Decoding CRISPR-Cas PAM recognition with UniDesign.

The critical first step in Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (CRISPR-Cas) protein-mediated gene editing is recognizing a preferred protospacer adjacent motif (PAM) on target DNAs by the protein's PAM-interacting amino acids (PIAAs). Thus, accurate computational modeling of PAM recognition is useful in assisting CRISPR-Cas engineering to relax or tighten PAM requirements for subsequent applications. Here, we describe a universal computational protein design framework (UniDesign) for designing protein-nucleic acid interactions. As a proof of concept, we applied UniDesign to decode the PAM-PIAA interactions for eight Cas9 and two Cas12a proteins. We show that, given native PIAAs, the UniDesign-predicted PAMs are largely identical to the natural PAMs of all Cas proteins. In turn, given natural PAMs, the computationally redesigned PIAA residues largely recapitulated the native PIAAs (74% and 86% in terms of identity and similarity, respectively). These results demonstrate that UniDesign faithfully captures the mutual preference between natural PAMs and native PIAAs, suggesting it is a useful tool for engineering CRISPR-Cas and other nucleic acid-interacting proteins. UniDesign is open-sourced at https://github.com/tommyhuangthu/UniDesign.

Regulatory variants in TCF7L2 are associated with thoracic aortic aneurysm.

Thoracic aortic aneurysm (TAA) is characterized by dilation of the aortic root or ascending/descending aorta. TAA is a heritable disease that can be potentially life threatening. While 10%-20% of TAA cases are caused by rare, pathogenic variants in single genes, the origin of the majority of TAA cases remains unknown. A previous study implicated common variants in FBN1 with TAA disease risk. Here, we report a genome-wide scan of 1,351 TAA-affected individuals and 18,295 control individuals from the Cardiovascular Health Improvement Project and Michigan Genomics Initiative at the University of Michigan. We identified a genome-wide significant association with TAA for variants within the third intron of TCF7L2 following replication with meta-analysis of four additional independent cohorts. Common variants in this locus are the strongest known genetic risk factor for type 2 diabetes. Although evidence indicates the presence of different causal variants for TAA and type 2 diabetes at this locus, we observed an opposite direction of effect. The genetic association for TAA colocalizes with an aortic eQTL of TCF7L2, suggesting a functional relationship. These analyses predict an association of higher expression of TCF7L2 with TAA disease risk. In vitro, we show that upregulation of TCF7L2 is associated with BCL2 repression promoting vascular smooth muscle cell apoptosis, a key driver of TAA disease.

Bicuspid Aortic Valve-Associated Regulatory Regions Reveal GATA4 Regulation and Function During Human-Induced Pluripotent Stem Cell-Based Endothelial-Mesenchymal Transition-Brief Report.

BACKGROUND: The endothelial-mesenchymal transition (EndoMT) is a fundamental process for heart valve formation and defects in EndoMT cause aortic valve abnormalities. Our previous genome-wide association study identified multiple variants in a large chromosome 8 segment as significantly associated with bicuspid aortic valve (BAV). The objective of this study is to determine the biological effects of this large noncoding segment in human induced pluripotent stem cell (hiPSC)-based EndoMT. METHODS: A large genomic segment enriched for BAV-associated variants was deleted in hiPSCs using 2-step CRISPR/Cas9 editing. To address the effects of the variants on GATA4 expression, we generated CRISPR repression hiPSC lines (CRISPRi) as well as hiPSCs from BAV patients. The resulting hiPSCs were differentiated to mesenchymal/myofibroblast-like cells through cardiovascular-lineage endothelial cells for molecular and cellular analysis. Single-cell RNA sequencing was also performed at different stages of EndoMT induction. RESULTS: The large deletion impaired hiPSC-based EndoMT in multiple biallelic clones compared with their isogenic control. It also reduced GATA4 transcript and protein levels during EndoMT, sparing the other genes nearby the deletion segment. Single-cell trajectory analysis revealed the molecular reprogramming during EndoMT. Putative GATA-binding protein targets during EndoMT were uncovered, including genes implicated in endocardial cushion formation and EndoMT process. Differentiation of cells derived from BAV patients carrying the rs117430032 variant as well as CRISPRi repression of the rs117430032 locus resulted in lower GATA4 expression in a stage-specific manner. TWIST1 was identified as a potential regulator of GATA4 expression, showing specificity to the locus tagged by rs117430032. CONCLUSIONS: BAV-associated distal regions regulate GATA4 expression during hiPSC-based EndoMT, which in turn promotes EndoMT progression, implicating its contribution to heart valve development.

Single-Molecule Spatial Transcriptomics of Human Thoracic Aortic Aneurysms Uncovers Calcification-Related CARTPT-Expressing Smooth Muscle Cells.

BACKGROUND: Although single-cell RNA-sequencing is commonly applied to dissect the heterogeneity in human tissues, it involves the preparation of single-cell suspensions via cell dissociation, causing loss of spatial information. In this study, we employed high-resolution single-cell transcriptome imaging to reveal rare smooth muscle cell (SMC) types in human thoracic aortic aneurysm (TAA) tissue samples. METHODS: Single-molecule spatial distribution of transcripts from 140 genes was analyzed in fresh-frozen human TAA samples with region and sex-matched controls. In vitro studies and tissue staining were performed to examine human CART prepropeptide (CARTPT) regulation and function. RESULTS: We captured thousands of cells per sample including a spatially distinct CARTPT-expressing SMC subtype enriched in male TAA samples. Immunoassays confirmed human CART (cocaine- and amphetamine-regulated transcript) protein enrichment in male TAA tissue and truncated CARTPT secretion into cell culture medium. Oxidized low-density lipoprotein, a cardiovascular risk factor, induced CARTPT expression, whereas CARTPT overexpression in human aortic SMCs increased the expression of key osteochondrogenic transcription factors and reduced contractile gene expression. Recombinant human CART treatment of human SMCs further confirmed this phenotype. Alizarin red staining revealed calcium deposition in male TAA samples showing similar localization with human CART staining. CONCLUSIONS: Here, we demonstrate the feasibility of single-molecule imaging in uncovering rare SMC subtypes in the diseased human aorta, a difficult tissue to dissociate. We identified a spatially distinct CARTPT-expressing SMC subtype enriched in male human TAA samples. Our functional studies suggest that human CART promotes osteochondrogenic switch of aortic SMCs, potentially leading to medial calcification of the thoracic aorta.

Lp-PLA2 (Lipoprotein-Associated Phospholipase A2) Deficiency Lowers Cholesterol Levels and Protects Against Atherosclerosis in Rabbits.

BACKGROUND: Elevated plasma Lp-PLA2 (lipoprotein-associated phospholipase A2) activity is closely associated with an increased risk of cardiovascular events. However, whether and how Lp-PLA2 is directly involved in the pathogenesis of atherosclerosis is still unclear. To examine the hypothesis that Lp-PLA2 could be a potential preventative target of atherosclerosis, we generated Lp-PLA2 knockout rabbits and investigated the pathophysiological functions of Lp-PLA2. METHODS: Lp-PLA2 knockout rabbits were generated using CRISPR/Cas9 system to assess the role of Lp-PLA2 in plasma lipids regulation and identify its underlying molecular mechanisms. Homozygous knockout rabbits along with wild-type rabbits were fed a cholesterol-rich diet for up to 14 weeks and their atherosclerotic lesions were compared. Moreover, the effects of Lp-PLA2 deficiency on the key cellular behaviors in atherosclerosis were assessed in vitro. RESULTS: When rabbits were fed a standard diet, Lp-PLA2 deficiency led to a significant reduction in plasma lipids. The decreased protein levels of SREBP2 (sterol regulatory element-binding protein 2) and HMGCR (3-hydroxy-3-methylglutaryl coenzyme A reductase) in livers of homozygous knockout rabbits indicated that the cholesterol biosynthetic pathway was impaired with Lp-PLA2 deficiency. In vitro experiments further demonstrated that intracellular Lp-PLA2 efficiently enhanced SREBP2-related cholesterol biosynthesis signaling independently of INSIGs (insulin-induced genes). When fed a cholesterol-rich diet, homozygous knockout rabbits exhibited consistently lower level of hypercholesterolemia, and their aortic atherosclerosis lesions were significantly reduced by 60.2% compared with those of wild-type rabbits. The lesions of homozygous knockout rabbits were characterized by reduced macrophages and the expression of inflammatory cytokines. Macrophages of homozygous knockout rabbits were insensitive to M1 polarization and showed reduced DiI-labeled lipoprotein uptake capacity compared with wild-type macrophages. Lp-PLA2 deficiency also inhibited the adhesion between monocytes and endothelial cells. CONCLUSIONS: These results demonstrate that Lp-PLA2 plays a causal role in regulating blood lipid homeostasis and Lp-PLA2 deficiency protects against dietary cholesterol-induced atherosclerosis in rabbits. Lp-PLA2 could be a potential target for the prevention of atherosclerosis.

Effect of Sodium Tanshinoneâ ¡A Sulfonate on Uric Acid, Sicam-1, ET-1 and FMD in Patients with Coronary Heart Disease Complicated with Hyperuricemia.

This study was to observe the effect of Sodium TanshinoneⅡA Sulfonate (ST-ⅡAS) on blood uric acid (UA), human Soluble Intercellular Adhesion Molecule-1 (sICAM-1), Endothelin-1 (ET-1) and percentage of brachial artery Flow-Mediated Dilatation (FMD) in individuals with Hyperuricemia Complicated Coronary Heart Disease (HC-CHD). The study's participants were 108 patients with HC-CHD who attended our hospital between January 2020 and June 2022. In the trial, the patients were split into two groups with 54 instances each: the general group and the observation group. The observation group received ST-IIAS therapy, while the general group received standard care. The experiment chose to observe and compare the difference of uric acid, sICAM-1, ET-1, FMD, therapeutic effectiveness and negative effects between the two groups at various times. Results showed that on the 14th day, the observation group's amounts of UA, sICAM-1, and ET-1 were inferior to the general group (P<0.05); On the 7th and 14th days, the observation group's amount of ET-1 was lower than that of the general group (P<0.05); The observation group's FMD of patients on the 14th day was inferior to the general group after treatment (P<0.05); The observation group's overall effective rate was 94.44% higher than the general group's (P<0.05); The observation group experienced fewer negative responses than the general group did (P<0.05). In conclusion, ST-ⅡAS can be used for uric acid, vascular endothelial systolic and diastolic function in patients with HC-CHD, and has better clinical efficacy and lower risk of adverse reactions.

Effect of fenofibrate on blood lipid, sICAM-1, ET-1 and prognosis in chronic heart failure patients complicated with diabetes.

This study was to observe the effects of fenofibrate on blood lipid, sICAM-1, ET-1 and prognosis in chronic heart failure patients complicated with diabetes. For this purpose, a total of 126 chronic heart failure patients complicated with diabetes admitted to our hospital from September 2020 to October 2021 were selected and divided into a control group and an observation group by random number table method, with 63 cases in each group. The control group received conventional drug treatment, and the observation group received fenofibrate treatment on the basis of the control group. After 12 months follow-up, the levels of blood lipid, sICAM-1, ET-1 were compared between the two groups at 3 months before and after treatment and 6, 12 months after treatment. Results showed that after 3 months of treatment, LDL-C, TG and TC were lower in the observation group than in the control group, showing a statistically significant difference (P<0.05). After 3 months of treatment, HDL-C was higher in the observation group than in the control group, showing a difference (P<0.05). After 3 months of treatment, sICAM-1 and ET-1 were lower in the observation group than in the control group, showing a difference (P<0.05). There was no significant difference in mortality after 6 months of treatment, re-hospitalization rate and mortality after 12 months of treatment between the two groups (P>0.05). The re-hospitalization rate of the observation group was 4.76% (3/63) after 6 months of treatment, which was lower than that of the control group in the same period, showing a significant difference (P<0.05). The conclusion was that fenofibrate can regulate blood lipids in chronic heart failure patients complicated with diabetes, inhibit sICAM-1 and ET-1, and reduce the re-hospitalization rate within 6 months after treatment. However, the effects on long-term re-hospitalization rate and mortality risk are consistent with those of conventional treatment.

hiPSC Modeling of Lineage-Specific Smooth Muscle Cell Defects Caused by TGFBR1A230T Variant, and Its Therapeutic Implications for Loeys-Dietz Syndrome.

BACKGROUND: Loeys-Dietz syndrome (LDS) is an inherited disorder predisposing individuals to thoracic aortic aneurysm and dissection. Currently, there are no medical treatments except surgical resection. Although the genetic basis of LDS is well-understood, molecular mechanisms underlying the disease remain elusive, impeding the development of a therapeutic strategy. In addition, aortic smooth muscle cells (SMCs) have heterogenous embryonic origins, depending on their spatial location, and lineage-specific effects of pathogenic variants on SMC function, likely causing regionally constrained LDS manifestations, have been unexplored. METHODS: We identified an LDS family with a dominant pathogenic variant in the TGFBR1 gene (TGFBR1A230T) causing aortic root aneurysm and dissection. To accurately model the molecular defects caused by this mutation, we used human induced pluripotent stem cells from a subject with normal aorta to generate human induced pluripotent stem cells carrying TGFBR1A230T, and corrected the mutation in patient-derived human induced pluripotent stem cells using CRISPR-Cas9 gene editing. After their lineage-specific SMC differentiation through cardiovascular progenitor cell (CPC) and neural crest stem cell lineages, we used conventional molecular techniques and single-cell RNA sequencing to characterize the molecular defects. The resulting data led to subsequent molecular and functional rescue experiments using activin A and rapamycin. RESULTS: Our results indicate the TGFBR1A230T mutation impairs contractile transcript and protein levels, and function in CPC-SMC, but not in neural crest stem cell-SMC. Single-cell RNA sequencing results implicate defective differentiation even in TGFBR1A230T/+ CPC-SMC including disruption of SMC contraction and extracellular matrix formation. Comparison of patient-derived and mutation-corrected cells supported the contractile phenotype observed in the mutant CPC-SMC. TGFBR1A230T selectively disrupted SMAD3 (SMAD family member 3) and AKT (AKT serine/threonine kinase) activation in CPC-SMC, and led to increased cell proliferation. Consistently, single-cell RNA sequencing revealed molecular similarities between a loss-of-function SMAD3 mutation (SMAD3c.652delA/+) and TGFBR1A230T/+. Last, combination treatment with activin A and rapamycin during or after SMC differentiation significantly improved the mutant CPC-SMC contractile gene expression and function, and rescued the mechanical properties of mutant CPC-SMC tissue constructs. CONCLUSIONS: This study reveals that a pathogenic TGFBR1 variant causes lineage-specific SMC defects informing the etiology of LDS-associated aortic root aneurysm. As a potential pharmacological strategy, our results highlight a combination treatment with activin A and rapamycin that can rescue the SMC defects caused by the variant.

Impaired learning and memory generated by hyperthyroidism is rescued by restoration of AMPA and NMDA receptors function.

Hyperthyroidism has been identified as a risk factor for cognitive disorders. The hippocampus is a key brain region associated with cognitive function, among which excitatory synapse transmission plays an important role in the process of learning and memory. However, the mechanism by which hyperthyroidism leads to cognitive dysfunction through a synaptic mechanism remains unknown. We investigated the synaptic mechanisms in the effects of hyperthyroidism in an animal model that involved repeated injection of triiodothyronine (T3). These mice displayed impaired learning and memory in the Novel object recognition test, Y-maze test, and Morris Water Maze test, as well as elevated anxiety in the elevated plus maze. Mature dendritic spines in the hippocampal CA1 region of hyperthyroid mice were significantly decreased, accompanied by decreased level of AMPA- and NMDA-type glutamate receptors in the hippocampus. In primary cultured hippocampal neurons, levels of AMPA- and NMDA-type glutamate receptors also decreased and whole-cell patch-clamp recording revealed that excitatory synaptic function was obviously attenuated after T3 treatment. Notably, pharmacological activation of AMPAR or NMDAR by intraperitoneal injection of CX546, an AMPAR agonist, or NMDA, an NMDAR agonist can restore excitatory synaptic function and corrected impaired learning and memory deficit in hyperthyroid mice. Together, our findings uncovered a previously unrecognized AMPAR and NMDAR-dependent mechanism involved in regulating hippocampal excitatory synaptic transmission and learning and memory disorders in hyperthyroidism.

Probing Molecular Interactions of Antibody Drugs, Silicone Oil, and Surfactant at Buried Interfaces In Situ.

Antibody drugs have been rapidly developed to cure many diseases including COVID-19 infection. Silicone oil is commonly used as a lubricant coating material for devices used in the pharmaceutical industry to store and administer antibody drug formulations. However, the interaction between silicone oil and antibody molecules could lead to the adsorption, denaturation, and aggregation of antibody molecules, impacting the efficacy of antibody drugs. Here, we studied the molecular interactions between antibodies and silicone oil in situ in real time. The effect of the surfactant on such interactions was also investigated. Specifically, the adsorption dynamics of a bispecific antibody (BsAb) onto a silicone oil surface without and with different concentrations of the surfactant PS80 in antibody solutions were monitored. Also the possible lowest effective PS80 concentrations that can prevent the adsorption of BsAb as well as a monoclonal antibody (mAb) onto silicone oil were measured. It was found that different concentrations of PS80 are required for preventing the adsorption of different antibodies. Both BsAB and mAB denature on silicone oil without a surfactant. However, for a low surfactant concentration in the solution, although the surfactant could not completely prevent the antibody from adsorption, it could maintain the native structures of adsorbed BsAb and mAb antibodies on silicone oil. This is important knowledge, showing that to prevent antibody aggregation on silicone oil it is not necessary to add surfactant to a concentration high enough to completely minimize protein adsorption.

Social isolation induces neuroinflammation and microglia overactivation, while dihydromyricetin prevents and improves them.

BACKGROUND: Anxiety disorders are the most prevalent mental illnesses in the U.S. and are estimated to consume one-third of the country's mental health treatment cost. Although anxiolytic therapies are available, many patients still exhibit treatment resistance, relapse, or substantial side effects. Further, due to the COVID-19 pandemic and stay-at-home order, social isolation, fear of the pandemic, and unprecedented times, the incidence of anxiety has dramatically increased. Previously, we have demonstrated dihydromyricetin (DHM), the major bioactive flavonoid extracted from Ampelopsis grossedentata, exhibits anxiolytic properties in a mouse model of social isolation-induced anxiety. Because GABAergic transmission modulates the immune system in addition to the inhibitory signal transmission, we investigated the effects of short-term social isolation on the neuroimmune system. METHODS: Eight-week-old male C57BL/6 mice were housed under absolute social isolation for 4 weeks. The anxiety-like behaviors after DHM treatment were examined using elevated plus-maze and open field behavioral tests. Gephyrin protein expression, microglial profile changes, NF-κB pathway activation, cytokine level, and serum corticosterone were measured. RESULTS: Socially isolated mice showed increased anxiety levels, reduced exploratory behaviors, and reduced gephyrin levels. Also, a dynamic alteration in hippocampal microglia were detected illustrated as a decline in microglia number and overactivation as determined by significant morphological changes including decreases in lacunarity, perimeter, and cell size and increase in cell density. Moreover, social isolation induced an increase in serum corticosterone level and activation in NF-κB pathway. Notably, DHM treatment counteracted these changes. CONCLUSION: The results suggest that social isolation contributes to neuroinflammation, while DHM has the ability to improve neuroinflammation induced by anxiety.

RNA sequencing reveals perivascular adipose tissue plasticity in response to angiotensin II.

Most blood vessels are surrounded by perivascular adipose tissue (PVAT), which is a unique adipose tissue that plays critical roles in vascular physiology and pathophysiology. PVAT displays regional differences that impact vascular homeostasis. Angiotensin II (Ang II) is the main biologically active component of the renin-angiotensin-aldosterone system (RAAS), which has been extensively studied in vascular biology. However, the effects of Ang II on PVAT are less explored and remain to be elucidated. In this study, we systematically investigated the regional heterogeneity of three portions of aortic PVAT, i.e., ascending thoracic aortic PVAT (ATA-PVAT), descending thoracic aortic PVAT (DTA-PVAT) and abdominal aortic PVAT (AA-PVAT), and their responses to 7-day Ang II infusion using RNA sequencing. We found that AA-PVAT is clearly distinguished from both ATA-PVAT and DTA-PVAT, with significantly down-regulated oxidative phosphorylation and up-regulated inflammatory response pathways. Furthermore, AA-PVAT expresses lower levels of brown adipocyte marker genes, such as Ucp1, Cidea, Cox8b, Dio2 and Pgc1α, but expresses higher levels of proinflammatory genes, such as Ccl2, Il1ß and Tnfα, and components of the RAAS, including Agt, Ace and Agtr1a. Ang II infusion significantly down-regulated oxidative phosphorylation in all regions of aortic PVAT and significantly up-regulated inflammatory response specifically in ATA-PVAT and DTA-PVAT. Moreover, ATA-PVAT was most responsive to Ang II induced inflammation. We further used CDGSH iron-sulfur domain-containing protein 1 (a.k.a. mitoNEET) transgenic mice that exhibit enhanced brown adipose tissue (BAT)-like phenotype in aortic PVAT, as indicated by elevated expression levels of brown adipocyte marker genes, and found that the enhanced BAT-like phenotype of aortic PVAT could counterbalance Ang II induced inflammatory and oxidative effects.

New Insight Into Metformin-Induced Cholesterol-Lowering Effect Crosstalk Between Glucose and Cholesterol Homeostasis via ChREBP (Carbohydrate-Responsive Element-Binding Protein)-Mediated PCSK9 (Proprotein Convertase Subtilisin/Kexin Type 9) Regulation.

[Figure: see text].

Endothelial TFEB (Transcription Factor EB) Improves Glucose Tolerance via Upregulation of IRS (Insulin Receptor Substrate) 1 and IRS2.

OBJECTIVE: Vascular endothelial cells (ECs) play a critical role in maintaining vascular homeostasis. Aberrant EC metabolism leads to vascular dysfunction and metabolic diseases. TFEB (transcription factor EB), a master regulator of lysosome biogenesis and autophagy, has protective effects on vascular inflammation and atherosclerosis. However, the role of endothelial TFEB in metabolism remains to be explored. In this study, we sought to investigate the role of endothelial TFEB in glucose metabolism and underlying molecular mechanisms. Approach and Results: To determine whether endothelial TFEB is critical for glucose metabolism in vivo, we utilized EC-selective TFEB knockout and EC-selective TFEB transgenic mice fed a high-fat diet. EC-selective TFEB knockout mice exhibited significantly impaired glucose tolerance compared with control mice. Consistently, EC-selective TFEB transgenic mice showed improved glucose tolerance. In primary human ECs, small interfering RNA-mediated TFEB knockdown blunts Akt (AKT serine/threonine kinase) signaling. Adenovirus-mediated overexpression of TFEB consistently activates Akt and significantly increases glucose uptake in ECs. Mechanistically, TFEB upregulates IRS1 and IRS2 (insulin receptor substrate 1 and 2). TFEB increases IRS2 transcription measured by reporter gene and chromatin immunoprecipitation assays. Furthermore, we found that TFEB increases IRS1 protein via downregulation of microRNAs (miR-335, miR-495, and miR-548o). In vivo, Akt signaling in the skeletal muscle and adipose tissue was significantly impaired in EC-selective TFEB knockout mice and consistently improved in EC-selective TFEB transgenic mice on high-fat diet. CONCLUSIONS: Our data revealed a critical role of TFEB in endothelial metabolism and suggest that TFEB constitutes a potential molecular target for the treatment of vascular and metabolic diseases.

A stepwise mechanism for aqueous two-phase system formation in concentrated antibody solutions.

Aqueous two-phase system (ATPS) formation is the macroscopic completion of liquid-liquid phase separation (LLPS), a process by which aqueous solutions demix into 2 distinct phases. We report the temperature-dependent kinetics of ATPS formation for solutions containing a monoclonal antibody and polyethylene glycol. Measurements are made by capturing dark-field images of protein-rich droplet suspensions as a function of time along a linear temperature gradient. The rate constants for ATPS formation fall into 3 kinetically distinct categories that are directly visualized along the temperature gradient. In the metastable region, just below the phase separation temperature, Tph , ATPS formation is slow and has a large negative apparent activation energy. By contrast, ATPS formation proceeds more rapidly in the spinodal region, below the metastable temperature, Tmeta , and a small positive apparent activation energy is observed. These region-specific apparent activation energies suggest that ATPS formation involves 2 steps with opposite temperature dependencies. Droplet growth is the first step, which accelerates with decreasing temperature as the solution becomes increasingly supersaturated. The second step, however, involves droplet coalescence and is proportional to temperature. It becomes the rate-limiting step in the spinodal region. At even colder temperatures, below a gelation temperature, Tgel , the proteins assemble into a kinetically trapped gel state that arrests ATPS formation. The kinetics of ATPS formation near Tgel is associated with a remarkably fragile solid-like gel structure, which can form below either the metastable or the spinodal region of the phase diagram.

Untargeted metabolomics identifies succinate as a biomarker and therapeutic target in aortic aneurysm and dissection.

AIMS: Aortic aneurysm and dissection (AAD) are high-risk cardiovascular diseases with no effective cure. Macrophages play an important role in the development of AAD. As succinate triggers inflammatory changes in macrophages, we investigated the significance of succinate in the pathogenesis of AAD and its clinical relevance. METHODS AND RESULTS: We used untargeted metabolomics and mass spectrometry to determine plasma succinate concentrations in 40 and 1665 individuals of the discovery and validation cohorts, respectively. Three different murine AAD models were used to determine the role of succinate in AAD development. We further examined the role of oxoglutarate dehydrogenase (OGDH) and its transcription factor cyclic adenosine monophosphate-responsive element-binding protein 1 (CREB) in the context of macrophage-mediated inflammation and established p38αMKOApoe-/- mice. Succinate was the most upregulated metabolite in the discovery cohort; this was confirmed in the validation cohort. Plasma succinate concentrations were higher in patients with AAD compared with those in healthy controls, patients with acute myocardial infarction (AMI), and patients with pulmonary embolism (PE). Moreover, succinate administration aggravated angiotensin II-induced AAD and vascular inflammation in mice. In contrast, knockdown of OGDH reduced the expression of inflammatory factors in macrophages. The conditional deletion of p38α decreased CREB phosphorylation, OGDH expression, and succinate concentrations. Conditional deletion of p38α in macrophages reduced angiotensin II-induced AAD. CONCLUSION: Plasma succinate concentrations allow to distinguish patients with AAD from both healthy controls and patients with AMI or PE. Succinate concentrations are regulated by the p38α-CREB-OGDH axis in macrophages.

Improving the genome assembly of rabbits with long-read sequencing.

The European rabbit (Oryctolagus cuniculus) is important as a biomedical model given its unique features in immunity and metabolism. The current reference genome OryCun2.0 established with whole-genome shotgun sequencing was quite fragmented and had not been updated for ten years. In this work, we provided a new rabbit genome assembly UM_NZW_1.0 to improve OryCun2.0 by leveraging the contig lengths based on long-read sequencing and a wealth of available Illumina paired-end sequence data. UM_NZW_1.0 showed a remarkable increase of continuity compared with OryCun2.0, with 5 times longer contig N50 and approximately 75% gaps closed. Many of the closed gaps were overlapped with protein-coding genes or transcriptional features, resulting in an enhancement of gene annotations. In particular, UM_NZW_1.0 presented a more complete landscape of the MHC region and the IGH locus, therefore provided a valuable resource for future researches on rabbits.

The use of a surgical boot camp combining anatomical education and surgical simulation for internship preparedness among senior medical students.

BACKGROUND: Senior medical students feel unprepared for surgical procedures and care for surgery patients when they begin their internship. This study sought to introduce and evaluate a surgical boot camp training for senior medical students. METHODS: A 44-h surgical boot camp program of lectures on clinical practice simulation, anatomical dissections, and simulated operation on cadavers was designed, implemented, and evaluated during the 2018 to 2019 academic year. A self-administered questionnaire was used to assess students' perceptions of the content, delivery, and self-confidence. The mini-Clinical Evaluation Exercise (mini-CEX) and the Operative Performance Rating System were used to assess skills essential to good clinical care and to facilitate feedback. RESULTS: Over 93% of the students were satisfied with the surgical boot camp, training equipment, and learning materials provided. After six sessions of training, 85.3% reported gaining self-confidence and performed better in some surgical procedures such as major gastrectomy. The mini-CEX scores suggested significant improvement in the students' clinical skills, attitudes, and behaviors (P < 0.01). Ninety-eight percent of students felt that the anatomical knowledge taught met their needs. The scores of the Operative Performance Rating System suggested that the students' surgical skills such as instruments handling, incising, treatment of surrounding tissues (blood vessels, nerves), and smoothness of the whole operation had increased significantly following the surgical boot camp (All P < 0.01). CONCLUSION: The surgical boot camp curriculum improved students' satisfaction and confidence in core clinical practice competencies. Therefore, medical schools the world over should continue to seek ways to bridge the gaps between pre-clinical, clinical, and internship training.

Time-series gene expression patterns and their characteristics of Beauveria bassiana in the process of infecting pest insects.

Beauveria bassiana has been widely used as an important biological control fungus for agricultural and forest pests, and clarifying the interaction mechanism between B. bassiana and its host will help to better exert the efficacy of the mycoinsecticide. Here, we proposed a novel pattern analysis (PA) method for analyzing time-series data and applied it to a transcriptomic data set of B. bassiana infecting Galleria mellonella. We screened out 14 patterns including 868 genes, which had some characteristics that were not inferior to differentially expressed genes (DEGs). Compared with the previous analysis of this data set, we had three novel discoveries during B. bassiana infection, including overall downregulation of gene expression, the more critical first 24 h, and enrichment of regulatory functions of downregulated genes. Our new PA method promises to be an important complement to DEGs analysis for time-series transcriptomic data, and our findings enrich our knowledge of molecular mechanisms of fungal-host interactions.

Structural Investigation of the Interaction Mechanism between Chlorogenic Acid and AMPA Receptor via In Silico Approaches.

Chlorogenic acid (CGA), an important metabolite in natural plant medicines such as honeysuckle and eucommia, has been shown to have potent antinociceptive effects. Nevertheless, the mechanism by which CGA relieves chronic pain remains unclear. α-amino-3-hydroxy-5-methyl-4-isooxazolpropionic acid receptor (AMPAR) is a major ionotropic glutamate receptor that mediates rapid excitatory synaptic transmission and its glutamate ionotropic receptor AMPA type subunit 1 (GluA1) plays a key role in nociceptive transmission. In this study, we used Western blot, surface plasmon resonance (SPR) assay, and the molecular simulation technologies to investigate the mechanism of interaction between CGA and AMPAR to relieve chronic pain. Our results indicate that the protein expression level of GluA1 showed a dependent decrease as the concentration of CGA increased (0, 50, 100, and 200 µM). The SPR assay demonstrates that CGA can directly bind to GluA1 (KD = 496 µM). Furthermore, CGA forms a stable binding interaction with GluA1, which is validated by molecular dynamics (MD) simulation. The binding free energy between CGA and GluA1 is -39.803 ± 14.772 kJ/mol, where van der Waals interaction and electrostatic interaction are the major contributors to the GluA1-CGA binding, and the key residues are identified (Val-32, Glu-33, Ala-36, Glu-37, Leu-48), which play a crucial role in the binding interaction. This study first reveals the structural basis of the stable interaction between CGA and GluA1 to form a binding complex for the relief of chronic pain. The research provides the structural basis to understand the treatment of chronic pain and is valuable to the design of novel drug molecules in the future.