RAGE mediates hippocampal pericyte responses and neurovascular unit lesions after TBI.

Traumatic brain injury impairs brain function through various mechanisms. Recent studies have shown that alterations in pericytes in various diseases affect neurovascular function, but the effects of TBI on hippocampal pericytes remain unclear. Here, we investigated the effects of RAGE activation on pericytes after TBI using male C57BL/6 J mice. Hippocampal samples were collected at different time points within 7 days after TBI, the expression of PDGFR-ß, NG2 and the HMGB1-S100B/RAGE signaling pathway was assessed by Western blotting, and the integrity of the hippocampal BBB at different time points was measured by immunofluorescence. RAGE-associated BBB damage in hippocampal pericytes occurred early after cortical impact. By culturing primary mouse brain microvascular pericytes, we determined the different effects of HMGB1-S100B on pericyte RAGE. To investigate whether RAGE blockade could protect neurological function after TBI, we reproduced the process of CCI by administering FPS-ZM1 to RAGE-/- mice. TEM images and BBB damage-related assays showed that inhibition of RAGE resulted in a significant improvement in the number of hippocampal vascular basement membranes and tight junctions and a reduction in perivascular oedema compared with those in the untreated group. In contrast, mouse behavioural testing and doublecortin staining indicated that targeting the HMGB1-S100B/RAGE axis after CCI could protect neurological function by reducing pericyte-associated BBB damage. In conclusion, the present study provides experimental evidence for the strong correlation between the pericyte HMGB1-S100B/RAGE axis and NVU damage in the hippocampus at the early stage of TBI and further demonstrates that pericyte RAGE serves as an important target for the protection of neurological function after TBI.

MeJA-induced hairy roots in Plumbago auriculata L. by RNA-seq profiling and key synthase provided new insights into the sustainable production of plumbagin and saponins.

Naturally synthesized secondary metabolites in plants are considered an important source of drugs, food additives, etc. Among them, research on natural plant medicinal components and their synthesis mechanisms has always been of high concern. We identified a novel medicinal floral crop, Plumbago auriculata L., that can be treated with methyl jasmonate (MeJA) for the rapid or sustainable production of natural bioactives from hairy roots. In the study, we globally analyzed the changes in the accumulation of plumbagin and others in the hairy roots of Plumbago auriculata L. hairy roots (PAHR) 15834 in P. auriculata L. based on 100 µmol/L of MeJA treatment by RNA-seq profiling, and we found that there was a significant increase in the accumulation of plumbagin and saponin before 24 h. To explain the principle of co-accumulation, it showed that MeJA induced JA signaling and the shikimic acid pathway, and the methylvaleric acid (MVA) pathway was activated downstream subsequently by the Mfuzz and weighted gene co-expression analysis. Under the shared metabolic pathway, the high expression of PAL3 and HMGR promoted the activity of the "gateway enzymes" phenylalanine ammonia lyase (PAL) and 3-hydroxy-3-methylglutaryl CoA reductase (HMGR), which respectively induced the high expression of key reaction enzyme genes, including chalcone synthase (CHS), isopentenyl diphosphate (IPP), and farnesyl pyrophosphate synthase (FPS), that led to the synthesis of plumbagin and saponin. We speculated that large amounts of ketones and/or aldehydes were formed under the action of these characteristic enzymes, ultimately achieving their co-accumulation through polyketone and high-level sugar and amino acid metabolism. The study results provided a theoretical basis for carrying out the factory refinement and biosynthesis of plumbagin and saponins and also provided new ideas for fully exploiting multifunctional agricultural crops and plants and developing new agricultural by-products.

Engineering a Self-Assembled Protein Cage for Targeted Dual Functionalization.

Self-assembled protein cages are attractive scaffolds for organizing various proteins of interest (POIs) toward applications in synthetic biology and medical science. However, specifically attaching multiple POIs to a single protein cage remains challenging, resulting in diversity among the functionalized particles. Here, we present the engineering of a self-assembled protein cage, DTMi3ST, capable of independently recruiting two different POIs using SpyCatcher (SC)/SpyTag (ST) and DogCatcher (DC)/DogTag (DT) chemistries, thereby reducing variability between assemblies. Using fluorescent proteins as models, we demonstrate controlled targeting of two different POIs onto DTMi3ST protein cages both in vitro and inside living cells. Furthermore, dual functionalization of the DTMi3ST protein cage with a membrane-targeting peptide and ß-galactosidase resulted in the construction of membrane-bound enzyme assemblies in Escherichia coli, leading to a 69.6% enhancement in substrate utilization across the membrane. This versatile protein cage platform provides dual functional nanotools for biological and biomedical applications.

Enriched environment treatment promotes neurofunctional recovery by regulating the ALK5/Smad2/3/Gadd45ß signaling pathway in rats with cerebral ischemia /reperfusion injury.

It has been demonstrated that an enriched environment (EE) treatment can alter neuroplasticity in neurodegenerative diseases. However, the role of EE treatment in ischemic stroke remains unclear. Previous findings have revealed that EE treatment can promote cerebral activin-receptor-like-kinase-5 (ALK5) expression after cerebral ischemia/reperfusion (I/R) injury. ALK5 has been identified as a potential mediator of neuroplasticity through its modulation of Smad2/3 and Gadd45ß. Therefore, the aim of this study was to investigate whether EE treatment could promote neurofunctional recovery by regulating the ALK5/Smad2/3/Gadd45ß pathway. The study utilized the rat model of middle cerebral artery occlusion/reperfusion (MCAO/R). The ALK5/Smad2/3/Gadd45ß signaling pathway changes were evaluated using western blotting (WB). Brain injury was assessed by infarct volume and neurobehavioral scores. The effect of EE treatment on neurogenesis was evaluated using Doublecortin (DCX) and Nestin, axonal plasticity with biotinylated dextran amine (BDA) nerve tracing, and dendritic plasticity was assessed using Golgi-Cox staining. EE treatment has been demonstrated to modulate the Smad2/3/Gadd45ß pathway by regulating the expression of ALK5. The protective effects of EE treatment on brain infarct volume, neurological function, newborn neurons, dendritic and axonal plasticity following cerebral I/R injury were counteracted by ALK5 silencing. EE treatment can enhance neurofunctional recovery after cerebral I/R injury, which is achieved by regulating the ALK5/Smad2/3/Gadd45ß signaling pathway to promote neuroplasticity.

Covalent organic frameworks-based materials for antibiotics fluorescence detection.

Antibiotics play a vital role in safeguarding people's health since most bacterial infection can be efficiently controlled and cured by treating with suitable antibiotics. However, excessive use of antibiotics in husbandry and aquaculture leaded to the pollution of eco-environment. Thus, it is important to develop simple facile methods and effective functional materials for quick on-site analysis of antibiotics. Covalent organic frameworks (COFs), as a kind of porous crystalline covalent bond linked polymers, have demonstrated its power in multiple fields. Herein, we will discuss COFs-based materials utilized as antibiotics sensors with fluorescence method. For each sensor, we will mainly discuss the mechanism for antibiotics recognition, the preparation, characterization and fluorescence sensing performance of specific antibiotics. The mechanism to illustrate the interaction between sensors and antibiotics analytes would also be stressed.

The mind wanders to dark places: Mind-wandering catalyzes rumination in the context of negative affect and impulsivity.

Spontaneous mind-wandering has been theorized to increase susceptibility for rumination, contributing to risk for major depressive disorder (MDD). Clarifying whether-and under what circumstances-mind-wandering leads to rumination could inform the development of targeted interventions to reduce risk for ruminative sequelae. Using intensively sampled data in 44 young adults with remitted MDD and 38 healthy volunteers with 1,558 total observations collected from 2018 to 2022, we conducted multilevel models to investigate temporal relationships between mind-wandering and rumination. Contextual factors (e.g., intensity of negative affect; momentary impulsivity) and individual factors (e.g., MDD history) were examined as moderators of these relationships. Mind-wandering predicted increased rumination, whereas rumination did not predict increased mind-wandering. When individuals experienced greater negative affect or acted more impulsively compared to their usual levels, they showed a stronger relationship between mind-wandering and subsequent rumination. Depression history did not significantly moderate temporal relationships between mind-wandering and rumination. Spontaneous mind-wandering may transition into rumination, particularly during moments when people experience more negative affect or impulsivity compared to usual. Delivering interventions in these moments could reduce risk for ruminative sequelae. The tendency to ruminate in response to mind-wandering is suggested to be consistent regardless of depression history, suggesting the transdiagnostic and dimensional nature of rumination as a possible consequence of mind-wandering. Future work is needed to determine whether these associations are generalizable across the lifespan. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

Optical-resolution photoacoustic microelastography system for elasticity mapping: Phantom study and practical application.

Elastography is a noninvasive technique for characterizing the mechanical properties of biological tissues. Conventional methods have limitations in resolution and sensitivity, hindering disease detection in clinical diagnostics. To address these issues, this study developed an optical-resolution photoacoustic microelastography (OR-PAME) system. Using an agar tissue phantom with varying agar concentrations and contrast agents, PAME evaluated elasticity distribution under compression in both lateral and axial dimensions. It indirectly measured elastic properties by correlating photoacoustic responses, temporal lags, and induced displacement. We also applied the system to the study of the distribution of elastic characteristics of the liver tissue after ablation, which confirmed the potential of OR-PAME in the study of elastic characteristics. Quantitative analysis showed greater lateral displacement in regions with reduced agar concentrations, indicating decreased stiffness. PAME also detected vertical displacement along the axial plane, validating its efficacy in elastographic imaging. By improving resolution and penetration, PAME provides superior visualization of elasticity distribution. Its methodology correlates microstructural alterations with tissue biomechanics, holding potential implications in medical diagnostics.

Interleukin-17A in diabetic retinopathy: The crosstalk of inflammation and angiogenesis.

Diabetic retinopathy (DR) is a severe ocular complication of diabetes which can leads to irreversible vision loss in its late-stage. Chronic inflammation results from long-term hyperglycemia contributes to the pathogenesis and progression of DR. In recent years, the interleukin-17 (IL-17) family have attracted the interest of researchers. IL-17A is the most widely explored cytokine in IL-17 family, involved in various acute and chronic inflammatory diseases. Growing body of evidence indicate the role of IL-17A in the pathogenesis of DR. However, the pro-inflammatory and pro-angiogenic effect of IL-17A in DR have not hitherto been reviewed. Gaining an understanding of the pro-inflammatory role of IL-17A, and how IL-17A control/impact angiogenesis pathways in the eye will deepen our understanding of how IL-17A contributes to DR pathogenesis. Herein, we aimed to thoroughly review the pro-inflammatory role of IL-17A in DR, with focus in how IL-17A impact inflammation and angiogenesis crosstalk.

[Clinical study of Bushen Culuan Formula in treatment of ovulatory disorder infertility caused by hyperprolactinemia].

This paper aims to study the therapeutic effect and safety of Bushen Culuan Formula in the treatment of patients with infertility caused by hyperprolactinemia. Sixty patients with infertility caused by hyperprolactinemia of kidney deficiency and blood stasis were divided into the treatment group(Bushen Culuan Formula + Bromocriptine Mesylate Tablets placebo) and the control group(Bromocriptine Mesylate Tablets + Bushen Culuan Formula placebo), and ovulation rate, pregnancy rate, serum sex hormones, basal body temperature(BBT), and traditional Chinese medicine(TCM) symptom scores were observed. The results showed the clinical effective rate was 90.00% in the treatment group and 80.00% in the control group. The treatment group was able to significantly reduce the PRL level and increase the pregnancy rate, and it was superior to the control group in increasing the BBT biphasic ratio, improving the TCM symptom scores, and enhancing the ovulation rate. The results show that Bushen Culuan Formula is safe and reliable in treating ovulatory disorder infertility caused by hyperprolactinemia, with remarkable effects.

[Examples of Professor MA Kun's treatment of infertility caused by uterine fibroids using method of tonifying kidney and activating blood].

Uterine fibroids are a prevalent factor that impacts fertility in women of reproductive age. This study discusses the theoretical foundation and formula principles of Professor MA Kun's clinical treatment for infertility caused by uterine fibroids. The kidney stores essence and is responsible for reproduction, while blood serves as a vital material basis for women's physiological functions. Kidney deficiency is the fundamental pathogenesis of infertility, and imbalances in kidney Qi and essence or deficiencies in kidney Yin and Yang can result in blood stasis. Blood stasis plays a significant role throughout this condition by impeding the flow of blood, which is crucial for nourishing Qi. Therefore, both kidney deficiency and blood stasis are key factors contributing to infertility caused by uterine fibroids. Professor MA Kun treats infertility caused by uterine fibroids using an approach that involves tonifying the kidneys and activating blood circulation based on changes in Qi and blood during the menstrual cycle as well as follicular growth processes. By identifying stage-specific evidence, appropriate treatments can be applied accordingly. During menstruation when the uterus opens and menstrual blood flows out, promoting follicular development through nourishing kidney Yin and activating blood circulation becomes essential. In later stages of menstruation, additional measures are taken to remove blood stasis, alleviate symptoms, disperse knots, attack pathogens while simultaneously replenishing vital energy. During intermenstrual periods when Yin holds greater importance than Yang, tonifying the kidneys and activating blood circulation helps facilitate smooth discharge of eggs by promoting transformation between Yin and Yang energies. Premenstrual period to warm kidney Yang to promote pregnant egg implantation, and at the same time to dredge the liver and regulate Qi, Qi elimination stagnation, complementary in the line, with the symptoms of additional subtractions. Clinical effect is remarkable, for the reference of colleagues.

[Study on mechanism of regulation of sex hormones in treatment of hyperprolactinemia-induced ovulatory disorder infertility with Bushen Culuan Formula].

Transcriptomics was used to investigate the mechanism of action of Bushen Culuan Formula in the treatment of infertility caused by hyperprolactinemia(HPRL), and animal experiments were carried out to verify the results. After establishing an animal model of HPRL-induced infertility, the mice were divided into normal group, model group, Bushen Culuan Formula groups with high-, medium-, and low-doses, and bromocriptine group, and they were observed in terms of the estrous cycle, gonadal index, serum sex hormones, morphology of ovary and mammary gland, follicle count, and fertility. The results showed that the Bushen Culuan Formula could effectively restore the estrous cycle, down-regulate the levels of prolactin(PRL), follicle-stimulating hormone(FSH), and luteinizing hormone(LH), up-regulate the level of estradiol(E_2), increase the number of primordial follicles and sinus follicles, and improve the ovulation rate and fertility of mice. Through RNA sequencing combined with biosignature analysis, Bushen Culuan Formula may regulate the metabolism of lipids, antioxidant enzymes, and other substances in the cells of the ovary and pituitary gland through the signaling pathways of cAMP-PKA, Kiss-1/GPR54, and Hippo and exert therapeutic effects. The results of animal experiments showed that Bushen Culuan Formula could up-regulate serum dopamine(DA) level and pituitary DRD2 expression, down-regulate hypothalamus and ovary cAMP levels, as well as protein expressions of the pituitary gland and ovary PKA, CREB, and p-CREB, and treat HPRL-induced infertility by regulating the cAMP-PKA signaling pathway.

[Bushen Culuan Formula in treatment of infertility caused by polycystic ovary syndrome].

This study aims to observe the efficacy and safety of Bushen Culuan Formula in the treatment of infertility caused by polycystic ovary syndrome(PCOS) and to explore the mechanism using metabolomics. Ninety-four patients with infertility caused by PCOS with the syndrome of kidney deficiency and blood stasis were selected and assigned into treatment and control groups(n=47). The basal body temperature(BBT) was measured, and B-ultrasonography was employed to monitor follicles, ovarian volume, endometrium, ovulation, and pregnancy. The serum levels of sex hormones including follicle-stimulating hormone(FSH), luteinizing hormone(LH), prolactin(PRL), estradiol(E_2), progestin(P), testosterone(T), free testosterone(FT), androstenedione(A2), inhibin B(INHB), and anti-Müllerian hormone(AMH) were measured. The coagulation function, traditional Chinese medicine(TCM) symptom scores, blood and urine routine, liver and kidney functions and other safety indicators were determined. Metabolomics was employed to comparatively analyze the serum metabolites of 26 patients(13 patients in each group) in the clinical study. The results showed that the total response rate and pregnancy rate of the treatment group were higher than those of the control group(P<0.001), suggesting that Bushen Culuan Formula regulated the sex hormones and ovarian function. Specifically, it reduced the levels of LH, T, FT, A2, and INHB(P<0.05 or P<0.01) and the LH/FSH ratio(P<0.05), elevated the level of P(P<0.05), promoted ovulation, increased endothelial thickness, and lowered TCM symptom scores without causing adverse reactions. A total of 24 differential metabolites were screened by metabolomics, and there were correlations between sex hormones and differential metabolites in the PCOS-induced infertility patients with kidney deficiency and blood stasis. In conclusion, Bushen Culuan Formula may regulate hormone levels through lipid and amino acid metabolism.

CCR5-overexpressing mesenchymal stem cells protect against experimental autoimmune uveitis: insights from single-cell transcriptome analysis.

Autoimmune uveitis is a leading cause of severe vision loss, and animal models provide unique opportunities for studying its pathogenesis and therapeutic strategies. Here we employ scRNA-seq, RNA-seq and various molecular and cellular approaches to characterize mouse models of classical experimental autoimmune uveitis (EAU), revealing that EAU causes broad retinal neuron degeneration and marker downregulation, and that Müller glia may act as antigen-presenting cells. Moreover, EAU immune response is primarily driven by Th1 cells, and results in dramatic upregulation of CC chemokines, especially CCL5, in the EAU retina. Accordingly, overexpression of CCR5, a CCL5 receptor, in mesenchymal stem cells (MSCs) enhances their homing capacity and improves their immunomodulatory outcomes in preventing EAU, by reducing infiltrating T cells and activated microglia and suppressing Nlrp3 inflammasome activation. Taken together, our data not only provide valuable insights into the molecular characteristics of EAU but also open an avenue for innovative MSC-based therapy.

Drugs targeting CMPK2 inhibit pyroptosis to alleviate severe pneumonia caused by multiple respiratory viruses.

Severe pneumonia caused by respiratory viruses has become a major threat to humans, especially with the SARS-CoV-2 outbreak and epidemic. The aim of this study was to investigate the universal molecular mechanism of severe pneumonia induced by multiple respiratory viruses and to search for therapeutic strategies targeting this universal molecular mechanism. The common differential genes of four respiratory viruses, including respiratory syncytial virus (RSV), rhinovirus, influenza, and SARS-CoV-2, were screened by GEO database, and the hub gene was obtained by Sytohubba in Cytoscape. Then, the effect of hub genes on inflammasome and pyrodeath was investigated in the model of RSV infection in vitro and in vivo. Finally, through virtual screening, drugs targeting the hub gene were obtained, which could alleviate severe viral pneumonia in vitro and in vivo. The results showed that CMPK2 is one of the hub genes after infection by four respiratory viruses. CMPK2 activates the inflammasome by activating NLRP3, and promotes the releases of inflammatory factors interleukin (IL)-1ß and IL-18 to induce severe viral pneumonia. Z25 and Z08 can reduce the expression level of CMPK2 mRNA and protein, thereby inhibiting NLRP3 and alleviating the development of severe viral pneumonia. In conclusion, the inflammatory response mediated by CMPK2 is the common molecular mechanism of severe pneumonia induced by viral infection, and Z25 and Z08 can effectively alleviate viral infection and severe pneumonia through this mechanism.

Proteomics coupled transcriptomics reveals Slc34a1 and Slc34a3 downregulation as potential features of nephrotoxin-induced acute kidney injury.

Acute kidney injury (AKI) stands as a prevalent and economically burdensome condition worldwide, yet its complex molecular mechanisms remain incompletely understood. To address this gap, our study employs a multifaceted approach, combining mass spectrometry and RNA sequencing technologies, to elucidate the intricate molecular landscape underlying nephrotoxin-induced AKI in mice by cisplatin- and LPS-induced. By examining the protein and RNA expression profiles, we aimed to uncover novel insights into the pathogenesis of AKI and identify potential diagnostic and therapeutic targets. Our results demonstrate significant down-regulation of Slc34a1 and Slc34a3, shedding light on their crucial roles in AKI pathology and highlighting their promise as actionable targets for diagnosis and treatment. This comprehensive analysis not only enhances our understanding of AKI pathophysiology but also offers valuable avenues for the development of targeted interventions to mitigate its clinical impact. SIGNIFICANCE: Nephrotoxicity acute kidney injury (AKI) is a common clinical condition whose pathogenesis is the process by which some drugs, chemicals or other factors cause damage to the kidneys, resulting in impaired kidney function. Although it has been proved that different nephrotoxic substances can affect the kidney through different pathways, whether they have a commonality has not been registered. Here, we combined transcriptomics and proteomics to study the molecular mechanism of LPS and cisplatin-induced nephrotoxic acute kidney injury finding that the down-regulation of Slc34a1 and Slc34a3 may be a critical link in nephrotoxic acute kidney injury, which can be used as a marker for its early diagnosis.

A bioinformatics approach to systematically analyze the molecular patterns of monkeypox virus-host cell interactions.

Monkeypox has been spreading worldwide since May 2022, when the World Health Organization (WHO) declared the outbreak a "public health emergency of international concern." The spread of monkeypox has posed a serious threat to the health of people around the world, but few studies have been conducted, and the molecular mechanism of monkeypox after infection remains unclear. We therefore implemented a transcriptome analysis to identify signaling pathways and biomarkers in monkeypox-infected cells to help understand monkeypox-host cell interactions. In this study, datasets GSE36854 and GSE11234 were obtained from GEO. Of these, 84 significantly different genes were identified in the dataset GSE36854, followed by KEGG, GO analysis protein-protein interaction (PPI) construction, and Hub gene extraction. We also analyzed the expression regulation of hub genes and screened for drugs targeting hub genes. The results showed that monkeypox-infected cells significantly activated the cellular immune response. The top 10 hub genes are IER3, IFIT2, IL11, ZC3H12A, EREG, IER2, NFKBIE, FST, IFIT1 and AREG. AP-26113 and itraconazole can be used to counteract the inhibitory effect of monkeypox on IFIT1 and IFIT2 and serve as candidate drugs for the treatment of monkeypox virus infection. IRF1 may also be a transcription factor of IFIT. Our results provide a new entry point for understanding how monkeypox virus interacts with its host.

Multidirectional Intervention of Chinese Herbal Medicine in the Prevention and Treatment of Atherosclerosis: From Endothelial Protection to Immunomodulation.

Atherosclerosis is a significant risk factor for developing cardiovascular disease and a leading cause of death worldwide. The occurrence of atherosclerosis is closely related to factors such as endothelial injury, lipid deposition, immunity, and inflammation. Conventional statins, currently used in atherosclerosis treatment, have numerous adverse side effects that limit their clinical utility, prompting the urgent need to identify safer and more effective therapeutic alternatives. Growing evidence indicates the significant potential of Chinese herbs in atherosclerosis treatment. Herbal monomer components, such as natural flavonoid compounds extracted from herbs like Coptis chinensis and Panax notoginseng, have been utilized for their lipid-lowering and inflammation-inhibiting effects in atherosclerosis treatment. These herbs can be used as single components in treating diseases and with other Chinese medicines to form herbal combinations. This approach targets the disease mechanism in multiple ways, enhancing the therapeutic effects. Thus, this review examines the roles of Chinese herbal medicine monomers and Chinese herbal compounds in inhibiting atherosclerosis, including regulating lipids, improving endothelial function, reducing oxidative stress, regulating inflammation and the immune response, and apoptosis. By highlighting these roles, our study offers new perspectives on atherosclerosis treatment with Chinese herbs and is anticipated to contribute to advancements in related research fields.

Increasing Ca2+ accumulation in salt glands under salt stress increases stronger selective secretion of Na+ in Plumbago auriculata tetraploids.

Under salt stress, recretohalophyte Plumbago auriculata tetraploids enhance salt tolerance by increasing selective secretion of Na+ compared with that in diploids, although the mechanism is unclear. Using non-invasive micro-test technology, the effect of salt gland Ca2+ content on Na+ and K+ secretion were investigated in diploid and tetraploid P. auriculata under salt stress. Salt gland Ca2+ content and secretion rates of Na+ and K+ were higher in tetraploids than in diploids under salt stress. Addition of exogenous Ca2+ increased the Ca2+ content of the salt gland in diploids and is accompanied by an increase in the rate of Na+ and K+ secretion. With addition of a Ca2+ channel inhibitor, diploid salt glands retained large amounts of Ca2+, leading to higher Ca2+ content and Na+ secretion rate than those of tetraploids. Inhibiting H2O2 generation and H+-ATPase activity altered Na+ and K+ secretion rates in diploids and tetraploids under salt stress, indicating involvement in regulating Na+ and K+ secretion. Our results indicate that the increased Na+ secretion rate of salt gland in tetraploids under salt stress was associated with elevated Ca2+ content in salt gland.

No Remdesivir Resistance Observed in the Phase 3 Severe and Moderate COVID-19 SIMPLE Trials.

Remdesivir (RDV) is a broad-spectrum nucleotide analog prodrug approved for the treatment of COVID-19 in hospitalized and non-hospitalized patients with clinical benefit demonstrated in multiple Phase 3 trials. Here we present SARS-CoV-2 resistance analyses from the Phase 3 SIMPLE clinical studies evaluating RDV in hospitalized participants with severe or moderate COVID-19 disease. The severe and moderate studies enrolled participants with radiologic evidence of pneumonia and a room-air oxygen saturation of ≤94% or >94%, respectively. Virology sample collection was optional in the study protocols. Sequencing and related viral load data were obtained retrospectively from participants at a subset of study sites with local sequencing capabilities (10 of 183 sites) at timepoints with detectable viral load. Among participants with both baseline and post-baseline sequencing data treated with RDV, emergent Nsp12 substitutions were observed in 4 of 19 (21%) participants in the severe study and none of the 2 participants in the moderate study. The following 5 substitutions emerged: T76I, A526V, A554V, E665K, and C697F. The substitutions T76I, A526V, A554V, and C697F had an EC50 fold change of ≤1.5 relative to the wildtype reference using a SARS-CoV-2 subgenomic replicon system, indicating no significant change in the susceptibility to RDV. The phenotyping of E665K could not be determined due to a lack of replication. These data reveal no evidence of relevant resistance emergence and further confirm the established efficacy profile of RDV with a high resistance barrier in COVID-19 patients.

Dynamic Metabolons Using Stimuli-Responsive Protein Cages.

Naturally evolved metabolons have the ability to assemble and disassemble in response to environmental stimuli, allowing for the rapid reorganization of chemical reactions in living cells to meet changing cellular needs. However, replicating such capability in synthetic metabolons remains a challenge due to our limited understanding of the mechanisms by which the assembly and disassembly of such naturally occurring multienzyme complexes are controlled. Here, we report the synthesis of chemical- and light-responsive protein cages for assembling synthetic metabolons, enabling the dynamic regulation of enzymatic reactions in living cells. Particularly, a chemically responsive domain was fused to a self-assembled protein cage subunit, generating engineered protein cages capable of displaying proteins containing cognate interaction domains on their surfaces in response to small molecular cues. Chemical-induced colocalization of sequential enzymes on protein cages enhances the specificity of the branched deoxyviolacein biosynthetic reactions by 2.6-fold. Further, by replacing the chemical-inducible domain with a light-inducible dimerization domain, we created an optogenetic protein cage capable of reversibly recruiting and releasing targeted proteins onto and from the exterior of the protein cages in tens of seconds by on-off of blue light. Tethering the optogenetic protein cages to membranes enables the formation of light-switchable, membrane-bound metabolons, which can repeatably recruit-release enzymes, leading to the manipulation of substrate utilization across membranes on demand. Our work demonstrates a powerful and versatile strategy for constructing dynamic metabolons in engineered living cells for efficient and controllable biocatalysis.