Structural insights into the ubiquitylation strategy of the oligomeric CRL2FEM1B E3 ubiquitin ligase.

Cullin-RING E3 ubiquitin ligase (CRL) family members play critical roles in numerous biological processes and diseases including cancer and Alzheimer's disease. Oligomerization of CRLs has been reported to be crucial for the regulation of their activities. However, the structural basis for its regulation and mechanism of its oligomerization are not fully known. Here, we present cryo-EM structures of oligomeric CRL2FEM1B in its unneddylated state, neddylated state in complex with BEX2 as well as neddylated state in complex with FNIP1/FLCN. These structures reveal that asymmetric dimerization of N8-CRL2FEM1B is critical for the ubiquitylation of BEX2 while FNIP1/FLCN is ubiquitylated by monomeric CRL2FEM1B. Our data present an example of the asymmetric homo-dimerization of CRL. Taken together, this study sheds light on the ubiquitylation strategy of oligomeric CRL2FEM1B according to substrates with different scales.

RNA-binding protein PTENα blocks RIG-I activation to prevent viral inflammation.

A timely inflammatory response is crucial for early viral defense, but uncontrolled inflammation harms the host. Retinoic acid-inducible gene I (RIG-I) has a pivotal role in detecting RNA viruses, yet the regulatory mechanisms governing its sensitivity remain elusive. Here we identify PTENα, an N-terminally extended form of PTEN, as an RNA-binding protein with a preference for the CAUC(G/U)UCAU motif. Using both in vivo and in vitro viral infection assays, we demonstrated that PTENα restricted the host innate immune response, relying on its RNA-binding capacity and phosphatase activity. Mechanistically, PTENα directly bound to viral RNA and enzymatically converted its 5'-triphosphate to 5'-monophosphate, thereby reducing RIG-I sensitivity. Physiologically, brain-intrinsic PTENα exerted protective effects against viral inflammation, while peripheral PTENα restricted host antiviral immunity and, to some extent, promoted viral replication. Collectively, our findings underscore the significance of PTENα in modulating viral RNA- and RIG-I-mediated immune recognition, offering potential therapeutic implications for infectious diseases.

Platelet-Rich Plasma in Young and Elderly Humans Exhibits a Different Proteomic Profile.

As people age, their ability to resist injury and repair damage decreases significantly. Platelet-rich plasma (PRP) has demonstrated diverse therapeutic effects on tissue repair. However, the inconsistency of patient outcomes poses a challenge to the practical application of PRP in clinical practice. Furthermore, a comprehensive understanding of the specific impact of aging on PRP requires a systematic investigation. We derived PRP from 6 young volunteers and 6 elderly volunteers, respectively. Subsequently, 95% of high-abundance proteins were removed, followed by mass spectrometry analysis. Data are available via ProteomeXchange with the identifier PXD050061. We detected a total of 739 proteins and selected 311 proteins that showed significant differences, including 76 upregulated proteins in the young group and 235 upregulated proteins in the elderly group. Functional annotation and enrichment analysis unveiled upregulation of proteins associated with cell apoptosis, angiogenesis, and complement and coagulation cascades in the elderly. Conversely, IGF1 was found to be upregulated in the young group, potentially serving as the central source of enhanced cell proliferation ability. Our investigation not only provides insights into standardizing PRP preparation but also offers novel strategies for augmenting the functionality of aging cells or tissues.

COVID-19 increases extracorporeal coagulation during hemodialysis associated with upregulation of vWF/FBLN5 signaling in patients with severe/critical symptoms.

BACKGROUND: COVID-19 has been shown to increase the risk of extracorporeal coagulation during hemodialysis in patients, but the underlying mechanism remains unclear. This study aimed to investigate the effect and mechanism of COVID-19 on the risk of extracorporeal coagulation in patients with chronic kidney disease undergoing hemodialysis. METHODS: A retrospective analysis of the extracorporeal coagulation status of 339 hemodialysis patients at our center before and after COVID-19 infection was performed, including subgroup analyses. Post-infection blood composition was analyzed by protein spectrometry and ELISA. RESULTS: Compared to the pre-COVID-19 infection period, COVID-19-induced extracorporeal coagulation predominantly occurred in patients with severe/critical symptoms. Further proteomic analysis demonstrated that in patients with severe/critical symptoms, the coagulation cascade reaction, platelet activation, inflammation, and oxidative stress-related pathways were significantly amplified compared to those in patients with no/mild symptoms. Notably, the vWF/FBLN5 pathway, which is associated with inflammation, vascular injury, and coagulation, was significantly upregulated. CONCLUSIONS: Patients with severe/critical COVID-19 symptoms are at a higher risk of extracorporeal coagulation during hemodialysis, which is associated with the upregulation of the vWF/FBLN5 signaling pathway. These findings highlight the importance of early anticoagulant therapy initiation in COVID-19 patients with severe/critical symptoms, particularly those undergoing hemodialysis. Additionally, vWF/FBLN5 upregulation may be a novel mechanism for virus-associated thrombosis/coagulation.

Discovery of 2,4-diarylaminopyrimidine derivatives bearing sulfonamide moiety as novel FAK inhibitors.

Two series of 2,4-diarylaminopyrimidine derivatives containing sulfonamide moiety were designed and synthesized for screening as inhibitors of focal adhesion kinase (FAK). Most compounds significantly inhibited the enzymatic activities of FAK, and the best compound was 7b (IC50 = 0.27 nM). A majority of aminoethyl sulfonamide derivatives could effectively inhibit the proliferation of human cancer cell lines (HCT116, A549, MDA-MB-231 and Hela) expressing high levels of FAK. Particularly, compounds 7b, 7c, and 7o exhibited more significant efficacy against all of four cancer cell lines within concentrations of 1.5 µM. Furthermore, these three compounds displayed higher selectivity of cancer cells over normal cells (SI value > 14), compared to the positive control TAE226 (SI value = 1.63). Interestingly, introduction of dithiocarbamate moiety to the aminoethyl sulfonamide derivatives can indeed improve the antiproliferative activities against A549 cells. Especially, compound 8d demonstrated most significant cytotoxicity activity against A549 cells with an IC50 value of 0.08 µM, which is 20-fold superior to parent compound 7k. Additionally, compound 7b, which display the best anti-FAK potency, can inhibit the clone formation and migration of HCT-116 cells, and cause cell cycle arrest at G2/M phase, inducing apoptosis by promoting ROS production. Overall, these results suggest that 7b is a valuable FAK inhibitor that deserves further optimization to improve its druggability.

Transcranial Ultrasonic Focusing by a Phased Array Based on Micro-CT Images.

In this paper, we utilize micro-computed tomography (micro-CT) to obtain micro-CT images with a resolution of 60 µm and establish a micro-CT model based on the k-wave toolbox, which can visualize the microstructures in trabecular bone, including pores and bone layers. The transcranial ultrasound phased array focusing field characteristics in the micro-CT model are investigated. The ultrasonic waves are multiply scattered in skull and time delays calculations from the transducer to the focusing point are difficult. For this reason, we adopt the pulse compression method and the linear frequency modulation Barker code to compute the time delay and implement phased array focusing in the micro-CT model. It is shown by the simulation results that ultrasonic loss is mainly caused by scattering from the microstructures of the trabecular bone. The ratio of main and side lobes of the cross-correlation calculation is improved by 5.53 dB using the pulse compression method. The focusing quality and the calculation accuracy of time delay are improved. Meanwhile, the beamwidth at the focal point and the sound pressure amplitude decrease with the increase in the signal frequency. Focusing at different depths indicates that the beamwidth broadens with the increase in the focusing depth, and beam deflection focusing maintains good consistency in the focusing effect at a distance of 9 mm from the focal point. This indicates that the phased-array method has good focusing results and focus tunability in deep cranial brain. In addition, the sound pressure at the focal point can be increased by 8.2% through amplitude regulation, thereby enhancing focusing efficiency. The preliminary experiment verification is conducted with an ex vivo skull. It is shown by the experimental results that the phased array focusing method using pulse compression to calculate the time delay can significantly improve the sound field focusing effect and is a very effective transcranial ultrasound focusing method.

CUT&Tag for Efficient Epigenomic Profiling of Frozen Tissues.

Cleavage Under Targets and Tagmentation (CUT&Tag) provides high-resolution sequencing libraries for profiling diverse chromatin components. This protocol details the steps to generate CUT&Tag libraries from fresh or frozen tissues. This CUT&Tag workflow has nine main steps: isolation of nuclei from tissues, binding of nuclei to Concanavalin A-coated beads, binding of the primary antibody, binding of the secondary antibody, binding pA-Tn5 adapter complex, tagmentation, DNA extraction, PCR, and post-PCR cleanup and size selection. This protocol enabled us to generate and sequence CUT&Tag libraries across a broad range of fresh and frozen tissue types.

Quantitative assessment and driving factors analysis of surface urban heat island of urban agglomerations in China based on GEE.

The urban heat island (UHI) effect generated by the development of high-speed urbanization has become one of the major problems affecting the urban ecological environment. As the main body of urbanization in China, China's urban agglomerations are the core areas of urban heat island effect. The purpose of this study is to study the spatial-temporal characteristics and driving factors of surface urban heat island in 19 urban agglomerations in China, with a view to providing theoretical references for the prevention of urban thermal environmental risks. Based on Google Earth Engine (GEE), this paper estimated the surface urban heat island intensity (SUHII) of 19 urban agglomerations in China from 2003 to 2019 using MODIS land surface temperature (LST) data. Correlation analysis and regression analysis were used to explore the correlation between the change of SUHII and driving factors. Finally, the driving factors of SUHII were detected by the geo-detector model. Results showed that (1) the SUHII of 19 urban agglomerations in arid and semi-arid areas of northwestern China is higher than that in humid areas of eastern and southeastern China. (2) The SUHII of 19 urban agglomerations in China generally shows a decreasing trend, and the spatial variation of the change trend is significant. (3) There are positive correlations between SUHII and reference evapotranspiration (ET0), population density (POP), gross domestic product (GDP), nitrogen dioxide (NO2), ozone (O3), and ultraviolet aerosol index (UVAI); negative correlations with normalized difference vegetation index (NDVI), DEM, sulfur dioxide (SO2), carbon monoxide (CO), and formaldehyde (HCHO); the correlations all pass the significance test of P < 0.05 and are statistically significant. (4) The factor detection results showed that NDVI, land cover type (LC), and UVAI were the main driving factors of SUHII. The interaction detection results showed that the interaction between O3 and UVAI had the most significant impact on SUHII.

Robust, stable cooling cellulose composite: Coupling nano-SiO2 and cellulose acetate in natural cellulose.

Passive radiative cooling material of cellulose by coupling inorganic nanoparticles, have demonstrated competitive advantages in sustainably cooling buildings and constructions due to their voluminous availability, biodegradability, renewability, and natural origin. However, the weak stability of cellulose-inorganic nanoparticle materials when exposed to water or external forces remains a significant challenge that impedes their practical application. In this study, we proposed an easy-to-prepare, scalable, and robust cooling cellulose composite by coupling nano-SiO2 and cellulose acetate (CA) within cellulose fibers, using the mature pulping and paper process (filling of inorganic particles of nano-SiO2 and subsequent sizing of polymer of CA). More importantly, the CA molecules form the strong bonding with the cellulose molecules due to the high similarity of their molecular structure, which makes CA function as a "glue" to effectively fasten nano-SiO2 on the cellulose fibers. Correspondingly, our cellulose composite features desirable robustness and structural stability even undergoing mechanical beating and water-soaking treatments, demonstrating its excellent robustness and desirable adaptability to natural environments, such as wind and rain. As a result, despite undergoing water-soaking (for 40 days) or environmental exposure (for 90 days), the cooling cellulose composite still exhibits excellent solar reflectance (>95 %) and infrared thermal emissivity (>0.95 at 8-13 µm), enabling sub-ambient temperature (∼6.5 °C during daytime and ∼8 °C at nighttime) throughout the day. Our cooling cellulose composite demonstrates promising potential as an environmentally friendly, low-cost, and stable cooling material in our low-carbon society.

Tumor-Associated Monocytes Reprogram CD8+ T Cells into Central Memory-Like Cells with Potent Antitumor Effects.

CD8+ T cells are critical for host antitumor responses, whereas persistent antigenic stimulation and excessive inflammatory signals lead to T cell dysfunction or exhaustion. Increasing early memory T cells can improve T cell persistence and empower T cell-mediated tumor eradication, especially for adoptive cancer immunotherapy. Here, it is reported that tumor-associated monocytes (TAMos) are highly correlated with the accumulation of CD8+ memory T cells in human cancers. Further analysis identifies that TAMos selectively reprogram CD8+ T cells into T central memory-like (TCM-like) cells with enhanced recall responses. L-NMMA, a pan nitric oxide synthase inhibitor, can mitigate TAMo-mediated inhibition of T cell proliferation without affecting TCM-like cell generation. Moreover, the modified T cells by TAMo exposure and L-NMMA treatment exhibit long-term persistence and elicit superior antitumor effects in vivo. Mechanistically, the transmembrane protein CD300LG is involved in TAMo-mediated TCM-like cell polarization in a cell-cell contact-dependent manner. Thus, the terminally differentiated TAMo subset (CD300LGhighACElow) mainly contributes to TCM-like cell development. Taken together, these findings establish the significance of TAMos in boosting T-cell antitumor immunity.

A conserved N-terminal motif of CUL3 contributes to assembly and E3 ligase activity of CRL3KLHL22.

The CUL3-RING E3 ubiquitin ligases (CRL3s) play an essential role in response to extracellular nutrition and stress stimuli. The ubiquitin ligase function of CRL3s is activated through dimerization. However, how and why such a dimeric assembly is required for its ligase activity remains elusive. Here, we report the cryo-EM structure of the dimeric CRL3KLHL22 complex and reveal a conserved N-terminal motif in CUL3 that contributes to the dimerization assembly and the E3 ligase activity of CRL3KLHL22. We show that deletion of the CUL3 N-terminal motif impairs dimeric assembly and the E3 ligase activity of both CRL3KLHL22 and several other CRL3s. In addition, we found that the dynamics of dimeric assembly of CRL3KLHL22 generates a variable ubiquitination zone, potentially facilitating substrate recognition and ubiquitination. These findings demonstrate that a CUL3 N-terminal motif participates in the assembly process and provide insights into the assembly and activation of CRL3s.

CircPTEN-MT from PTEN regulates mitochondrial energy metabolism.

Phosphatase and tensin homolog (PTEN) is a multifunctional gene involved in a variety of physiological and pathological processes. Circular RNAs (circRNAs) are generated from back-splicing events during mRNA processing and participate in cell biological processes through binding to RNAs or proteins. However, PTEN-related circRNAs are largely unknown. Here, we report that circPTEN- mitochondria (MT) (hsa_circ_0002934) is a circular RNA encoded by exons 3, 4, and 5 of PTEN and is a critical regulator of mitochondrial energy metabolism. CircPTEN-MT is localized to mitochondria and physically associated with leucine-rich pentatricopeptide repeat-containing protein (LRPPRC), which regulates posttranscriptional gene expression in mitochondria. Knocking down circPTEN-MT reduces the interaction of LRPPRC and steroid receptor RNA activator (SRA) stem-loop interacting RNA binding protein (SLIRP) and inhibits the polyadenylation of mitochondrial mRNA, which decreases the mRNA level of the mitochondrial complex I subunit and reduces mitochondrial membrane potential and adenosine triphosphate production. Our data demonstrate that circPTEN-MT is an important regulator of cellular energy metabolism. This study expands our understanding of the role of PTEN, which produces both linear and circular RNAs with different and independent functions.

Structural and functional insights into the lipid regulation of human anion exchanger 2.

Anion exchanger 2 (AE2) is an electroneutral Na+-independent Cl-/HCO3- exchanger belongs to the SLC4 transporter family. The widely expressed AE2 participates in a variety of physiological processes, including transepithelial acid-base secretion and osteoclastogenesis. Both the transmembrane domains (TMDs) and the N-terminal cytoplasmic domain (NTD) are involved in regulation of AE2 activity. However, the regulatory mechanism remains unclear. Here, we report a 3.2 Å cryo-EM structure of the AE2 TMDs in complex with PIP2 and a 3.3 Å full-length mutant AE2 structure in the resting state without PIP2. We demonstrate that PIP2 at the TMD dimer interface is involved in the substrate exchange process. Mutation in the PIP2 binding site leads to the displacement of TM7 and further stabilizes the interaction between the TMD and the NTD. Reduced substrate transport activity and conformation similar to AE2 in acidic pH indicating the central contribution of PIP2 to the function of AE2.

HLA-DPB1 genotype variants predict DP molecule cell surface expression and DP donor specific antibody binding capacity.

The contribution of alloresponses to mismatched HLA-DP in solid organ transplantation and hematopoietic stem cell transplantation (HCT) has been well documented. Exploring the regulatory mechanisms of DPB1 alleles has become an important question to be answered. In this study, our initial investigation focused on examining the correlation between the rs9277534G/A SNP and DPB1 mRNA expression. The result showed that there was a significant increase in DPB1 mRNA expression in B lymphoblastoid cell lines (BLCLs) with the rs9277534GG genotype compared to rs9277534AA genotype. In addition, B cells with the rs9277534GG exhibited significantly higher DP protein expression than those carrying the rs9277534AA genotype in primary B cells. Furthermore, we observed a significant upregulation of DP expression in B cells following treatment with Interleukin 13 (IL-13) compared to untreated B cells carrying rs9277534GG-linked DPB1 alleles. Fluorescence in situ hybridization (FISH) analysis of DPB1 in BLCL demonstrated significant differences in both the cytoplasmic (p=0.0003) and nuclear (p=0.0001) localization of DP mRNA expression comparing DPB1*04:01 (rs9277534AA) and DPB1*05:01 (rs9277534GG) homozygous cells. The study of the correlation between differential DPB1 expression and long non-coding RNAs (lncRNAs) showed that lnc-HLA-DPB1-13:1 is strongly associated with DP expression (r=0.85), suggesting the potential involvement of lncRNA in regulating DP expression. The correlation of DP donor specific antibody (DSA) with B cell flow crossmatch (B-FCXM) results showed a better linear correlation of DP DSA against GG and AG donor cells (R2 = 0.4243, p=0.0025 and R2 = 0.6172, p=0.0003, respectively), compared to DSA against AA donor cells (R2 = 0.0649, p=0.4244). This explained why strong DP DSA with a low expression DP leads to negative B-FCXM. In conclusion, this study provides evidence supporting the involvement of lncRNA in modulating HLA-DP expression, shedding lights on the intricate regulatory mechanisms of DP, particularly under inflammatory conditions in transplantation.