Single-cell RNA-sequencing analysis reveals enhanced non-canonical neurotrophic factor signaling in the subacute phase of traumatic brain injury.

BACKGROUND: Traumatic brain injury (TBI) is a leading cause of long-term disability in young adults and induces complex neuropathological processes. Cellular autonomous and intercellular changes during the subacute phase contribute substantially to the neuropathology of TBI. However, the underlying mechanisms remain elusive. In this study, we explored the dysregulated cellular signaling during the subacute phase of TBI. METHODS: Single-cell RNA-sequencing data (GSE160763) of TBI were analyzed to explore the cell-cell communication in the subacute phase of TBI. Upregulated neurotrophic factor signaling was validated in a mouse model of TBI. Primary cell cultures and cell lines were used as in vitro models to examine the potential mechanisms affecting signaling. RESULTS: Single-cell RNA-sequencing analysis revealed that microglia and astrocytes were the most affected cells during the subacute phase of TBI. Cell-cell communication analysis demonstrated that signaling mediated by the non-canonical neurotrophic factors midkine (MDK), pleiotrophin (PTN), and prosaposin (PSAP) in the microglia/astrocytes was upregulated in the subacute phase of TBI. Time-course profiling showed that MDK, PTN, and PSAP expression was primarily upregulated in the subacute phase of TBI, and astrocytes were the major source of MDK and PTN after TBI. In vitro studies revealed that the expression of MDK, PTN, and PSAP in astrocytes was enhanced by activated microglia. Moreover, MDK and PTN promoted the proliferation of neural progenitors derived from human-induced pluripotent stem cells (iPSCs) and neurite growth in iPSC-derived neurons, whereas PSAP exclusively stimulated neurite growth. CONCLUSION: The non-canonical neurotrophic factors MDK, PTN, and PSAP were upregulated in the subacute phase of TBI and played a crucial role in neuroregeneration.

Forensic identification of sudden cardiac death: a new approach combining metabolomics and machine learning.

The determination of sudden cardiac death (SCD) is one of the difficult tasks in the forensic practice, especially in the absence of specific morphological changes in the autopsies and histological investigations. In this study, we combined the metabolic characteristics from corpse specimens of cardiac blood and cardiac muscle to predict SCD. Firstly, ultra-high performance liquid chromatography coupled with high-resolution mass spectrometry (UPLC-HRMS)-based untargeted metabolomics was applied to obtain the metabolomic profiles of the specimens, and 18 and 16 differential metabolites were identified in the cardiac blood and cardiac muscle from the corpses of those who died of SCD, respectively. Several possible metabolic pathways were proposed to explain these metabolic alterations, including the metabolism of energy, amino acids, and lipids. Then, we validated the capability of these combinations of differential metabolites to distinguish between SCD and non-SCD through multiple machine learning algorithms. The results showed that stacking model integrated differential metabolites featured from the specimens showed the best performance with 92.31% accuracy, 93.08% precision, 92.31% recall, 91.96% F1 score, and 0.92 AUC. Our results revealed that the SCD metabolic signature identified by metabolomics and ensemble learning in cardiac blood and cardiac muscle has potential in SCD post-mortem diagnosis and metabolic mechanism investigations.

Chinese Medicinal Herb-Derived Carbon Dots for Common Diseases: Efficacies and Potential Mechanisms.

The management of hemorrhagic diseases and other commonly refractory diseases (including gout, inflammatory diseases, cancer, pain of various forms and causes) are very challenging in clinical practice. Charcoal medicine is a frequently used complementary and alternative drug therapy for hemorrhagic diseases. However, studies (other than those assessing effects on hemostasis) on charcoal-processed medicines are limited. Carbon dots (CDs) are quasi-spherical nanoparticles that are biocompatible and have high stability, low toxicity, unique optical properties. Currently, there are various studies carried out to evaluate their efficacy and safety. The exploration of using traditional Chinese medicine (TCM) -based CDs for the treatment of common diseases has received great attention. This review summarizes the literatures on medicinal herbs-derived CDs for the treatment of the difficult-to-treat diseases, and explored the possible mechanisms involved in the process of treatment.

Synergistic Effect of Granular Seed Substrates and Soluble Additives in Structural Control of Prismatic CaCO3 Thin Films.

In biomineralization and bioinspired mineralization, substrates and additives function synergistically in providing structural control of the mineralized layers including their orientation, polymorph, morphology, hierarchical architecture, etc. Herein, a novel type of granular aragonitic CaCO3-poly(acrylic acid) substrate guides the mineralization of prismatic CaCO3 thin films of distinct morphology and polymorph in the presence of different additives including organic compounds and polymers. For instance, weakly charged amino acids lead to columnar aragonite overlayers, while their charged counterparts and organic acids/bases inhibit the overgrowth. Employment of several specific soluble polymer additives in overgrowth instead results in calcitic overlayers with distinct hierarchical architecture, good hardness/Young's modulus, and under-water superoleophobicity. Interestingly, self-organized patterns in the CaCO3-poly(l-glutamic acid) overlayer are obtained under proper mineralization conditions. We demonstrate that the granular seed comprised of mineralized and polymeric constituents is a versatile platform for obtaining prismatic CaCO3 thin films, where structural control can be realized by the employment of different types of additives in overgrowth. We expect the methodology to be applied to a broad spectrum of bioinspired, prismatic-type crystalline products, aiming for the development of high-performance hybrids.

Seeded Mineralization Leads to Hierarchical CaCO3 Thin Coatings on Fibers for Oil/Water Separation Applications.

Like their biogenic counterparts, synthetic minerals with hierarchical architectures should exhibit multiple structural functions, which nicely bridge the boundaries between engineering and functional materials. Nevertheless, design of bioinspired mineralization approaches to thin coatings with distinct micro/nanotextures remains challenging in the realm of materials chemistry. Herein, a general morphosynthetic method based on seeded mineralization was extended to achieve prismatic-type thin CaCO3 coatings on fibrous substrates for oil/water separation applications. Distinct micro/nanotextures of the overlayers could be obtained in mineralization processes in the presence of different soluble (bio)macromolecules. These hierarchical thin coatings therefore exhibit multiple structural functions including underwater superoleophobicity, ultralow adhesion force of oil in water, and comparable stiffness/strength to the prismatic-type biominerals found in mollusk shells. Moreover, this controllable approach could proceed on fibrous substrates to obtain robust thin coatings, so that a modified nylon mesh could be employed for oil/water separation driven by gravity. Our bioinspired approach based on seeded mineralization opens the door for the deposition of hierarchical mineralized thin coatings exhibiting multiple structural functions on planar and fibrous substrates. This bottom-up strategy could be readily extended for the syntheses of advanced thin coatings with a broad spectrum of engineering and functional constituents.

Total morphosynthesis of biomimetic prismatic-type CaCO3 thin films.

Biomimetic mineralization can lead to advanced crystalline composites with common chemicals under ambient conditions. An exceptional example is biomimetic nacre with its superior fracture toughness. The synthesis of the prismatic layer with stiffness and wear resistance nonetheless remains an elusive goal. Herein, we apply a biomimetic mineralization method to grow prismatic-type CaCO3 thin films, mimicking their biogenic counterparts found in mollusk shells with a three-step pathway: coating a polymer substrate, deposition of a granular transition layer, and mineralization of a prismatic overlayer. The synthetic prismatic overlayers exhibit structural similarity and comparable hardness and Young's modulus to their biogenic counterparts. Furthermore, employment of a biomacromolecular soluble additive, silk fibroin, in fabrication of the prismatic thin films leads to micro-/nano-textures with enhanced toughness and emerging under-water superoleophobicity. This study highlights the crucial role of the granular transition layer in promoting competition growth of the prismatic layer.

Proteasome inhibition in vivo promotes survival in a lethal murine model of severe acute respiratory syndrome.

Ubiquitination is a critical regulator of the host immune response to viral infection, and many viruses, including coronaviruses, encode proteins that target the ubiquitination system. To explore the link between coronavirus infection and the ubiquitin system, we asked whether protein degradation by the 26S proteasome plays a role in severe coronavirus infections using a murine model of SARS-like pneumonitis induced by murine hepatitis virus strain 1 (MHV-1). In vitro, the pretreatment of peritoneal macrophages with inhibitors of the proteasome (pyrrolidine dithiocarbamate [PDTC], MG132, and PS-341) markedly inhibited MHV-1 replication at an early step in its replication cycle, as evidenced by inhibition of viral RNA production. Proteasome inhibition also blocked viral cytotoxicity in macrophages, as well as the induction of inflammatory mediators such as IP-10, gamma interferon (IFN-γ), and monocyte chemoattractant protein 1 (MCP-1). In vivo, intranasal inoculation of MHV-1 results in a lethal pneumonitis in A/J mice. Treatment of A/J mice with the proteasome inhibitor PDTC, MG132, or PS-341 led to 40% survival (P < 0.01), with a concomitant improvement of lung histology, reduced pulmonary viral replication, decreased pulmonary STAT phosphorylation, and reduced pulmonary inflammatory cytokine expression. These data demonstrate that inhibition of the cellular proteasome attenuates pneumonitis and cytokine gene expression in vivo by reducing MHV-1 replication and the resulting inflammatory response. The results further suggest that targeting the proteasome may be an effective new treatment for severe coronavirus infections.

Targeted deletion of fgl2 leads to impaired regulatory T cell activity and development of autoimmune glomerulonephritis.

Mice with targeted deletion of fibrinogen-like protein 2 (fgl2) spontaneously developed autoimmune glomerulonephritis with increasing age, as did wild-type recipients reconstituted with fgl2-/- bone marrow. These data implicate FGL2 as an important immunoregulatory molecule and led us to identify the underlying mechanisms. Deficiency of FGL2, produced by CD4+CD25+ regulatory T cells (Treg), resulted in increased T cell proliferation to lectins and alloantigens, Th 1 polarization, and increased numbers of Ab-producing B cells following immunization with T-independent Ags. Dendritic cells were more abundant in fgl2-/- mice and had increased expression of CD80 and MHCII following LPS stimulation. Treg cells were also more abundant in fgl2-/- mice, but their suppressive activity was significantly impaired. Ab to FGL2 completely inhibited Treg cell activity in vitro. FGL2 inhibited dendritic cell maturation and induced apoptosis of B cells through binding to the low-affinity FcgammaRIIB receptor. Collectively, these data suggest that FGL2 contributes to Treg cell activity and inhibits the development of autoimmune disease.

[The study of cis-element HNF4 in the regulation of mfg12 prothrombinase/fibroleukin gene expression in response to nucleocapsid protein of MHV-3].

OBJECTIVE: To identify the transcription factor(s) that is essential for activation of mfgl2 prothrombinase/fibroleukin gene in response to nucleocapsid protein of murine hepatitis virus type 3 (MHV-3). METHODS: Western blotting was performed to investigate whether HNF4 is expressed in macrophages of Ba1b/c mice where mfgl2 is expressed. Confocus microscope immunofluorescence was performed to show whether N protein of MHV enters into the nucleus of infected cells, which is a critical step for the N protein to facilitate its transactivation property. To facilitate the identification of three candidate factor(s) including hepatocyte nuclear factor 4 (HNF4)/liver factor A1 (LF-A1), cytomegalovirus immediate early gene 1.2 (IE1.2) regulatory element and granulocyte- macrophage colony stimulating factor (GM-CSF) in response to mfgl2 activation upon the stimulation of MHV-A59 N protein, gel mobility shift assay (GMSA), competition experiments and site directed mutagenesis were performed. RESULTS: Western blotting displayed that HNF4 was constitutively expressed in macrophages and did not show significant change under the stimulation of different MHV. Confocus microscope immunofluorescence clearly showed that N protein of MHV entered into the nucleus of infected cells. GMSA and competition experiments demonstrated binding to both HNF4 and IE1.2 fragments could be competed with the cold specific oligonucleotides but not with the same amount of non-specific oligos nucleotides. A super shift band was observed when HNF4 antibody was pre-incubated with the nuclear extracts indicating the interaction between the HNF4 element and mfgl2 promoter. Site directed mutagenesis of cis-elements HNF4 (pfgl2HNF4mut) and HNF4/IE1.2 (pfgl2HNF4/IE1.2mut) mutations abolished over 75% of transcription from wild-type mfgl2 promoter. However the pfgl2IE1.2mut displayed almost wild-type promoter activity (75% approximately 80%). CONCLUSIONS: The factor HNF4 binds to mfgl2 promoter and serves as an essential transcription factor for mfgl2/fibroleukin expression in response to MHV-3 N protein.

[Detection of cDNA end sequence of porcine fibrinogen like protein2 and it's structure analysis].

The purpose of this study is to detect the end sequence of porcine FGL2 gene cDNA and make a preliminary analysis of it's structure. Porcine DNA library was screened by a cDNA probe labeled with radioactive isotope alpha-32P dCTP. Rapid amplification of cDNA end (RACE): Retroverse transcription product of total RNA extracted from normal porcine tissue was used as the template, gene specific primers were designed and advantage 2 polymerase mix was used in PCR, of which using porcine genomic DNA as the template:forward primer was designed according to the acquired consensus region of human and pig FGL2 3' sequences while reverse primer was designed from human FGL2 3' end downstream sequence; TA cloning. Screening library failed to get any specific positive clone; the specific transcription initiation site and first poly A signal were successfully detected by RACE reaction although it fails again to detect the second poly A signal. The unknown sequence of porcine FGL2 3' end including the second poly A signal was successfully detected by PCR using genomic DNA as the template. RACE reaction can be applied as an effective method to detect the specific transcription initiation and termination sites. Using advantage 2 polymerase mix instead of regular DNA polymerase may significantly improve the sensitivity,accuracy and specificity of PCR reaction. If it met particular difficulties in the regular screening of DNA library, PCR reaction utilizing primers designed from the known consensus sequence and genomic DNA as template may be considered as an appropriate alternative.