Age-associated changes in immune cells have been linked to an increased risk for infection. However, a global and detailed characterization of the changes that human circulating immune cells undergo with age is lacking. Here, we combined scRNA-seq, mass cytometry and scATAC-seq to compare immune cell types in peripheral blood collected from young and old subjects and patients with COVID-19. We found that the immune cell landscape was reprogrammed with age and was characterized by T cell polarization from naive and memory cells to effector, cytotoxic, exhausted and regulatory cells, along with increased late natural killer cells, age-associated B cells, inflammatory monocytes and age-associated dendritic cells. In addition, the expression of genes, which were implicated in coronavirus susceptibility, was upregulated in a cell subtypespecific manner with age. Notably, COVID-19 promoted age-induced immune cell polarization and gene expression related to inflammation and cellular senescence. Therefore, these findings suggest that a dysregulated immune system and increased gene expression associated with SARS-CoV-2 susceptibility may at least partially account for COVID-19 vulnerability in the elderly.
A major barrier to the use of antimicrobial peptides as antibiotics is the toxicity or ability to lyse eukaryotic cells. In this study, a 26-residue amphipathic α-helical antimicrobial peptide A12L/A20L (Ac-KWKSFLKTFKSLK KTVLHTLLKAISS-amide) was used as the framework to design a series of D- and L-diastereomeric peptides and study the relationships of helicity and biological activities of α-helical antimicrobial peptides. Peptide helicity was measured by circular dichroism spectroscopy and demonstrated to correlate with the hydrophobicity of peptides and the numbers of D-amino acid substitutions. Therapeutic index was used to evaluate the selectivity of peptides against prokaryotic cells. By introducing D-amino acids to replace the original L-amino acids on the non-polar face or the polar face of the helix, the hemolytic activity of peptide analogs have been significantly reduced. Compared to the parent peptide, the therapeutic indices were improved of 44-fold and 22-fold against Gram-negative and Grampositive bacteria, respectively. In addition, D- and L-diastereomeric peptides exhibited lower interaction with zwitterionic eukaryotic membrane and showed the significant membrane damaging effect to bacterial cells. Helicity was proved to play a crucial role on peptide specificity and biological activities. By simply replacing the hydrophobic or the hydrophilic amino acid residues on the non-polar or the polar face of these amphipathic derivatives of the parent peptide with D-amino acids, we demonstrated that this method could have excellent potential for the rational design of antimicrobial peptides with enhanced specificity.
Transcriptional activators are required to turn on the expression of genes in a eukaryotic cell. Activators bound to the enhancer can facilitate either the recruitment of RNA polymerase II to the promoter or its elongation. This article examines a few selected issues in understanding activator functions and activation mechanisms.
Catalases are well studied enzymes that play critical roles in protecting cells against the toxic effects of hydrogen peroxide. The ubiquity of the enzyme and the availability of substrates made heme catalases the focus of many biochemical and molecular biology studies over 100 years. In human, this has been implicated in various physiological and pathological conditions. Advancement in proteomics revealed many of novel and previously unknown features of this mysterious enzyme, but some functional aspects are yet to be explained. Along with discussion on future research area, this mini-review compile the information available on the structure, function and mechanism of action of human catalase.
Genome editing tools such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated system (Cas) have been widely used to modify genes in model systems including animal zygotes and human cells, and hold tremendous promise for both basic research and clinical applications. To date, a serious knowledge gap remains in our understanding of DNA repair mechanisms in human early embryos, and in the efficiency and potential off-target effects of using technologies such as CRISPR/Cas9 in human pre-implantation embryos. In this report, we used tripronuclear (3PN) zygotes to further investigate CRISPR/Cas9-mediated gene editing in human cells. We found that CRISPR/Cas9 could effectively cleave the endogenous β-globin gene (HBB). However, the efficiency of homologous recombination directed repair (HDR) of HBB was low and the edited embryos were mosaic. Off-target cleavage was also apparent in these 3PN zygotes as revealed by the T7E1 assay and whole-exome sequencing. Furthermore, the endogenous delta-globin gene (HBD), which is homologous to HBB, competed with exogenous donor oligos to act as the repair template, leading to untoward mutations. Our data also indicated that repair of the HBB locus in these embryos occurred preferentially through the non-crossover HDR pathway. Taken together, our work highlights the pressing need to further improve the fidelity and specificity of the CRISPR/Cas9 platform, a prerequisite for any clinical applications of CRSIPR/Cas9-mediated editing.
Atherosclerosis is a leading cause of death worldwide, and its mechanisms are still unclear. However, various animal models have significantly advanced our understanding of the mechanisms involved in atherosclerosis and have allowed the evaluation of therapeutic options. The aim of this paper is to review those animal models (i.e., rabbits, mice, rats, guinea pigs, hamsters, avian, carnivores, swine, and, non-human primates) that have been used to study atherosclerosis. Though there is no single perfect animal model that completely replicates the stages of human atherosclerosis, cholesterol feeding and mechanical endothelial injury are two common features shared by most models of atherosclerosis. Further, with the development of genetically modified animals, these models are significantly broadening our understanding of the pathogenesis of atherosclerosis.
Recent advances in the understanding of the molecular processes contributing to autophagy have provided insight into the relationship between autophagy and apoptosis. In contrast to the concept of “autophagic cell death,” accumulating evidence suggests that autophagy serves a largely cytoprotective role in physiologically relevant conditions. The cytoprotective function of autophagy is mediated in many circumstances by negative modulation of apoptosis. Apoptotic signaling, in turn, serves to inhibit autophagy. While the mechanisms mediating the complex counter-regulation of apoptosis and autophagy are not yet fully understood, important points of crosstalk include the interactions between Beclin-1 and Bcl-2/Bcl-xL and between FADD and Atg5, caspase- and calpain-mediated cleavage of autophagy-related proteins, and autophagic degradation of caspases. Continued investigation of these and other means of crosstalk between apoptosis and autophagy is necessary to elucidate the mechanisms controlling the balance between survival and death both under normal conditions and in diseases including cancer.
Ribosomal RNAs are important because they catalyze the synthesis of peptides and proteins. Comparative studies of the secondary structure of 18S rRNA have revealed the basic locations of its many length-conserved and length-variable regions. In recent years, many more sequences of 18S rDNA with unusual lengths have been documented in GenBank. These data make it possible to recognize the diversity of the secondary and tertiary structures of 18S rRNAs and to identify the length-conserved parts of 18S rDNAs. The longest 18S rDNA sequences of almost every known eukaryotic phylum were included in this study. We illustrated the bioinformatics-based structure to show that, the regions that are more length-variable, regions that are less length-variable, the splicing sites for introns, and the sites of A-minor interactions are mostly distributed in different parts of the 18S rRNA. Additionally, this study revealed that some length-variable regions or insertion positions could be quite close to the functional part of the 18S rRNA of Foraminifera organisms. The tertiary structure as well as the secondary structure of 18S rRNA can be more diverse than what was previously supposed. Besides revealing how this interesting gene evolves, it can help to remove ambiguity from the alignment of eukaryotic 18S rDNAs and to improve the performance of 18S rDNA in phylogenetic reconstruction. Six nucleotides shared by Archaea and Eukaryota but rarely by Bacteria are also reported here for the first time, which might further support the supposed origin of eukaryote from archaeans.
Ultraviolet-B (UV-B) is a relatively minor component of sunlight, but can induce stress-related physiological processes or UV-B-specific photomorphogenic responses in plants. In the last decade, significant progress has been made in understanding the UV-B photomorphogenic pathway, including identification of the key components in the pathway, molecular characterization of UV-B photoreceptor and perception mechanism, and elucidation of the signal transduction mechanisms from the photoactivated UV-B receptor to downstream gene expression. This review summarizes the key players identified to date in the UV-B photomorphogenic pathway and their roles in mediating UV-B signal transduction.
Transient receptor potential (TRP) channels are widely found throughout the animal kingdom. By serving as cellular sensors for a wide spectrum of physical and chemical stimuli, they play crucial physiological roles ranging from sensory transduction to cell cycle modulation. TRP channels are tetrameric protein complexes. While most TRP subunits can form functional homomeric channels, heteromerization of TRP channel subunits of either the same subfamily or different subfamilies has been widely observed. Heteromeric TRP channels exhibit many novel properties compared to their homomeric counterparts, indicating that co-assembly of TRP channel subunits has an important contribution to the diversity of TRP channel functions.
The WD40 domain exhibits a β-propeller architecture, often comprising seven blades. The WD40 domain is one of the most abundant domains and also among the top interacting domains in eukaryotic genomes. In this review, we will discuss the identification, definition and architecture of the WD40 domains. WD40 domain proteins are involved in a large variety of cellular processes, in which WD40 domains function as a protein-protein or protein-DNA interaction platform. WD40 domain mediates molecular recognition events mainly through the smaller top surface, but also through the bottom surface and sides. So far, no WD40 domain has been found to display enzymatic activity. We will also discuss the different binding modes exhibited by the large versatile family of WD40 domain proteins. In the last part of this review, we will discuss how post-translational modifications are recognized by WD40 domain proteins.
Inhibition of Mycobacterium tuberculosis (Mtb) cell wall assembly is an established strategy for anti-TB chemotherapy. Arabinosyltransferase EmbB, which catalyzes the transfer of arabinose from the donor decaprenyl-phosphate-arabinose (DPA) to its arabinosyl acceptor is an essential enzyme for Mtb cell wall synthesis. Analysis of drug resistance mutations suggests that EmbB is the main target of the front-line anti-TB drug, ethambutol. Herein, we report the cryo-EM structures of Mycobacterium smegmatis EmbB in its “resting state” and DPA-bound “active state”. EmbB is a fifteentransmembrane-spanning protein, assembled as a dimer. Each protomer has an associated acyl-carrierprotein (AcpM) on their cytoplasmic surface. Conformational changes upon DPA binding indicate an asymmetric movement within the EmbB dimer during catalysis. Functional studies have identified critical residues in substrate recognition and catalysis, and demonstrated that ethambutol inhibits transferase activity of EmbB by competing with DPA. The structures represent the first step directed towards a rational approach for anti-TB drug discovery.
Sodium-glucose cotransporter 2 (SGLT2) inhibitors reduce cardiovascular mortality in patients with diabetes mellitus but the protective mechanism remains elusive. Here we demonstrated that the SGLT2 inhibitor, Empagliflozin (EMPA), suppresses cardiomyocytes autosis (autophagic cell death) to confer cardioprotective effects. Using myocardial infarction (MI) mouse models with and without diabetes mellitus, EMPA treatment significantly reduced infarct size, and myocardial fibrosis, thereby leading to improved cardiac function and survival. In the context of ischemia and nutritional glucose deprivation where autosis is already highly stimulated, EMPA directly inhibits the activity of the Na+/H+ exchanger 1 (NHE1) in the cardiomyocytes to regulate excessive autophagy. Knockdown of NHE1 significantly rescued glucose deprivation-induced autosis. In contrast, overexpression of NHE1 aggravated the cardiomyocytes death in response to starvation, which was effectively rescued by EMPA treatment. Furthermore, in vitro and in vivo analysis of NHE1 and Beclin 1 knockout mice validated that EMPA’s cardioprotective effects are at least in part through downregulation of autophagic flux. These findings provide new insights for drug development, specifically targeting NHE1 and autosis for ventricular remodeling and heart failure after MI in both diabetic and non-diabetic patients.
Emerging and re-emerging RNA viruses occasionally cause epidemics and pandemics worldwide, such as the on-going outbreak of the novel coronavirus SARS-CoV-2. Herein, we identified two potent inhibitors of human DHODH, S312 and S416, with favorable drug-likeness and pharmacokinetic profiles, which all showed broadspectrum antiviral effects against various RNA viruses, including influenza A virus, Zika virus, Ebola virus, and particularly against SARS-CoV-2. Notably, S416 is reported to be the most potent inhibitor so far with an EC50 of 17 nmol/L and an SI value of 10,505.88 in infected cells. Our results are the first to validate that DHODH is an attractive host target through high antiviral efficacy invivoand low virus replication in DHODH knock-out cells. This work demonstrates that both S312/S416 and old drugs (Leflunomide/Teriflunomide) with dual actions of antiviral and immuno-regulation may have clinical potentials to cure SARS-CoV-2 or other RNA viruses circulating worldwide, no matter such viruses are mutated or not.
Many human genetic diseases, including Hutchinson-Gilford progeria syndrome (HGPS), are caused by single point mutations. HGPS is a rare disorder that causes premature aging and is usually caused by a de novo point mutation in the LMNA gene. Base editors (BEs) composed of a cytidine deaminase fused to CRISPR/Cas9 nickase are highly efficient at inducing C to T base conversions in a programmable manner and can be used to generate animal disease models with single amino-acid substitutions. Here, we generated the first HGPS monkey model by delivering a BE mRNA and guide RNA (gRNA) targeting the LMNA gene via microinjection into monkey zygotes. Five out of six newborn monkeys carried the mutation specifically at the target site. HGPS monkeys expressed the toxic form of lamin A, progerin, and recapitulated the typical HGPS phenotypes including growth retardation, bone alterations, and vascular abnormalities. Thus, this monkey model genetically and clinically mimics HGPS in humans, demonstrating that the BE system can efficiently and accurately generate patient-specific disease models in non-human primates.
Common γ chain cytokines are important for immune memory formation. Among them, the role of IL-2 remains to be fully explored. It has been suggested that this cytokine is critically needed in the late phase of primary CD4 T cell activation. Lack of IL-2 at this stage sets for a diminished recall response in subsequent challenges. However, as IL-2 peak production is over at this point, the source and the exact mechanism that promotes its production remain elusive. We report here that resting, previously antigen-stimulated CD4 T cells maintain a minimalist response to dendritic cells after their peak activation in vitro. This subtle activation event may be induced by DCs without overt presence of antigen and appears to be stronger if IL-2 comes from the same dendritic cells. This encounter reactivates a miniature IL-2 production and leads a gene expression profile change in these previously activated CD4 T cells. The CD4 T cells so experienced show enhanced reactivation intensity upon secondary challenges later on. Although mostly relying on in vitro evidence, our work may implicate a subtle programing for CD4 T cell survival after primary activation in vitro.
Fertilization is a process involving multiple steps that lead to the final fusion of one sperm and the oocyte to form the zygote. One of the steps, acrosome reaction (AR), is an exocytosis process, during which the outer acrosome membrane fuses with the inner sperm membrane, leading to the release of acrosome enzymes that facilitate sperm penetration of the egg investments. Though AR has been investigated for decades, the initial steps of AR
Autophagy and apoptosis are both highly regulated biological processes that play essential roles in tissue homeostasis, development and diseases. Autophagy is also described as a mechanism of death pathways, however, the precise mechanism of how autophagy links to cell death remains to be fully understood. Beclin 1 is a dual regulator for both autophagy and apoptosis. In this study we found that Beclin 1 was a substrate of caspase-3 with two cleavage sites at positions 124 and 149, respectively. Furthermore, the autophagosome formation occurred, followed by the appearance of morphological hallmarks of apoptosis after staurosporine treatment. The cleavage products of Beclin 1 reduced autophagy and promoted apoptosis in HeLa cells and the cells in which Beclin 1 was stably knocked down by specific shRNA. In addition, the cleavage of Beclin 1 resulted in abrogating the interaction between Bcl-2 with Beclin 1, which could be blocked by z-VAD-fmk. Thus, our results suggest that the cleavage of Beclin 1 by caspase-3 may contribute to inactivate autophagy leading towards augmented apoptosis.
Brain and the gastrointestinal (GI) tract are intimately connected to form a bidirectional neurohumoral communication system. The communication between gut and brain, knows as the gut-brain axis, is so well established that the functional status of gut is always related to the condition of brain. The researches on the gut-brain axis were traditionally focused on the psychological status affecting the function of the GI tract. However, recent evidences showed that gut microbiota communicates with the brain via the gut-brain axis to modulate brain development and behavioral phenotypes. These recent fi ndings on the new role of gut microbiota in the gut-brain axis implicate that gut microbiota could associate with brain functions as well as neurological diseases via the gut-brain axis. To elucidate the role of gut microbiota in the gut-brain axis, precise identification of the composition of microbes constituting gut microbiota is an essential step. However, identifi cation of microbes constituting gut microbiota has been the main technological challenge currently due to massive amount of intestinal microbes and the diffi culties in culture of gut microbes. Current methods for identifi cation of microbes constituting gut microbiota are dependent on omics analysis methods by using advanced high tech equipment. Here, we review the association of gut microbiota with the gut-brain axis, including the pros and cons of the current high throughput methods for identifi cation of microbes constituting gut microbiota to elucidate the role of gut microbiota in the gut-brain axis.
Prostate cancer is the most commonly diagnosed noncutaneous cancers in North American men. While androgen deprivation has remained as the cornerstone of prostate cancer treatment, resistance ensues leading to lethal disease. Forkhead box A1 (FOXA1) encodes a pioneer factor that induces open chromatin conformation to allow the binding of other transcription factors. Through direct interactions with the Androgen Receptor (AR), FOXA1 helps to shape AR signaling that drives the growth and survival of normal prostate and prostate cancer cells. FOXA1 also possesses an AR-independent role of regulating epithelial-to-mesenchymal transition (EMT). In prostate cancer, mutations converge onto the coding sequence and cis-regulatory elements (CREs) of FOXA1, leading to functional alterations. In addition, FOXA1 activity in prostate cancer can be modulated post-translationally through various mechanisms such as LSD1-mediated protein demethylation. In this review, we describe the latest discoveries related to the function and regulation of FOXA1 in prostate cancer, pointing to their relevance to guide future clinical interventions.
Two-dimensional gel electrophoresis (2-DE) is a gel-based technique widely used for analyzing the protein composition of biological samples. It is capable of resolving complex mixtures containing more than a thousand protein components into individual protein spots through the coupling of two orthogonal biophysical separation techniques: isoelectric focusing (first dimension) and polyacrylamide gel electrophoresis (second dimension). 2-DE is ideally suited for analyzing the entire expressed protein complement of a bacterial cell: its proteome. Its relative simplicity and good reproducibility have led to 2-DE being widely used for exploring proteomics within a wide range of environmental and medically-relevant bacteria. Here we give a broad overview of the basic principles and historical development of gel-based proteomics, and how this powerful approach can be applied for studying bacterial biology and physiology. We highlight specific 2-DE applications that can be used to analyze when, where and how much proteins are expressed. The links between proteomics, genomics and mass spectrometry are discussed. We explore how proteomics involving tandem mass spectrometry can be used to analyze (post-translational) protein modifications or to identify proteins of unknown origin by
17β-estradiol (E2) treatment of cells results in an upregulation of SIRT1 and a down-regulation of PPARγ. The decrease in PPARγ expression is mediated by increased degradation of PPARγ. Here we report that PPARγ is ubiquitinated by HECT E3 ubiquitin ligase NEDD4-1 and degraded, along with PPARγ, in response to E2 stimulation. The PPARγ interacts with ubiquitin ligase NEDD4-1 through a conserved PPXY-WW binding motif. The WW3 domain in NEDD4-1 is critical for binding to PPARγ. NEDD4-1 overexpression leads to PPARγ ubiquitination and reduced expression of PPARγ. Conversely, knockdown of NEDD4-1 by specific siRNAs abolishes PPARγ ubiquitination. These data indicate that NEDD4-1 is the E3 ubiquitin ligase responsible for PPARγ ubiquitination. Here, we show that NEDD4-1 delays cellular senescence by degrading PPARγ expression. Taken together, our data show that E2 could upregulate SIRT1 expression via promoting the PPARγ ubiquitination-proteasome degradation pathway to delay the process of cell senescence.
c-Jun, the most extensively studied protein of the activator protein-1 (AP-1) complex, is involved in numerous cell activities, such as proliferation, apoptosis, survival, tumorigenesis and tissue morphogenesis. Earlier studies focused on the structure and function have led to the identification of c-Jun as a basic leucine zipper (bZIP) transcription factor that acts as homo- or hetero-dimer, binding to DNA and regulating gene transcription. Later on, it was shown that extracellular signals can induce post-translational modifications of c-Jun, resulting in altered transcriptional activity and target gene expression. More recent work has uncovered multiple layers of a complex regulatory scheme in which c-Jun is able to crosstalk, amplify and integrate different signals for tissue development and disease. One example of such scheme is the autocrine amplification loop, in which signal-induced AP-1 activates the