toxin that has retained its activity, but have lost the antidote through pseudogenization. We show that the N2 toxin allele has acquired mutations that enable piRNA binding to initiate MUT-16-dependent 22G small RNA amplification that targets the transcript for degradation. The N2 haplotype represents the first naturally occurring unlinked toxin-antidote system where the toxin is post-transcriptionally suppressed by endogenous small RNA pathways.

The Impact of Alcohol-Induced Epigenetic Modifications in the Treatment of Alcohol use Disorders.

Alcohol use disorders are responsible for 5.9% of all death annually and 5.1% of the global disease burden. It has been suggested that alcohol abuse can modify gene expression through epigenetic processes, namely DNA and histone methylation, histone acetylation, and microRNA expression. The alcohol influence on epigenetic mechanisms leads to molecular adaptation of a wide number of brain circuits, including the hypothalamus-hypophysis-adrenal axis, the prefrontal cortex, the mesolimbic-dopamine pathways and the endogenous opioid pathways. Epigenetic regulation represents an important level of alcohol-induced molecular adaptation in the brain. It has been demonstrated that acute and chronic alcohol exposure can induce opposite modifications in epigenetic mechanisms: acute alcohol exposure increases histone acetylation, decreases histone methylation and inhibits DNA methyltransferase activity, while chronic alcohol exposure induces hypermethylation of DNA. Some studies investigated the chromatin status during the withdrawal period and the craving period and showed that craving was associated with low methylation status, while the withdrawal period was associated with elevated activity of histone deacetylase and decreased histone acetylation. Given the effects exerted by ethanol consumption on epigenetic mechanisms, chromatin structure modifiers, such as histone deacetylase inhibitors and DNA methyltransferase inhibitors, might represent a new potential strategy to treat alcohol use disorder. Further investigations on molecular modifications induced by ethanol might be helpful to develop new therapies for alcoholism and drug addiction targeting epigenetic processes.

Abstract 4125992: Eicosapentaenoic Acid (EPA) Increases Expression of Nrf2-mediated Antioxidant Response Element and Heme Oxygenase-1 in Cytokine-Activated Endothelial Cells

Background: Endothelial cell (EC) nuclear factor erythroid 2-related factor (Nrf2) translocates to the nucleus during inflammation to mediate transcription of genes in the antioxidant response elements (ARE). An ARE regulates mRNA encoding HMOX-1, thioredoxin (TXN), and NAD(P)H quinone oxidoreductase-1 (NQO1), among others. Parkinson disease protein 7 (PARK7) and p62 work independently to prevent Keap1-mediated degradation of Nrf2 during oxidative stress. Eicosapentaenoic acid (EPA) administered as icosapent ethyl reduced CV events (REDUCE-IT). We tested the effects of EPA on ARE protein expression in ECs from multiple tissues during cytokine challenge. Methods: Human brain, pulmonary and vascular ECs were treated with IL-6 (12 ng/mL) for 2 hours and then incubated with EPA (40 µM) for 24 h. Global proteomic analysis performed by LC-MS measured relative protein expression. Significant (p<0.05) changes between treatment groups >1-fold were analyzed by differential enrichment analysis of proteomics data (DEP) and included in gene set enrichment analyses (GSEA). Significantly modulated pathways within the Gene Ontology (GO) database were identified as those with an adjusted-p value <0.05. Results: Proteomic analysis revealed that EPA significantly modulated ≥600 proteins in ECs distinct from IL-6 challenge alone. In EC, EPA significantly modulated the “cellular response to oxidative stress” pathway (GO: 0034599) relative to IL-6 alone. Within this GO pathway, EPA increased levels of catalase (1.2-fold, 1.1-fold, 1.1-fold) and mitochondrial glutathione reductase (1.1-fold, 1.1-fold, 1.1-fold) in brain, pulmonary, and vascular ECs, respectively. We also observed a consistent increase in brain, pulmonary, and vascular ECs of p62 (1.3-fold, 1.2-fold, and 1.2-fold, respectively), and ARE members HMOX-1 (1.5-fold, 1.7-fold, and 2.1-fold, respectively), and NQO1 (1.1-fold, 1.2-fold, and 1.3-fold, respectively). TXN was increased in pulmonary and vascular ECs (1.1-fold and 1.2-fold, respectively) with EPA. Mitochondrial TXN (also called TXN2) increased in brain ECs with EPA 1.1-fold. EPA also significantly increased levels of PARK7 1.1-fold. Conclusions: During inflammation, EPA enhanced expression of cytoprotective proteins in the ARE, notably HMOX-1, along with its regulators, in ECs from multiple tissues. Augmented endogenous antioxidant pathways may contribute to EPA’s clinical benefits.

Regulatory T cells require peripheral CCL2-CCR2 signaling to facilitate the resolution of medication overuse headache-related behavioral sensitization

BackgroundMedication overuse headache (MOH) is the most common secondary headache disorder, resulting from chronic and excessive use of medication to treat headaches, for example, sumatriptan. In a recent study, we have shown that the peripheral C-C motif ligand 2 (CCL2), C-C motif chemokine receptor 2 (CCR2) and calcitonin-gene-related peptide (CGRP) signaling pathways interact with each other and play critical roles in the development of chronic migraine-related behavioral and cellular sensitization. In the present study, we investigated whether CCL2-CCR2 and CGRP signaling pathways play a role in the development of sumatriptan overuse-induced sensitization, and whether they are involved in its resolution by the low-dose interleukin-2 (LD-IL-2) treatment.MethodsMice received daily sumatriptan administration for 12 days. MOH-related behavioral sensitization was assessed by measuring changes of periorbital mechanical thresholds for 3 weeks. CCL2-CCR2 and CGRP signaling pathways were inhibited by targeted gene deletion or with an anti-CCL2 antibody. Ca2+-imaging was used to examine whether repetitive sumatriptan treatment enhances CGRP and pituitary adenylate cyclase–activating polypeptide (PACAP) signaling in trigeminal ganglion (TG) neurons. LD-IL-2 treatment was initiated after the establishment of sumatriptan-induced sensitization. Immunohistochemistry and flow cytometry analyses were used to examine whether CCL2-CCR2 signaling controls regulatory T (Treg) cell proliferation and/or trafficking.ResultsCCL2, CCR2 and CGRPα global KO mice exhibited robust sumatriptan-induced behavioral sensitization comparable to wild-type controls. Antibody neutralization of peripheral CCL2 did not affect sumatriptan-induced behaviors either. Repeated sumatriptan administration did not enhance the strength of CGRP or PACAP signaling in TG neurons. Nevertheless, LD-IL-2 treatment, which facilitated the resolution of sumatriptan-induced sensitization in wild-type and CGRPα KO mice, was completely ineffective in mice with compromised CCL2-CCR2 signaling. In CCL2 KO mice, we observed normal LD-IL-2-induced Treg expansion in peripheral blood, but the increase of Treg cells in dura and TG tissues was significantly reduced in LD-IL-2-treated CCL2 KO mice relative to wild-type controls.ConclusionsThese results indicate that the endogenous CCL2-CCR2 and CGRP signaling pathways are not involved in sumatriptan-induced behavioral sensitization, suggesting that distinct molecular mechanisms underlie chronic migraine and MOH. On the other hand, peripheral CCL2-CCR2 signaling is required for LD-IL-2 to reverse chronic headache-related sensitization.Graphical abstract

A conserved two-component system senses intracellular iron levels and regulates redox balance in Mycobacterium spp.

For bacteria, an intricate coordination between sensing and regulating iron levels and managing oxidative stress is required as their levels are tightly interlinked. While various oxidative stress and heme-based redox sensors have been reported for both pathogenic and non-pathogenic bacteria, the mechanisms governing the modulation of intracellular iron levels in response to changes in redox status remain unclear. In this study, a gene-inactivated strain of mycobacterial sensor kinase PdtaS showed dysregulated expression of genes associated with iron metabolism, including Fe-S clusters, NADH dehydrogenases, and iron uptake. The strain showed poor growth in nutrient-limiting conditions, a defect rescuable by heme but not by Fe3+ supplementation. This observation was associated with the PAS domain of the PdtaS sensor kinase. Biochemical and biophysical experiments established heme-binding to the PAS domain and its inhibitory effect on PdtaS auto-kinase activity, suggesting that the absence of heme induces activation of this sensor kinase. Interestingly, despite having an endogenous heme biosynthetic pathway or even external heme supplementation, the ∆pdtaS mutant exhibited persistent low intracellular iron levels concomitant with elevated oxidative stress. Antioxidant supplementation mitigated growth defects, emphasizing the link between oxidative stress, intracellular iron levels, and PdtaS activity. RNA-IP identified key targets associated with redox homeostasis and iron metabolism as targets of the PdtaR response regulator. The study proposes a novel role for the PdtaS-PdtaR TCS in sensing heme, regulation of intracellular iron levels, and redox balance.IMPORTANCEThe research article investigates the intricate interplay between bacteria's ability to take and utilize iron without inducing excess iron's toxic effects, including oxidative stress. The study shows that bacteria achieve this by sensing intracellular iron available as heme through a sensory protein PdtaS, which turns off when heme is in excess and prevents iron uptake and iron efflux. The process shields bacteria from generating Fe-dependent free radicals and allows it to maintain viability. The absence of sensor kinase abrogates all these processes, increasing bacteria susceptibility to ROS and thereby slowing growth. This feature of the sensor kinase PdtaS makes it an attractive co-therapeutic target for tuberculosis therapy, where its inhibition will prevent iron uptake, even in the presence of low iron, thereby halting bacterial proliferation.

The angiogenic role of the alpha 9-nicotinic acetylcholine receptor in triple-negative breast cancers.

Nicotine acts as an angiogenic factor by stimulating endogenous cholinergic pathways. Several subtypes of nicotinic acetylcholine receptors (nAChRs) have been demonstrated to be closely correlated to the formation and progression of different types of cancers. Recently, several studies have found that nicotinic acetylcholine receptors α9 (α9-nAChRs) are highly expressed in breast tumors, especially in tumors derived from patients diagnosed at advanced stages. In vitro studies have demonstrated that activation of α9-nAChRs is associated with increased proliferation and migration of breast cancer. To study the tumor-promoting role of α9-nAChRs in breast cancers, we generated a novel anti-α9-nAChR and methoxy-polyethylene glycol (mPEG) bispecific antibody (α9 BsAb) for dissecting the molecular mechanism on α9-nAChR-mediated tumor progression. Unexpectedly, we discovered the angiogenic role of α9-nAChR in nicotine-induced neovascularization of tumors. It revealed α9 BsAbs reduced nicotine-induced endothelial cell tube formation, blood vessel development in Matrigel plug assay and angiogenesis in microtube array membrane murine model (MTAMs). To unbraid the molecular mechanism of α9-nAChR in nicotine-mediated angiogenesis, the α9 BsAbs were applied and revealed the inhibitory roles in nicotine-induced production of hypoxia-inducible factor-2 alpha (HIF-2α), vascular endothelial growth factor A (VEGF-A), phosphorylated vascular endothelial growth factor receptor 2 (p-VEGFR2), vascular endothelial growth factor receptor 2 (VEGFR2) and matrix metalloproteinase-9 (MMP9) from triple-negative breast cancer cells (MDA-MB-231), suggesting α9-nAChRs played an important role in nicotine-induced angiogenesis. To confirm our results, the shRNA targeting α9-nAChRs was designed and used to silence α9-nAChR expression and then evaluated the angiogenic role of α9-nAChRs. The results showed α9 shRNA also played an inhibitory effect in blocking the nicotine-induced angiogenic signaling. Taken together, α9-nAChR played a critical role in nicotine-induced angiogenesis and this bispecific antibody (α9 BsAb) may serve as a potential therapeutic candidate for treatments of the α9 positive cancers.

A major role of class III HD-ZIPs in promoting sugar beet cyst nematode parasitism in Arabidopsis.

Cyst nematodes use a stylet to secrete CLE-like peptide effector mimics into selected root cells of their host plants to hijack endogenous plant CLE signaling pathways for feeding site (syncytium) formation. Here, we identified ATHB8, encoding a HD-ZIP III family transcription factor, as a downstream component of the CLE signaling pathway in syncytium formation. ATHB8 is expressed in the early stages of syncytium initiation, and then transitions to neighboring cells of the syncytium as it expands; an expression pattern coincident with auxin response at the infection site. Conversely, MIR165a, which expresses in endodermal cells and moves into the vasculature to suppress HD-ZIP III TFs, is down-regulated near the infection site. Knocking down HD-ZIP III TFs by inducible over-expression of MIR165a in Arabidopsis dramatically reduced female development of the sugar beet cyst nematode (Heterodera schachtii). HD-ZIP III TFs are known to function downstream of auxin to promote cellular quiescence and define stem cell organizer cells in vascular patterning. Taken together, our results suggest that HD-ZIP III TFs function together with a CLE and auxin signaling network to promote syncytium formation, possibly by inducing root cells into a quiescent status and priming them for initial syncytial cell establishment and/or subsequent cellular incorporation.

Mechanisms underlying mitochondrial dysfunction and intestinal damage induced by ingestion of microplastics in Leuciscus waleckii: The role of the NF-κB/Nrf2 signaling pathway

This study investigates the impact of polystyrene microplastics (PS-MPs) on the growth, immunity, oxidative stress, mitochondrial function, and intestinal health of Leuciscus waleckii (3.00±0.02 g) juveniles over 8 weeks. Our findings indicate that exposure to PS-MPs negatively affected the growth of Leuciscus waleckii, resulting in digestive disturbances. Furthermore, PS-MPs triggered immune dysfunction and oxidative stress through the activation of the NF-κB pathway and suppression of the Nrf2 signaling cascade. PS-MPs damaged intestinal tissue and compromised the intestinal barrier. Additionally, mitochondrial homeostasis was disrupted, activating endogenous mitochondrial apoptotic pathways, ultimately leading to cell apoptosis. High-throughput 16S rRNA sequencing disclosed that PS-MPs provoked shifts in the intestinal microbiota. Mantel analysis indicates significant correlations between growth parameters and the activities of enzymes related to antioxidant defense, immunity, apoptosis, and mitochondria, as well as with the gut microbiota. In summary, our study reveals that PS-MPs induce intestinal inflammation and oxidative stress in Leuciscus waleckii by activating the NF-κB pathway and the intrinsic mitochondrial apoptotic pathway while repressing the Nrf2 signaling, ultimately leading to cellular apoptosis, mitochondrial dysfunction, intestinal microbiota imbalance, and growth inhibition.

The Effect of Time-Restricted Eating on Cardiometabolic Risk Factors: A Systematic Review and Meta-Analysis.

Background/Objectives: Endogenous metabolic pathways periodically adjust with fluctuations in day and night, a biological process known as circadian rhythm. Time-restricted eating (TRE) aligns the time of food intake with the circadian rhythm. This study aims to investigate the effects of TRE on body weight, body composition and cardiometabolic risk factors. Methods: We reviewed articles from PubMed and Cochrane libraries for clinical trials that compare TRE with regular diet without calorie restriction. We conducted a meta-analysis of 26 studies. Results: Participants who followed TRE demonstrated reduction in body weight [mean-MD: -1.622 kg, (95% confidence interval (CI -2.302 to -0.941)], body mass index (BMI) [MD: -0.919 kg/m2 (95% CI: -1.189 to -0.650)], waist circumference [MD: -2.015 cm (95% CI: -3.212 to -0.819] and whole-body fat mass (WBFM) [MD: -0.662 kg (95% CI: -0.795 to -0.530)]. Improvements in cardiometabolic risk factors such as a decrease in insulin concentrations [MD: -0.458 mIU/L, (95% CI: -0.843 to -0.073)], total cholesterol [MD: -2.889 mg/dL (95% CI: -5.447 to -0.330) and LDL concentrations [MD: -2.717 mg/dL (95% CI: -4.412 to -1.021)] were observed. Conclusions: TRE is beneficial for weight loss and improvements in cardiometabolic risk factors. Further large-scale clinical trials are needed to confirm these findings.

Inflammation and resolution in obesity.

Inflammation is an essential physiological defence mechanism, but prolonged or excessive inflammation can cause disease. Indeed, unresolved systemic and adipose tissue inflammation drives obesity-related cardiovascular diseaseand type 2 diabetes mellitus. Drugs targeting pro-inflammatory cytokine pathways or inflammasome activation have been approved for clinical use for the past two decades. However, potentially serious adverse effects, such as drug-induced weight gain and increased susceptibility to infections, prevented their wider clinical implementation. Furthermore, these drugs do not modulate the resolution phase of inflammation. This phase is an active process orchestrated by specialized pro-resolving mediators, such as lipoxins, and other endogenous resolution mechanisms. Pro-resolving mediators mitigate inflammation and development of obesity-related disease, for instance, alleviating insulin resistance and atherosclerosis in experimental disease models, so mechanisms to modulate their activity are, therefore, of great therapeutic interest. Here, we review current clinical attempts to either target pro-inflammatory mediators (IL-1β,NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3)inflammasome,tumour necrosis factor (TNF)and IL-6) or utilize endogenous resolution pathways to reduce obesity-related inflammation and improve cardiometabolic outcomes. A remaining challenge in the field is to establish more precise biomarkers that can differentiate between acute and chronic inflammation and to assess the functionality of individual leukocyte populations. Such advancements would improve the monitoring of drug effects and support personalized treatment strategies that battle obesity-related inflammation and cardiometabolic disease.

CRISPR-based molecule-regulatory expression platform for specific immunotherapy of cancer.

Cancer is still a major challenge of human health. The abnormality of intracellular cancer-related signal pathways is an important mechanism for the occurrence of cancer. We used a molecular-senor to act on the endogenous signal molecules within the cell to redirect the abnormal signal flows in the cell to treat cancer. Based on CRISPR-dCas12f procedures, we combined aptamers and ribozymes to construct riboswitches, which served as molecular switches to reprogram sgRNAs, so that CRISPR-dCas12f redirected the intracellular anti-cancer signal flows after sensing specific input signal molecules. In addition, the activated molecular sensors and the inhibitory molecular sensors were constructed by combining transcription factors (VP64) and transcription inhibitors (KRAB) to specifically activate and inhibit target genes of interest. Our experimental results showed that the molecular sensors that we designed and constructed specifically sensed the endogenous signal molecules and then redirect the cancer related signal networks of cancer cells. In addition, corresponding logic gates were constructed to distinguish cancer cells from normal cells and redirect anticancer signal flows to trigger specific cancer immunotherapy. The constructed molecular sensors constructed specifically recognized the signal molecules within the cell and redirected the endogenous signal pathway to reprogram the fate of cancer cells.

Treating Alzheimer's Disease: Focusing on Neurodegenerative Consequences.

Neurodegenerative disorders involve progressive dysfunction and loss of synapses and neurons and brain atrophy, slowly declining memories and cognitive skills, throughout a long process. Alzheimer's disease (AD), the leading neurodegenerative disorder, suffers from a lack of effective therapeutic drugs. Decades of efforts targeting its pathologic hallmarks, amyloid plaques and neurofibrillary tangles, in clinical trials have produced therapeutics with marginal benefits that lack meaningful clinical improvements in cognition. Delivering meaningful clinical therapeutics to treat or prevent neurodegenerative disorders thus remains a great challenge to scientists and clinicians. Emerging evidence, however, suggests that dysfunction of various synaptogenic signaling pathways participates in the neurodegenerative progression, resulting in deterioration of operation/structure of the synaptic networks involved in cognition. These derailed endogenous signaling pathways and disease processes are potential pharmacological targets for the therapies. Therapeutics with meaningful clinical benefit in cognition may depend on the effectiveness of arresting and reversing the neurodegenerative process through these targets. In essence, promoting neuro-regeneration may represent the only option to recover degenerated synapses and neurons. These potential directions in clinical trials for AD therapeutics with meaningful clinical benefit in cognitive function are summarized and discussed.

Methylarginine targeting chimeras for lysosomal degradation of intracellular proteins.

A paradigm shift in drug development is the discovery of small molecules that harness the ubiquitin-proteasomal pathway to eliminate pathogenic proteins. Here we provide a modality for targeted protein degradation in lysosomes. We exploit an endogenous lysosomal pathway whereby protein arginine methyltransferases (PRMTs) initiate substrate degradation via arginine methylation. We developed a heterobifunctional small molecule, methylarginine targeting chimera (MrTAC), that recruits PRMT1 to a target protein for induced degradation in lysosomes. MrTAC compounds degraded substrates across cell lines, timescales and doses. MrTAC degradation required target protein methylation for subsequent lysosomal delivery via microautophagy. A library of MrTAC molecules exemplified the generality of MrTAC to degrade known targets and neo-substrates-glycogen synthase kinase 3β, MYC, bromodomain-containing protein 4 and histone deacetylase 6. MrTAC selectively degraded target proteins and drove biological loss-of-function phenotypes in survival, transcription and proliferation. Collectively, MrTAC demonstrates the utility of endogenous lysosomal proteolysis in the generation of a new class of small molecule degraders.

Identification and Characterization of a Novel Insulin-like Receptor (LvRTK2) Involved in Regulating Growth and Glucose Metabolism of the Pacific White Shrimp Litopenaeus vannamei.

The insulin receptor (IR) plays a crucial role in the growth and metabolism of animals. However, there are still many questions regarding the IR in crustaceans, particularly their role in shrimp growth and glucose metabolism. In this study, we identified a novel insulin-like receptor gene in Litopenaeus vannamei and cloned its full length of 6439 bp. This gene exhibited a highly conserved sequence and structural characteristics. Phylogenetic analysis confirmed it as an unreported RTK2-type IR, namely, LvRTK2. Expression pattern analysis showed that LvRTK2 is primarily expressed in female reproductive and digestive organs. Through a series of in vivo and in vitro experiments, including glucose treatment, exogenous insulin treatment, and starvation treatment, LvRTK2 was confirmed to be involved in the endogenous glucose metabolic pathway of shrimp under different glucose variations. Moreover, long-term and short-term interference experiments with LvRTK2 revealed that the interference significantly reduced the shrimp growth rate and serum glucose clearance rate. Further studies indicated that LvRTK2 may regulate shrimp growth by modulating the downstream PI3K/AKT signaling pathway and a series of glucose metabolism events, such as glycolysis, gluconeogenesis, glycogen synthesis, and glycogenolysis. This report on the characteristics and functions of LvRTK2 confirms the important role of RTK2-type IRs in regulating shrimp growth and glucose metabolism.

Kukoamine A alleviates nephrolithiasis by inhibiting endogenous oxalate synthesis via the IL-6/JAK/STAT3/DAO signaling pathway

BackgroundThe recurrent nature and socioeconomic burden of nephrolithiasis demand effective treatments. Delineating the crosstalk between inflammatory processes and the endogenous oxalate metabolism pathway, which underpins nephrolithiasis pathogenesis, is essential for advancing treatment strategies. PurposeWe aim to screen therapeutic Chinese herbal remedies and their bioactive constituents for kidney stone treatment using a fruit fly model, followed by efficacy and mechanism validation in a rodent nephrolithiasis model as well as in vitro human cell culture model. Study design and methodsWe developed a fruit fly model to screen for efficient traditional Chinese medicine herbs and their active compounds for kidney stone treatment. Candidate active compounds from efficient herbs were separated and identified by solid-phase chromatography coupled with LC-MS analysis. Fruit fly genetic tools were employed to manipulate the expression of related genes to explore the therapeutic mechanisms of the Lycii Cortex and kukoamine A (KuA). To confirm the therapeutic effects and mechanisms of KuA for mammalian nephrolithiasis, mouse model of glyoxylate-induced kidney stone and human renal tubular cells were used. The therapeutic role of kukoamine A in nephrolithiasis was evaluated by assessing tubular injury, crystal deposition, and adhesion. The level of expression and phosphorylation in cells and mice was assessed using RT-qPCR and western blot. ResultsOur findings indicate that Lycii Cortex potently inhibits calcium oxalate stone formation via activation of the JNK/Upd3/JAK/STAT signaling cascade, resulting in diminished endogenous oxalate synthesis by downregulating D-amino acid oxidase (DAO) gene expression, predominantly in fruit fly Malpighian tube stellate cells. KuA was identified as the principal bioactive constituent mediating these effects. Both mouse models and human cell assays confirmed KuA's efficacy in preventing calcium oxalate nephrolithiasis in mammals, through hepatic JAK/STAT3 pathway activation and upregulation of IL-6, culminating in reduced urinary crystal deposition. ConclusionOur research underscores the potential of kukoamine A as a lead compound in treating nephrolithiasis and reveals the interplay between the IL-6/JAK/STAT3 inflammatory pathway and endogenous oxalate metabolism in nephrolithiasis pathogenesis. Additionally, it highlights the utility of the fruit fly model as a powerful tool for deciphering the therapeutic mechanisms of traditional Chinese herbs.

High-resolution and programmable RNA-IN and RNA-OUT genetic circuit in living mammalian cells

RNAs and their encoded proteins intricately regulate diverse cell types and states within the human body. Dysregulated RNA expressions or mutations can lead to various diseased cell states, including tumorigenesis. Detecting and manipulating these endogenous RNAs offers significant promise for restoring healthy cell states and targeting tumors both in research and clinical contexts. This study presents an RNA-IN and RNA-OUT genetic circuit capable dynamically sensing and manipulating any RNA target in a programmable manner. The RNA-IN module employes a programmable CRISPR-associated protease (CASP) complex for RNA detection, while the RNA-OUT module utilizes an engineered protease-responsive dCas9-VPR activator. Additionally, the CASP module can detect point mutations by harnessing an uncovered dual-nucleotide synergistic switching effect within the CASP complex, resulting in the amplification of point-mutation signals from initially undetectable levels (1.5-fold) to a remarkable 94-fold. We successfully showcase the circuit’s ability to rewire endogenous RNA-IN signals to activate endogenous progesterone biosynthesis pathway, dynamically monitor adipogenic differentiation of mesenchymal stem cells (MSCs) and the epithelial-to-mesenchmal trans-differentiation, as well as selective killing of tumor cells. The programmable RNA-IN and RNA-OUT circuit exhibits tremendous potential for applications in gene therapy, biosensing and design of synthetic regulatory networks.

Multifunctional injectable oxidized sodium alginate/carboxymethyl chitosan hydrogel for rapid hemostasis

Uncontrolled bleeding from incompressible or irregularly shaped wounds is a major factor in the death of people in the battlefield or surgery process. Ideal rapid hemostatic materials should have the performance of rapid hemostasis and at the same time can be applied to a variety of complex wound trauma types, in addition, excellent antimicrobial properties, adhesion, biocompatibility, degradation, and the non-toxicity of degradation products are also necessary, but there are fewer hemostatic materials that meet these requirements. Herein, we prepared an injectable hemostatic hydrogel based on the natural products sodium alginate (SA) and carboxymethyl chitosan (CMC). Oxidized sodium alginate (OSA) was prepared by the oxidation reaction of NaIO4 with SA, and OSA with aldehyde group was mixed with CMC with amino group to rapidly form an in situ injectable hemostatic hydrogel (OSA/CMC) by the Schiff base reaction. OSA/CMC hydrogel exhibited excellent antimicrobial and adhesion properties by the Schiff base reaction. In addition, OSA/CMC hydrogel directly activate the endogenous coagulation pathway through the synergistic effect of OSA, CMC to enhance the hemostatic effect. The results of in vivo hemostasis study showed that OSA/CMC hydrogel significantly accelerated hemostasis and reduced blood loss in liver hemorrhage model and tail amputation model. Therefore, OSA/CMC hydrogel is expected to be a potential material in the direction of rapid clinical hemostasis due to its good adhesion properties, antimicrobial properties, biocompatibility, blood compatibility, and efficient rapid hemostasis.

AKR7A5 knockout promote acute liver injury by inducing inflammatory response, oxidative stress and apoptosis in mice.

Alcohol liver disease has become a worldwide critical health problem. The ingested alcohol could be converted into acetaldehyde or combined with free fatty acids to induce the endoplasmic reticulum oxidative stress in the liver. Coincidentally, AKR7A5 has both aldehyde detoxification and antioxidant effects. Therefore, we discuss the possible role and mechanism of AKR7A5 in the acute alcohol injury of mice liver. There were four experiment groups, the C57BL/6 mice of wild-type mice (WT) or AKR7A5-/- mice (KO) were intragastrically administrated with saline or 50% ethanol at 14 mL/kg, respectively. Compared to the WT + alcohol group, abnormal liver function, disordered hepatic cord, severe congestion in the hepatic sinus and the space of the hepatic cord, occurrence of oxidative stress, DNA damage and different expressions of apoptosis-related proteins were detected in the KO + alcohol group. Meanwhile, the biological process enrichment analysis showed that the down-regulated proteins were related to the metabolism of fatty acid, the up-regulated proteins positive regulation of reactive oxygen species metabolic process, negative regulation of coagulation and haemostasis. In conclusion, single ethanol binge combined with the absence of AKR7A5 caused more severe inflammatory response, oxidative stress, apoptosis of endogenous pathways, abnormal lipids metabolism and disordered coagulation in mice liver.

One-pot bioconversion of lignin to 4-vinylphenol derivatives

Lignin, the largest renewable aromatic resource in nature, is a promising feedstock to produce aromatic fine chemicals. The bioconversion of lignin into 4-vinylphenol derivatives (4VPs) is a sustainable route to promote both lignin valorization and bioeconomy. However, challenges such as heterologous lignin and an inefficient synthetic pathway for 4VPs restrict the bioconversion performance. In this work, microbial cell factories were designed to valorize lignin into 4VPs, emphasizing an atom-economic route. Rewriting endogenous pathways and heterogeneously expressing phenolic acid decarboxylases successfully produced 4VPs from lignin-derived aromatic precursors. Enzymatic kinetics revealed that the screened phenolic acid decarboxylases possessed outstanding catalytic activity against lignin derivatives. A biphasic glyceryl tributyrate/water system was developed for efficient in situ extraction of 4VPs, successfully removing the product-feedback inhibition effect. Accordingly, the microbial cell factory produced the record titers of 4-vinylphenol, 4-vinylguaiacol and 4-vinylsyringol at 235.3, 203.0 and 35.8 g/L, respectively, with the productivity of 3.0, 2.7, and 0.8 g/L/h. The microbial cell factory was further engineered to simultaneously convert three-type aromatic of lignin into 4VPs through a one-pot bioconversion. The microbial cell factory also exhibited the remarkable bioconversion capacity of actual lignin hydrolysate by producing 12.4 g/L and 2.3 g/L of 4-vinylphenol and 4-vinylguaiacol, respectively. The molecular mechanism suggested that corresponding polymers were successfully produced and displayed good thermal stability. Overall, the microbial cell factory demonstrated an impressive capacity to convert lignin derivatives and achieve record titers of 4VPs, showcasing its potential to revolutionize lignin valorization and the production of functional polystyrene materials.

Preclinical Development of a Genetically Engineered Albumin‐Binding Nanoparticle of Paclitaxel

Nab‐paclitaxel (Abraxane), an albumin‐bound solvent‐free paclitaxel (PTX) formulation that takes advantage of the endogenous albumin transport pathway, is the current gold standard for treatment of solid tumors with PTX. However, nab‐paclitaxel has several limitations, including complex manufacturing, immunogenicity, slow drug‐release, and a narrow therapeutic window. Nevertheless, no other PTX formulation has gained the Food and Drug Administration approval since Abraxane's 18‐year reign. Addressing these concerns, herein, a PTX‐loaded nanoparticle of a recombinant polypeptide that—like nab‐paclitaxel—capitalizes on the long in vivo half‐life of albumin is reported. This genetically engineered nanoparticle packages PTX in the core of the nanoparticle and displays an albumin‐binding domain on the exterior of the nanoparticle. Upon in vivo administration, the drug‐loaded nanoparticle binds albumin with nanomolar affinity, and acquires an albumin‐corona, which eliminates the need to use exogenous albumin. The nanoparticles can be stored at subzero temperature as lyophilized powder without any cryoprotectants for upto a year and can be reconstituted on‐demand in aqueous buffer at high concentration, thus greatly simplifying formulation processes. These albumin‐binding nanoparticles improve the therapeutic window by at least twofold compared to nonalbumin‐binding counterpart and outperform nab‐paclitaxel in multiple murine tumor models, results that have been independently replicated by a contract research organization.